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Sample records for histidine kinase inhibitor

  1. Discovery of inhibitors of bacterial histidine kinases

    NARCIS (Netherlands)

    Velikova, N.R.

    2014-01-01

    Discovery of Inhibitors of Bacterial Histidine Kinases Summary

    The thesis is on novel antibacterial drug discovery (http://youtu.be/NRMWOGgeysM). Using structure-based and fragment-based drug discovery approach, we have identified small-molecule histidine-kinase

  2. Structure-based discovery of inhibitors of the YycG histidine kinase

    DEFF Research Database (Denmark)

    Qin, X.; Zhang, J.; Xu, B.

    2006-01-01

    inhibitors of YycG histidine kinase thus are of potential value as leads for developing new antibiotics against infecting staphylococci. The structure-based virtual screening (SBVS) technology can be widely used in screening potential inhibitors of other bacterial TCSs, since it is more rapid and efficacious...... than traditional screening technology....

  3. Structure of the sporulation histidine kinase inhibitor Sda from Bacillus subtilis and insights into its solution state

    Energy Technology Data Exchange (ETDEWEB)

    Jacques, David A.; Streamer, Margaret [School of Molecular and Microbial Biosciences, University of Sydney (Australia); Rowland, Susan L.; King, Glenn F. [Institute of Molecular Biology, University of Queensland (Australia); Guss, J. Mitchell; Trewhella, Jill; Langley, David B., E-mail: d.langley@usyd.edu.au [School of Molecular and Microbial Biosciences, University of Sydney (Australia)

    2009-06-01

    The crystal structure of Sda, a DNA-replication/damage checkpoint inhibitor of sporulation in B. subtilis, has been solved via the MAD method. The subunit arrangement in the crystal has enabled a reappraisal of previous biophysical data, resulting in a new model for the behaviour of the protein in solution. The crystal structure of the DNA-damage checkpoint inhibitor of sporulation, Sda, from Bacillus subtilis, has been solved by the MAD technique using selenomethionine-substituted protein. The structure closely resembles that previously solved by NMR, as well as the structure of a homologue from Geobacillus stearothermophilus solved in complex with the histidine kinase KinB. The structure contains three molecules in the asymmetric unit. The unusual trimeric arrangement, which lacks simple internal symmetry, appears to be preserved in solution based on an essentially ideal fit to previously acquired scattering data for Sda in solution. This interpretation contradicts previous findings that Sda was monomeric or dimeric in solution. This study demonstrates the difficulties that can be associated with the characterization of small proteins and the value of combining multiple biophysical techniques. It also emphasizes the importance of understanding the physical principles behind these techniques and therefore their limitations.

  4. Structural Insights into the HWE Histidine Kinase Family: The Brucella Blue Light-Activated Histidine Kinase Domain.

    Science.gov (United States)

    Rinaldi, Jimena; Arrar, Mehrnoosh; Sycz, Gabriela; Cerutti, María Laura; Berguer, Paula M; Paris, Gastón; Estrín, Darío Ariel; Martí, Marcelo Adrián; Klinke, Sebastián; Goldbaum, Fernando Alberto

    2016-03-27

    In response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism. The Brucella histidine kinase (HK) domain belongs to the HWE family, for which there is no structural information. The HWE family is exclusively present in proteobacteria and usually coupled to a wide diversity of light sensor domains. This work reports the crystal structure of the Brucella HK domain, which presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. Contrary to these crystallographic structures and unlike other HKs, in solution, the Brucella HK domain is monomeric and still active, showing an astonishing instability of the dimeric interface. Despite this instability, using cross-linking experiments, we show that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Broadening the antibacterial spectrum of histidine kinase autophosphorylation inhibitors via the use of ε-poly-L-lysine capped mesoporous silica-based nanoparticles

    NARCIS (Netherlands)

    Velikova, Nadya; Mas, Nuria; Miguel-Romero, Laura; Polo, Lorena; Stolte, Ellen; Zaccaria, Edoardo; Cao, Rui; Taverne, Nico; Murguía, José Ramón; Martinez-Manez, Ramon; Marina, Alberto; Wells, Jerry

    2017-01-01

    Two-component systems (TCS) regulate diverse processes such as virulence, stress responses, metabolism and antibiotic resistance in bacteria but are absent in humans, making them promising targets for novel antibacterials. By incorporating recently described TCS histidine kinase autophosphorylation

  6. The identification of four histidine kinases that influence sporulation in Clostridium thermocellum.

    Science.gov (United States)

    Mearls, Elizabeth B; Lynd, Lee R

    2014-08-01

    In this study, we sought to identify genes involved in the onset of spore formation in Clostridium thermocellum via targeted gene deletions, gene over-expression, and transcriptional analysis. We determined that three putative histidine kinases, clo1313_0286, clo1313_2735 and clo1313_1942 were positive regulators of sporulation, while a fourth kinase, clo1313_1973, acted as a negative regulator. Unlike Bacillus or other Clostridium species, the deletion of a single positively regulating kinase was sufficient to abolish sporulation in this organism. Sporulation could be restored in these asporogenous strains via overexpression of any one of the positive regulators, indicating a high level of redundancy between these kinases. In addition to having a sporulation defect, deletion of clo1313_2735 produced L-forms. Thus, this kinase may play an additional role in repressing L-form formation. This work suggests that C. thermocellum enters non-growth states based on the sensory input from multiple histidine kinases. The ability to control the development of non-growth states at the genetic level has the potential to inform strategies for improved strain development, as well as provide valuable insight into C. thermocellum biology. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Structural and Functional Aspects of the Sensor Histidine Kinase PrrB from Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Nowak, E.; Panjikar, S.; Morth, J.P.

    2006-01-01

    We describe the solution structures of two- and three-domain constructs of the sensor histidine kinase PrrB from Mycobacterium tuberculosis, which allow us to locate the HAMP linker relative to the ATP binding and dimerization domains. We show that the three-domain construct is active both...

  8. Tyrosine kinase inhibitors: Multi-targeted or single-targeted?

    Science.gov (United States)

    Broekman, Fleur; Giovannetti, Elisa; Peters, Godefridus J

    2011-02-10

    Since in most tumors multiple signaling pathways are involved, many of the inhibitors in clinical development are designed to affect a wide range of targeted kinases. The most important tyrosine kinase families in the development of tyrosine kinase inhibitors are the ABL, SCR, platelet derived growth factor, vascular endothelial growth factor receptor and epidermal growth factor receptor families. Both multi-kinase inhibitors and single-kinase inhibitors have advantages and disadvantages, which are related to potential resistance mechanisms, pharmacokinetics, selectivity and tumor environment. In different malignancies various tyrosine kinases are mutated or overexpressed and several resistance mechanisms exist. Pharmacokinetics is influenced by interindividual differences and differs for two single targeted inhibitors or between patients treated by the same tyrosine kinase inhibitor. Different tyrosine kinase inhibitors have various mechanisms to achieve selectivity, while differences in gene expression exist between tumor and stromal cells. Considering these aspects, one type of inhibitor can generally not be preferred above the other, but will depend on the specific genetic constitution of the patient and the tumor, allowing personalized therapy. The most effective way of cancer treatment by using tyrosine kinase inhibitors is to consider each patient/tumor individually and to determine the strategy that specifically targets the consequences of altered (epi)genetics of the tumor. This strategy might result in treatment by a single multi kinase inhibitor for one patient, but in treatment by a couple of single kinase inhibitors for other patients.

  9. Alkali metals in addition to acidic pH activate the EvgS histidine kinase sensor in Escherichia coli.

    Science.gov (United States)

    Eguchi, Yoko; Utsumi, Ryutaro

    2014-09-01

    Two-component signal transduction systems (TCSs) in bacteria perceive environmental stress and transmit the information via phosphorelay to adjust multiple cellular functions for adaptation. The EvgS/EvgA system is a TCS that confers acid resistance to Escherichia coli cells. Activation of the EvgS sensor initiates a cascade of transcription factors, EvgA, YdeO, and GadE, which induce the expression of a large group of acid resistance genes. We searched for signals activating EvgS and found that a high concentration of alkali metals (Na(+), K(+)) in addition to low pH was essential for the activation. EvgS is a histidine kinase, with a large periplasmic sensor region consisting of two tandem PBPb (bacterial periplasmic solute-binding protein) domains at its N terminus. The periplasmic sensor region of EvgS was necessary for EvgS activation, and Leu152, located within the first PBPb domain, was involved in the activation. Furthermore, chimeras of EvgS and PhoQ histidine kinases suggested that alkali metals were perceived at the periplasmic sensor region, whereas the cytoplasmic linker domain, connecting the transmembrane region and the histidine kinase domain, was required for low-pH perception. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  10. A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA.

    Science.gov (United States)

    Schaenzer, Adam J; Wlodarchak, Nathan; Drewry, David H; Zuercher, William J; Rose, Warren E; Striker, Rob; Sauer, John-Demian

    2017-10-13

    Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial P enicillin-binding-protein A nd S erine/ T hreonine kinase- A ssociated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition.

  11. Comparative analysis of LytS/LytTR-type histidine kinase/response regulator systems in γ-proteobacteria.

    Directory of Open Access Journals (Sweden)

    Stefan Behr

    Full Text Available Bacterial histidine kinase/response regulator systems operate at the interface between environmental cues and physiological states. Escherichia coli contains two LytS/LytTR-type histidine kinase/response regulator systems, BtsS/BtsR (formerly YehU/YehT and YpdA/YpdB, which have been identified as pyruvate-responsive two-component systems. Since they exhibit remarkable similarity, we analyzed their phylogenetic distribution within the γ-proteobacteria, and experimentally characterized them in a set of representative species. We found that BtsS/BtsR is the predominant LytS/LytTR-type two-component system among γ-proteobacteria, whereas YpdA/YpdB primarily appears in a supplementary role. Based on our observations in E. coli, we used the highly conserved DNA-binding motifs to test the in vivo functionality of both systems in various genera, including Salmonella, Enterobacter, Citrobacter, Xenorhabdus, Yersinia, Aeromonas and Vibrio. The results suggest that, in all cases tested, BtsS/BtsR and YpdA/YpdB respond to different levels of pyruvate in the environment.

  12. Janus kinase inhibitors: jackpot or potluck?

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    Pavithran Keechilat

    2012-06-01

    Full Text Available The reports of a unique mutation in the Janus kinase-2 gene (JAK2 in polycythemia vera by several independent groups in 2005 quickly spurred the development of the Janus kinase inhibitors. In one of the great victories of translational research in recent times, the first smallmolecule Janus kinase inhibitor ruxolitinib entered a phase I trial in 2007. With the approval of ruxolitinib by the US Federal Drug Administration in November 2011 for high-risk and intermediate-2 risk myelofibrosis, a change in paradigm has occurred in the management of a subset of myeloproliferative neoplasms (MPN: primary myelofibrosis, post-polycythemia vera myelofibrosis, and post-essential thrombocythemia myelofibrosis. Whereas the current evidence for ruxolitinib only covers high-risk and intermediate-2 risk myelofibrosis, inhibitors with greater potency are likely to offer better disease control and survival advantage in patients belonging to these categories, and possibly to the low-risk and intermediate-1 risk categories of MPN as well. But use of the Janus kinase inhibitors also probably has certain disadvantages, such as toxicity, resistance, withdrawal phenomenon, non-reversal of histology, and an implausible goal of disease clone eradication, some of which could offset the gains. In spite of this, Janus kinase inhibitors are here to stay, and for use in more than just myeloproliferative neoplasms.

  13. Allosteric small-molecule kinase inhibitors

    DEFF Research Database (Denmark)

    Wu, Peng; Clausen, Mads Hartvig; Nielsen, Thomas E.

    2015-01-01

    current barriers of kinase inhibitors, including poor selectivity and emergence of drug resistance. In spite of the small number of identified allosteric inhibitors in comparison with that of inhibitors targeting the ATP pocket, encouraging results, such as the FDA-approval of the first small...

  14. Janus Associated Kinases Inhibitors in the Pharmacological Thera

    Directory of Open Access Journals (Sweden)

    Daniela Santos1

    2017-01-01

    Full Text Available Janus associated kinases inhibitors are a new strategy for the treatment of different clinical conditions like immunologic, inflammatory and oncology disorders. The aim of this study was to perform a review of all Janus associated kinases inhibitors available in national and international pharmaceutical market, their therapeutic indications and adverse effects, and the potential indications for investigation of those already available in the pharmaceutical market. It was also performed a review of the main new Janus associated kinases inhibitors that are still in clinical research. A literature review was conducted by consulting the summary of product characteristics of Janus associated kinases inhibitors available in the pharmaceutical market and a research in the bibliographic database PubMed using the terms «JAK inhibitors», «Janus associated kinases inhibitors» and «Janus kinases inhibitors». Ninety-five publications were included in the present review, published from January 2014 to January 2015. Drug databases of the European Medicines Agency and United States Food and Drug Administration were also consulted to search for Janus associated kinases inhibitors authorized in clinical practice. Currently, ruxolitinib and tofacitinib are available in the pharmaceutical market and oclatinib is approved as a veterinary medicinal product. Both drugs approved for human use have major adverse effects at hematological and immunological levels, which enhance the importance of the pharmacist’s role in the monitoring of patients involved in these treatments. However, several molecules are in pre-clinical and clinical studies trying to prove its potential in the treatment of several immunologic, inflammatory and oncology disorders. Thus, it is still necessary to deepen the knowledge in this area in order to overcome the risks of therapy with these agents. These risks weighed against the benefits of its clinical use have compromised the progress of

  15. The Essential WalK Histidine Kinase and WalR Regulator Differentially Mediate Autolysis of Staphylococcus aureus RN4220.

    Science.gov (United States)

    Zheng, Li; Yan, Meiying; Fan, Frank; Ji, Yinduo

    2015-06-01

    The two-component regulatory system, WalR/WalK is necessary for growth of different gram-positive bacteria, including Staphylococcus aureus . In present study, we confirmed the essentiality of both the histidine kinase protein WalK and the response regulator WalR for growth using S. aureus RN4220 strain and demonstrated that the histidine kinase protein WalK and the response regulator WalR function differently in regulation of staphylococcal autolysis. The down-regulation of walR expression effectively inhibited Triton X-100-induced lysis and had a weak impact on bacterial tolerance to penicillin induced cell lysis. In contrast, the down-regulation of walK expression had no influence on either Triton X-100- or penicillin-caused autolysis. Moreover, we determined the effect of WalR and WalK on bacterial hydrolase activity using a zymogram analysis. The results showed that the cell lysate of down-regulated walR expression mutant displayed several bands of decreased cell wall hydrolytic activities; however, the down-regulation of WalK had no dramatic impact on the hydrolytic activities. Furthermore, we examined the impact of WalR on the transcription of cidA associated with staphylococcal autolysis, and the results showed that the down-regulation of WalR led to decreased transcription of cidA in the log phase of growth. Taken together, the above results suggest that the essential WalR response regulator and the essential WalK histidine kinase might differently control bacterial lysis in RN4220 strain.

  16. Potassium sensing histidine kinase in Bacillus subtilis.

    Science.gov (United States)

    López, Daniel; Gontang, Erin A; Kolter, Roberto

    2010-01-01

    The soil-dwelling organism Bacillus subtilis is able to form multicellular aggregates known as biofilms. It was recently reported that the process of biofilm formation is activated in response to the presence of various, structurally diverse small-molecule natural products. All of these small-molecule natural products made pores in the membrane of the bacterium, causing the leakage of potassium cations from the cytoplasm of the cell. The potassium cation leakage was sensed by the membrane histidine kinase KinC, triggering the genetic pathway to the production of the extracellular matrix that holds cells within the biofilm. This chapter presents the methodology used to characterize the leakage of cytoplasmic potassium as the signal that induces biofilm formation in B. subtilis via activation of KinC. Development of novel techniques to monitor activation of gene expression in microbial populations led us to discover the differentiation of a subpopulation of cells specialized to produce the matrix that holds all cells together within the biofilm. This phenomenon of cell differentiation was previously missed by conventional techniques used to monitor transcriptional gene expression. Copyright © 2010 Elsevier Inc. All rights reserved.

  17. The putative sensor histidine kinase CKI1 is involved in female gametophyte development in Arabidopsis

    Czech Academy of Sciences Publication Activity Database

    Hejátko, Jan; Pernisová, M.; Eneva, T.; Palme, K.; Brzobohatý, Břetislav

    2003-01-01

    Roč. 269, č. 4 (2003), s. 443-453 ISSN 1617-4615 R&D Projects: GA MŠk VS96096; GA MŠk LN00A081 Grant - others:INCO-Copernicus(XE) ERB3512-PL966135; QLRT(XE) 2000-0020 Institutional research plan: CEZ:AV0Z5004920 Keywords : female gametophyte development * two-component signaling * sensor histidine kinase Subject RIV: BO - Biophysics Impact factor: 2.240, year: 2003

  18. Proton NMR Studies of a Large Protein. pH, Substrate Titrations, and NOESY Experiments with Perdeuterated Yeast Phosphoglycerate Kinase Containing [ 1H]Histidine Residues

    Science.gov (United States)

    Pappu, K. M.; Serpersu, E. H.

    Fully deuterated yeast phosphoglycerate kinase ([ 2H]PGK) was prepared biosynthetically with only histidine side chains of normal ( 1H) isotopic composition. The 1H NMR spectrum of this enzyme([ 1H]His[ 2H]PGK) showed that the histidine side chains are clearly visible as sharp signals. Thus detailed structural studies by 1H NMR became feasible with isotope-hybrid phosphoglycerate kinase which is otherwise too large ( Mr ˜ 46,000) for conventional 1H NMR studies. Proton signals of bound substrates were visible in the 1H NMR spectrum even with a substrate-to-enzyme ratio of less than 1/2 (mol/mol). The 2D NOESY spectrum of enzyme-MgdATP-glycerol 3-phosphate complex showed that, although protein concentration was very high (1.5 m M), no intraprotein cross peaks were observed other than those of intraresidue histidine NOE cross peaks. In addition, intrasubstrate NOEs and intermolecular NOEs between histidine and substrate protons were visible at a 1.5/1 substrate/enzyme (mol/mol) ratio. Paramagnetic effects of a substrate analog, Cr(III)ATP, on some of the histidine side chains indicated that the formation of the ternary enzyme-substrate complex causes large conformational changes in the enzyme.

  19. Sensor histidine kinase is a β-lactam receptor and induces resistance to β-lactam antibiotics.

    Science.gov (United States)

    Li, Lu; Wang, Qiyao; Zhang, Hui; Yang, Minjun; Khan, Mazhar I; Zhou, Xiaohui

    2016-02-09

    β-Lactams disrupt bacterial cell wall synthesis, and these agents are the most widely used antibiotics. One of the principle mechanisms by which bacteria resist the action of β-lactams is by producing β-lactamases, enzymes that degrade β-lactams. In Gram-negative bacteria, production of β-lactamases is often induced in response to the antibiotic-associated damage to the cell wall. Here, we have identified a previously unidentified mechanism that governs β-lactamase production. In the Gram-negative enteric pathogen Vibrio parahaemolyticus, we found a histidine kinase/response regulator pair (VbrK/VbrR) that controls expression of a β-lactamase. Mutants lacking either VbrK or VbrR do not produce the β-lactamase and are no longer resistant to β-lactam antibiotics. Notably, VbrK autophosphorylation is activated by β-lactam antibiotics, but not by other lactams. However, single amino acid substitutions in the putative periplasmic binding pocket of VbrK leads its phosphorylation in response to both β-lactam and other lactams, suggesting that this kinase is a β-lactam receptor that can directly detect β-lactam antibiotics instead of detecting the damage to cell wall resulting from β-lactams. In strong support of this idea, we found that purified periplasmic sensor domain of VbrK binds penicillin, and that such binding is critical for VbrK autophosphorylation and β-lactamase production. Direct recognition of β-lactam antibiotics by a histidine kinase receptor may represent an evolutionarily favorable mechanism to defend against β-lactam antibiotics.

  20. The 'retro-design' concept for novel kinase inhibitors.

    Science.gov (United States)

    Müller, Gerhard; Sennhenn, Peter C; Woodcock, Timothy; Neumann, Lars

    2010-07-01

    Protein kinases are among the most attractive therapeutic targets for a broad range of diseases. This feature review highlights and classifies the main design principles employed to generate active and selective kinase inhibitors. In particular, emphasis is focused on a fragment-based lead-generation approach, which constitutes a novel design method for developing type II kinase inhibitors with distinct binding kinetic attributes. This 'retro-design' strategy relies on a customized fragment library, and contrasts the traditional approach used in the design of type II inhibitors.

  1. Oncogenic Receptor Tyrosine Kinases Directly Phosphorylate Focal Adhesion Kinase (FAK) as a Resistance Mechanism to FAK-Kinase Inhibitors.

    Science.gov (United States)

    Marlowe, Timothy A; Lenzo, Felicia L; Figel, Sheila A; Grapes, Abigail T; Cance, William G

    2016-12-01

    Focal adhesion kinase (FAK) is a major drug target in cancer and current inhibitors targeted to the ATP-binding pocket of the kinase domain have entered clinical trials. However, preliminary results have shown limited single-agent efficacy in patients. Despite these unfavorable data, the molecular mechanisms that drive intrinsic and acquired resistance to FAK-kinase inhibitors are largely unknown. We have demonstrated that receptor tyrosine kinases (RTK) can directly bypass FAK-kinase inhibition in cancer cells through phosphorylation of FAK's critical tyrosine 397 (Y397). We also showed that HER2 forms a direct protein-protein interaction with the FAK-FERM-F1 lobe, promoting direct phosphorylation of Y397. In addition, FAK-kinase inhibition induced two forms of compensatory RTK reprogramming: (i) the rapid phosphorylation and activation of RTK signaling pathways in RTK High cells and (ii) the long-term acquisition of RTKs novel to the parental cell line in RTK Low cells. Finally, HER2 +: cancer cells displayed resistance to FAK-kinase inhibition in 3D growth assays using a HER2 isogenic system and HER2 + cancer cell lines. Our data indicate a novel drug resistance mechanism to FAK-kinase inhibitors whereby HER2 and other RTKs can rescue and maintain FAK activation (pY397) even in the presence of FAK-kinase inhibition. These data may have important ramifications for existing clinical trials of FAK inhibitors and suggest that individual tumor stratification by RTK expression would be important to predict patient response to FAK-kinase inhibitors. Mol Cancer Ther; 15(12); 3028-39. ©2016 AACR. ©2016 American Association for Cancer Research.

  2. Sensor histidine kinase is a β-lactam receptor and induces resistance to β-lactam antibiotics

    OpenAIRE

    Li, Lu; Wang, Qiyao; Zhang, Hui; Yang, Minjun; Khan, Mazhar I.; Zhou, Xiaohui

    2016-01-01

    Bacteria can produce β-lactamases, enzymes that destroy β-lactam antibiotics and thereby resist these potent antibiotics that target cell wall synthesis. Production of β-lactamases is often controlled by β-lactam-induced perturbations in the cell wall. Here, we have identified a new mechanism controlling β-lactamase production. We found a signaling system in which a membrane-associated histidine kinase directly binds β-lactams, triggering the expression of a β-lactamase and resistance to β-la...

  3. Identification of novel bacterial histidine biosynthesis inhibitors using docking, ensemble rescoring, and whole-cell assays

    DEFF Research Database (Denmark)

    Henriksen, Signe Teuber; Liu, J.; Estiu, G.

    2010-01-01

    histidine biosynthesis pathway, which is predicted to be essential for bacterial biomass productions. Virtual screening of a library of similar to 10(6) compounds identified 49 potential inhibitors of three enzymes of this pathway. Eighteen representative compounds were directly tested on three S. aureus......-and two Escherichia coli strains in standard disk inhibition assays. Thirteen compounds are inhibitors of some or all of the S. aureus strains, while 14 compounds weakly inhibit growth in one or both E. coli strains. The high hit rate obtained from a fast virtual screen demonstrates the applicability...

  4. Kinase inhibitors: a new class of antirheumatic drugs

    Directory of Open Access Journals (Sweden)

    Kyttaris VC

    2012-09-01

    Full Text Available Vasileios C KyttarisDivision of Rheumatology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USAAbstract: The outlook for patients with rheumatoid arthritis has improved significantly over the last three decades with the use of disease-modifying antirheumatic drugs. However, despite the use of methotrexate, cytokine inhibitors, and molecules targeting T and B cells, a percentage of patients do not respond or lose their response over time. The autoimmune process in rheumatoid arthritis depends on activation of immune cells, which utilize intracellular kinases to respond to external stimuli such as cytokines, immune complexes, and antigens. In the past decade, small molecules targeting several kinases, such as p38 MAPK, Syk, and JAK have been developed. Several p38 MAPK inhibitors proved ineffective in treating rheumatoid arthritis. The Syk inhibitor, fostamatinib, proved superior to placebo in Phase II trials and is currently under Phase III investigation. Tofacitinib, a JAK1/3 inhibitor, was shown to be efficacious in two Phase III trials, while VX-509, a JAK3 inhibitor, showed promising results in a Phase II trial. Fostamatinib and tofacitinib were associated with increased rates of infection, elevation of liver enzymes, and neutropenia. Moreover, fostamatinib caused elevations of blood pressure and diarrhea, while tofacitinib was associated with an increase in creatinine and elevation of lipid levels.Keywords: rheumatoid arthritis, kinase inhibitors, mitogen-activated phosphokinase p38, spleen tyrosine kinase, Janus kinases

  5. Second-generation inhibitors of Bruton tyrosine kinase

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    Jingjing Wu

    2016-09-01

    Full Text Available Abstract Bruton tyrosine kinase (BTK is a critical effector molecule for B cell development and plays a major role in lymphoma genesis. Ibrutinib is the first-generation BTK inhibitor. Ibrutinib has off-target effects on EGFR, ITK, and Tec family kinases, which explains the untoward effects of ibrutinib. Resistance to ibrutinib was also reported. The C481S mutation in the BTK kinase domain was reported to be a major mechanism of resistance to ibrutinib. This review summarizes the clinical development of novel BTK inhibitors, ACP-196 (acalabrutinib, ONO/GS-4059, and BGB-3111.

  6. The protein histidine phosphatase LHPP is a tumour suppressor.

    Science.gov (United States)

    Hindupur, Sravanth K; Colombi, Marco; Fuhs, Stephen R; Matter, Matthias S; Guri, Yakir; Adam, Kevin; Cornu, Marion; Piscuoglio, Salvatore; Ng, Charlotte K Y; Betz, Charles; Liko, Dritan; Quagliata, Luca; Moes, Suzette; Jenoe, Paul; Terracciano, Luigi M; Heim, Markus H; Hunter, Tony; Hall, Michael N

    2018-03-29

    Histidine phosphorylation, the so-called hidden phosphoproteome, is a poorly characterized post-translational modification of proteins. Here we describe a role of histidine phosphorylation in tumorigenesis. Proteomic analysis of 12 tumours from an mTOR-driven hepatocellular carcinoma mouse model revealed that NME1 and NME2, the only known mammalian histidine kinases, were upregulated. Conversely, expression of the putative histidine phosphatase LHPP was downregulated specifically in the tumours. We demonstrate that LHPP is indeed a protein histidine phosphatase. Consistent with these observations, global histidine phosphorylation was significantly upregulated in the liver tumours. Sustained, hepatic expression of LHPP in the hepatocellular carcinoma mouse model reduced tumour burden and prevented the loss of liver function. Finally, in patients with hepatocellular carcinoma, low expression of LHPP correlated with increased tumour severity and reduced overall survival. Thus, LHPP is a protein histidine phosphatase and tumour suppressor, suggesting that deregulated histidine phosphorylation is oncogenic.

  7. A Duo of Potassium-Responsive Histidine Kinases Govern the Multicellular Destiny of Bacillus subtilis.

    Science.gov (United States)

    Grau, Roberto R; de Oña, Paula; Kunert, Maritta; Leñini, Cecilia; Gallegos-Monterrosa, Ramses; Mhatre, Eisha; Vileta, Darío; Donato, Verónica; Hölscher, Theresa; Boland, Wilhelm; Kuipers, Oscar P; Kovács, Ákos T

    2015-07-07

    Multicellular biofilm formation and surface motility are bacterial behaviors considered mutually exclusive. However, the basic decision to move over or stay attached to a surface is poorly understood. Here, we discover that in Bacillus subtilis, the key root biofilm-controlling transcription factor Spo0A~Pi (phosphorylated Spo0A) governs the flagellum-independent mechanism of social sliding motility. A Spo0A-deficient strain was totally unable to slide and colonize plant roots, evidencing the important role that sliding might play in natural settings. Microarray experiments plus subsequent genetic characterization showed that the machineries of sliding and biofilm formation share the same main components (i.e., surfactin, the hydrophobin BslA, exopolysaccharide, and de novo-formed fatty acids). Sliding proficiency was transduced by the Spo0A-phosphorelay histidine kinases KinB and KinC. We discovered that potassium, a previously known inhibitor of KinC-dependent biofilm formation, is the specific sliding-activating signal through a thus-far-unnoticed cytosolic domain of KinB, which resembles the selectivity filter sequence of potassium channels. The differential expression of the Spo0A~Pi reporter abrB gene and the different levels of the constitutively active form of Spo0A, Sad67, in Δspo0A cells grown in optimized media that simultaneously stimulate motile and sessile behaviors uncover the spatiotemporal response of KinB and KinC to potassium and the gradual increase in Spo0A~Pi that orchestrates the sequential activation of sliding, followed by sessile biofilm formation and finally sporulation in the same population. Overall, these results provide insights into how multicellular behaviors formerly believed to be antagonistic are coordinately activated in benefit of the bacterium and its interaction with the host. Alternation between motile and sessile behaviors is central to bacterial adaptation, survival, and colonization. However, how is the collective

  8. 2-Aminobenzimidazoles as potent Aurora kinase inhibitors.

    Science.gov (United States)

    Zhong, Min; Bui, Minna; Shen, Wang; Baskaran, Subramanian; Allen, Darin A; Elling, Robert A; Flanagan, W Michael; Fung, Amy D; Hanan, Emily J; Harris, Shannon O; Heumann, Stacey A; Hoch, Ute; Ivy, Sheryl N; Jacobs, Jeffrey W; Lam, Stuart; Lee, Heman; McDowell, Robert S; Oslob, Johan D; Purkey, Hans E; Romanowski, Michael J; Silverman, Jeffrey A; Tangonan, Bradley T; Taverna, Pietro; Yang, Wenjin; Yoburn, Josh C; Yu, Chul H; Zimmerman, Kristin M; O'Brien, Tom; Lew, Willard

    2009-09-01

    This Letter describes the discovery and key structure-activity relationship (SAR) of a series of 2-aminobenzimidazoles as potent Aurora kinase inhibitors. 2-Aminobenzimidazole serves as a bioisostere of the biaryl urea residue of SNS-314 (1c), which is a potent Aurora kinase inhibitor and entered clinical testing in patients with solid tumors. Compared to SNS-314, this series of compounds offers better aqueous solubility while retaining comparable in vitro potency in biochemical and cell-based assays; in particular, 6m has also demonstrated a comparable mouse iv PK profile to SNS-314.

  9. Approved and Experimental Small-Molecule Oncology Kinase Inhibitor Drugs: A Mid-2016 Overview.

    Science.gov (United States)

    Fischer, Peter M

    2017-03-01

    Kinase inhibitor research is a comparatively recent branch of medicinal chemistry and pharmacology and the first small-molecule kinase inhibitor, imatinib, was approved for clinical use only 15 years ago. Since then, 33 more kinase inhibitor drugs have received regulatory approval for the treatment of a variety of cancers and the volume of reports on the discovery and development of kinase inhibitors has increased to an extent where it is now difficult-even for those working in the field-easily to keep an overview of the compounds that are being developed, as currently there are 231 such compounds, targeting 38 different protein and lipid kinases (not counting isoforms), in clinical use or under clinical investigation. The purpose of this review is thus to provide an overview of the biomedical rationales for the kinases being targeted on the one hand, and the design principles, as well as chemical, pharmacological, pharmaceutical, and toxicological kinase inhibitor properties, on the other hand. Two issues that are especially important in kinase inhibitor research, target selectivity and drug resistance, as well as the underlying structural concepts, are discussed in general terms and in the context of relevant kinases and their inhibitors. © 2016 Wiley Periodicals, Inc.

  10. Computational methods for analysis and inference of kinase/inhibitor relationships

    Directory of Open Access Journals (Sweden)

    Fabrizio eFerrè

    2014-06-01

    Full Text Available The central role of kinases in virtually all signal transduction networks is the driving motivation for the development of compounds modulating their activity. ATP-mimetic inhibitors are essential tools for elucidating signaling pathways and are emerging as promising therapeutic agents. However, off-target ligand binding and complex and sometimes unexpected kinase/inhibitor relationships can occur for seemingly unrelated kinases, stressing that computational approaches are needed for learning the interaction determinants and for the inference of the effect of small compounds on a given kinase. Recently published high-throughput profiling studies assessed the effects of thousands of small compound inhibitors, covering a substantial portion of the kinome. This wealth of data paved the road for computational resources and methods that can offer a major contribution in understanding the reasons of the inhibition, helping in the rational design of more specific molecules, in the in silico prediction of inhibition for those neglected kinases for which no systematic analysis has been carried yet, in the selection of novel inhibitors with desired selectivity, and offering novel avenues of personalized therapies.

  11. Discovery of novel inhibitors for Leishmania nucleoside diphosphatase kinase (NDK) based on its structural and functional characterization

    Science.gov (United States)

    Mishra, Arjun K.; Singh, Nidhi; Agnihotri, Pragati; Mishra, Shikha; Singh, Saurabh P.; Kolli, Bala K.; Chang, Kwang Poo; Sahasrabuddhe, Amogh A.; Siddiqi, M. I.; Pratap, J. Venkatesh

    2017-06-01

    Nucleoside diphosphate kinases (NDKs) are ubiquitous enzymes that catalyze the transfer of the γ-phosphate moiety from an NTP donor to an NDP acceptor, crucial for maintaining the cellular level of nucleoside triphosphates (NTPs). The inability of trypanosomatids to synthesize purines de novo and their dependence on the salvage pathway makes NDK an attractive target to develop drugs for the diseases they cause. Here we report the discovery of novel inhibitors for Leishmania NDK based on the structural and functional characterization of purified recombinant NDK from Leishmania amazonensis. Recombinant LaNDK possesses auto-phosphorylation, phosphotransferase and kinase activities with Histidine 117 playing an essential role. LaNDK crystals were grown by hanging drop vapour diffusion method in a solution containing 18% PEG-MME 500, 100 mM Bis-Tris propane pH 6.0 and 50 mM MgCl2. It belongs to the hexagonal space group P6322 with unit cell parameters a = b = 115.18, c = 62.18 Å and α = β = 90°, γ = 120°. The structure solved by molecular replacement methods was refined to crystallographic R-factor and Rfree values of 22.54 and 26.52%, respectively. Molecular docking and dynamics simulation -based virtual screening identified putative binding compounds. Protein inhibition studies of selected hits identified five inhibitors effective at micromolar concentrations. One of the compounds showed 45% inhibition of Leishmania promastigotes proliferation. Analysis of inhibitor-NDK complexes reveals the mode of their binding, facilitating design of new compounds for optimization of activities as drugs against leishmaniasis.

  12. Canine osteosarcoma cells exhibit resistance to aurora kinase inhibitors.

    Science.gov (United States)

    Cannon, C M; Pozniak, J; Scott, M C; Ito, D; Gorden, B H; Graef, A J; Modiano, J F

    2015-03-01

    We evaluated the effect of Aurora kinase inhibitors AZD1152 and VX680 on canine osteosarcoma cells. Cytotoxicity was seen in all four cell lines; however, half-maximal inhibitory concentrations were significantly higher than in human leukaemia and canine lymphoma cells. AZD1152 reduced Aurora kinase B phosphorylation, indicating resistance was not because of failure of target recognition. Efflux mediated by ABCB1 and ABCG2 transporters is one known mechanism of resistance against these drugs and verapamil enhanced AZD1152-induced apoptosis; however, these transporters were only expressed by a small percentage of cells in each line and the effects of verapamil were modest, suggesting other mechanisms contribute to resistance. Our results indicate that canine osteosarcoma cells are resistant to Aurora kinase inhibitors and suggest that these compounds are unlikely to be useful as single agents for this disease. Further investigation of these resistance mechanisms and the potential utility of Aurora kinase inhibitors in multi-agent protocols is warranted. © 2013 Blackwell Publishing Ltd.

  13. Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies.

    Science.gov (United States)

    Duong, Minh Ngoc; Matera, Eva-Laure; Mathé, Doriane; Evesque, Anne; Valsesia-Wittmann, Sandrine; Clémenceau, Béatrice; Dumontet, Charles

    2015-01-01

    Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated.

  14. Clinical Pharmacology of Kinase Inhibitors in Oncology : Personalized and Optimzed Dosing

    NARCIS (Netherlands)

    Verheijen, Remy B.

    2017-01-01

    Kinase inhibitors are an important category of molecularly targeted therapies used for cancer. Verheijen’s doctoral thesis describes several clinical pharmacological studies to optimize and personalize the treatment of cancer with kinase inhibitors, using pharmacokinetics, molecular imaging and

  15. Novel Bruton's tyrosine kinase inhibitors currently in development

    Directory of Open Access Journals (Sweden)

    D'Cruz OJ

    2013-03-01

    Full Text Available Osmond J D'Cruz,1 Fatih M Uckun1,21Children's Center for Cancer and Blood Diseases, Children's Hospital Los Angeles, Los Angeles, CA, USA; 2Department of Pediatrics, University of Southern California, Los Angeles, CA, USAAbstract: Bruton's tyrosine kinase (Btk is intimately involved in multiple signal-transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. Btk is overexpressed and constitutively active in several B-lineage lymphoid malignancies. Btk has emerged as a new antiapoptotic molecular target for treatment of B-lineage leukemias and lymphomas. Preclinical and early clinical results indicate that Btk inhibitors may be useful in the treatment of leukemias and lymphomas.Keywords: tyrosine kinase, personalized therapy, kinase inhibitors, Btk, leukemia, lymphoma

  16. ATP forms a stable complex with the essential histidine kinase WalK (YycG) domain

    Energy Technology Data Exchange (ETDEWEB)

    Celikel, Reha; Veldore, Vidya Harini [University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205 (United States); Mathews, Irimpan [Stanford Synchrotron Radiation Lightsource, 2575 Sand Hill Road, Menlo Park, CA 94025 (United States); Devine, Kevin M., E-mail: kdevine@tcd.ie [Trinity College Dublin, Dublin 2 (Ireland); Varughese, Kottayil I., E-mail: kdevine@tcd.ie [University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205 (United States)

    2012-07-01

    The histidine WalK (YycG) plays a crucial role in coordinating murein synthesis with cell division and the crystal structure of its ATP binding domain has been determined. Interestingly the bound ATP was not hydrolyzed during crystallization and remains intact in the crystal lattice. In Bacillus subtilis, the WalRK (YycFG) two-component system coordinates murein synthesis with cell division. It regulates the expression of autolysins that function in cell-wall remodeling and of proteins that modulate autolysin activity. The transcription factor WalR is activated upon phosphorylation by the histidine kinase WalK, a multi-domain homodimer. It autophosphorylates one of its histidine residues by transferring the γ-phosphate from ATP bound to its ATP-binding domain. Here, the high-resolution crystal structure of the ATP-binding domain of WalK in complex with ATP is presented at 1.61 Å resolution. The bound ATP remains intact in the crystal lattice. It appears that the strong binding interactions and the nature of the binding pocket contribute to its stability. The triphosphate moiety of ATP wraps around an Mg{sup 2+} ion, providing three O atoms for coordination in a near-ideal octahedral geometry. The ATP molecule also makes strong interactions with the protein. In addition, there is a short contact between the exocyclic O3′ of the sugar ring and O2B of the β-phosphate, implying an internal hydrogen bond. The stability of the WalK–ATP complex in the crystal lattice suggests that such a complex may exist in vivo poised for initiation of signal transmission. This feature may therefore be part of the sensing mechanism by which the WalRK two-component system is so rapidly activated when cells encounter conditions conducive for growth.

  17. Recent Advances in the Development and Application of Radiolabeled Kinase Inhibitors for PET Imaging

    Directory of Open Access Journals (Sweden)

    Vadim Bernard-Gauthier

    2015-12-01

    Full Text Available Over the last 20 years, intensive investigation and multiple clinical successes targeting protein kinases, mostly for cancer treatment, have identified small molecule kinase inhibitors as a prominent therapeutic class. In the course of those investigations, radiolabeled kinase inhibitors for positron emission tomography (PET imaging have been synthesized and evaluated as diagnostic imaging probes for cancer characterization. Given that inhibitor coverage of the kinome is continuously expanding, in vivo PET imaging will likely find increasing applications for therapy monitoring and receptor density studies both in- and outside of oncological conditions. Early investigated radiolabeled inhibitors, which are mostly based on clinically approved tyrosine kinase inhibitor (TKI isotopologues, have now entered clinical trials. Novel radioligands for cancer and PET neuroimaging originating from novel but relevant target kinases are currently being explored in preclinical studies. This article reviews the literature involving radiotracer design, radiochemistry approaches, biological tracer evaluation and nuclear imaging results of radiolabeled kinase inhibitors for PET reported between 2010 and mid-2015. Aspects regarding the usefulness of pursuing selective vs. promiscuous inhibitor scaffolds and the inherent challenges associated with intracellular enzyme imaging will be discussed.

  18. Resorufin: a lead for a new protein kinase CK2 inhibitor

    DEFF Research Database (Denmark)

    Sandholt, Iben Skjøth; Olsen, Birgitte Brinkmann; Guerra, Barbara

    2009-01-01

    Screening a natural compound library led to the identification of resorufin as a highly selective and potent inhibitor of protein kinase CK2. Out of 52 kinases tested, only CK2 was inhibited, in contrast to emodin, a structurally related, known CK2 inhibitor that, in addition to CK2, inhibited te...

  19. FDA-approved small-molecule kinase inhibitors

    DEFF Research Database (Denmark)

    Wu, Peng; Nielsen, Thomas E.; Clausen, Mads Hartvig

    2015-01-01

    Kinases have emerged as one of the most intensivelypursued targets in current pharmacological research,especially for cancer, due to their critical roles in cellularsignaling. To date, the US FDA has approved 28 smallmoleculekinase inhibitors, half of which were approvedin the past 3 years. While...

  20. Mechanisms of High Temperature Resistance of Synechocystis sp. PCC 6803: An Impact of Histidine Kinase 34

    Directory of Open Access Journals (Sweden)

    Jan Červený

    2015-03-01

    Full Text Available Synechocystis sp. PCC 6803 is a widely used model cyanobacterium for studying responses and acclimation to different abiotic stresses. Changes in transcriptome, proteome, lipidome, and photosynthesis in response to short term heat stress are well studied in this organism, and histidine kinase 34 (Hik34 is shown to play an important role in mediating such response. Corresponding data on long term responses, however, are fragmentary and vary depending on parameters of experiments and methods of data collection, and thus are hard to compare. In order to elucidate how the early stress responses help cells to sustain long-term heat stress, as well as the role of Hik34 in prolonged acclimation, we examined the resistance to long-term heat stress of wild-type and ΔHik34 mutant of Synechocystis. In this work, we were able to precisely control the long term experimental conditions by cultivating Synechocystis in automated photobioreactors, measuring selected physiological parameters within a time range of minutes. In addition, morphological and ultrastructural changes in cells were analyzed and western blotting of individual proteins was used to study the heat stress-affected protein expression. We have shown that the majority of wild type cell population was able to recover after 24 h of cultivation at 44 °C. In contrast, while ΔHik34 mutant cells were resistant to heat stress within its first hours, they could not recover after 24 h long high temperature treatment. We demonstrated that the early induction of HspA expression and maintenance of high amount of other HSPs throughout the heat incubation is critical for successful adaptation to long-term stress. In addition, it appears that histidine kinase Hik34 is an essential component for the long term high temperature resistance.

  1. Mechanisms of High Temperature Resistance of Synechocystis sp. PCC 6803: An Impact of Histidine Kinase 34.

    Science.gov (United States)

    Červený, Jan; Sinetova, Maria A; Zavřel, Tomáš; Los, Dmitry A

    2015-03-02

    Synechocystis sp. PCC 6803 is a widely used model cyanobacterium for studying responses and acclimation to different abiotic stresses. Changes in transcriptome, proteome, lipidome, and photosynthesis in response to short term heat stress are well studied in this organism, and histidine kinase 34 (Hik34) is shown to play an important role in mediating such response. Corresponding data on long term responses, however, are fragmentary and vary depending on parameters of experiments and methods of data collection, and thus are hard to compare. In order to elucidate how the early stress responses help cells to sustain long-term heat stress, as well as the role of Hik34 in prolonged acclimation, we examined the resistance to long-term heat stress of wild-type and ΔHik34 mutant of Synechocystis. In this work, we were able to precisely control the long term experimental conditions by cultivating Synechocystis in automated photobioreactors, measuring selected physiological parameters within a time range of minutes. In addition, morphological and ultrastructural changes in cells were analyzed and western blotting of individual proteins was used to study the heat stress-affected protein expression. We have shown that the majority of wild type cell population was able to recover after 24 h of cultivation at 44 °C. In contrast, while ΔHik34 mutant cells were resistant to heat stress within its first hours, they could not recover after 24 h long high temperature treatment. We demonstrated that the early induction of HspA expression and maintenance of high amount of other HSPs throughout the heat incubation is critical for successful adaptation to long-term stress. In addition, it appears that histidine kinase Hik34 is an essential component for the long term high temperature resistance.

  2. Aurora B kinase inhibition in mitosis: strategies for optimising the use of aurora kinase inhibitors such as AT9283.

    Science.gov (United States)

    Curry, Jayne; Angove, Hayley; Fazal, Lynsey; Lyons, John; Reule, Matthias; Thompson, Neil; Wallis, Nicola

    2009-06-15

    Aurora kinases play a key role in regulating mitotic division and are attractive oncology targets. AT9283, a multi-targeted kinase inhibitor with potent activity against Aurora A and B kinases, inhibited growth and survival of multiple solid tumor cell lines and was efficacious in mouse xenograft models. AT9283-treatment resulted in endoreduplication and ablation of serine-10 histone H3 phosphorylation in both cells and tumor samples, confirming that in these models it acts as an Aurora B kinase inhibitor. In vitro studies demonstrated that exposure to AT9283 for one complete cell cycle committed an entire population of p53 checkpoint-compromised cells (HCT116) to multinucleation and death whereas treatment of p53 checkpoint-competent cells (HMEC, A549) for a similar length of time led to a reversible arrest of cells with 4N DNA. Further studies in synchronized cell populations suggested that exposure to AT9283 during mitosis was critical for optimal cytotoxicity. We therefore investigated ways in which these properties might be exploited to optimize the efficacy and therapeutic index of Aurora kinase inhibitors for p53 checkpoint compromised tumors in vivo. Combining Aurora B kinase inhibition with paclitaxel, which arrests cells in mitosis, in a xenograft model resulted in promising efficacy without additional toxicity. These findings have implications for optimizing the efficacy of Aurora kinase inhibitors in clinical practice.

  3. Chemical Proteomics Reveals Ferrochelatase as a Common Off-target of Kinase Inhibitors.

    Science.gov (United States)

    Klaeger, Susan; Gohlke, Bjoern; Perrin, Jessica; Gupta, Vipul; Heinzlmeir, Stephanie; Helm, Dominic; Qiao, Huichao; Bergamini, Giovanna; Handa, Hiroshi; Savitski, Mikhail M; Bantscheff, Marcus; Médard, Guillaume; Preissner, Robert; Kuster, Bernhard

    2016-05-20

    Many protein kinases are valid drug targets in oncology because they are key components of signal transduction pathways. The number of clinical kinase inhibitors is on the rise, but these molecules often exhibit polypharmacology, potentially eliciting desired and toxic effects. Therefore, a comprehensive assessment of a compound's target space is desirable for a better understanding of its biological effects. The enzyme ferrochelatase (FECH) catalyzes the conversion of protoporphyrin IX into heme and was recently found to be an off-target of the BRAF inhibitor Vemurafenib, likely explaining the phototoxicity associated with this drug in melanoma patients. This raises the question of whether FECH binding is a more general feature of kinase inhibitors. To address this, we applied a chemical proteomics approach using kinobeads to evaluate 226 clinical kinase inhibitors for their ability to bind FECH. Surprisingly, low or submicromolar FECH binding was detected for 29 of all compounds tested and isothermal dose response measurements confirmed target engagement in cells. We also show that Vemurafenib, Linsitinib, Neratinib, and MK-2461 reduce heme levels in K562 cells, verifying that drug binding leads to a loss of FECH activity. Further biochemical and docking experiments identified the protoporphyrin pocket in FECH as one major drug binding site. Since the genetic loss of FECH activity leads to photosensitivity in humans, our data strongly suggest that FECH inhibition by kinase inhibitors is the molecular mechanism triggering photosensitivity in patients. We therefore suggest that a FECH assay should generally be part of the preclinical molecular toxicology package for the development of kinase inhibitors.

  4. Structural Biology Insight for the Design of Sub-type Selective Aurora Kinase Inhibitors.

    Science.gov (United States)

    Sarvagalla, Sailu; Coumar, Mohane Selvaraj

    2015-01-01

    Aurora kinase A, B and C, are key regulators of mitosis and are over expressed in many of the human cancers, making them an ideal drug target for cancer chemotherapy. Currently, over a dozen of Aurora kinase inhibitors are in various phases of clinical development. The majority of the inhibitors (VX-680/MK-0457, PHA-739358, CYC116, SNS-314, AMG 900, AT-9283, SCH- 1473759, ABT-348, PF-03814735, R-763/AS-703569, KW-2449 and TAK-901) are pan-selective (isoform non-selective) and few are Aurora A (MLN8054, MLN8237, VX-689/MK5108 and ENMD 2076) and Aurora B (AZD1152 and GSK1070916) sub-type selective. Despite the intensive research efforts in the past decade, no Aurora kinase inhibitor has reached the market. Recent evidence suggests that the sub-type selective Aurora kinase A inhibitor could possess advantages over pan-selective Aurora inhibitors, by avoiding Aurora B mediated neutropenia. However, sub-type selective Aurora kinase A inhibitor design is very challenging due to the similarity in the active site among the isoforms. Structural biology and computational aspects pertaining to the design of Aurora kinase inhibitors were analyzed and found that a possible means to develop sub-type selective inhibitor is by targeting Aurora A specific residues (Leu215, Thr217 and Arg220) or Aurora B specific residues (Arg159, Glu161 and Lys164), near the solvent exposed region of the protein. Particularly, a useful strategy for the design of sub-type selective Aurora A inhibitor could be by targeting Thr217 residue as in the case of MLN8054. Further preclinical and clinical studies with the sub-type selective Aurora inhibitors could help bring them to the market for the treatment of cancer.

  5. Exploiting Chemical Libraries, Structure, and Genomics in the Search for Kinase Inhibitors

    NARCIS (Netherlands)

    Gray, Nathanael S.; Wodicka, Lisa; Thunnissen, Andy-Mark W.H.; Norman, Thea C.; Kwon, Soojin; Espinoza, F. Hernan; Morgan, David O.; Barnes, Georjana; LeClerc, Sophie; Meijer, Laurent; Kim, Sung-Hou; Lockhart, David J.; Schultz, Peter G.

    1998-01-01

    Selective protein kinase inhibitors were developed on the basis of the unexpected binding mode of 2,6,9-trisubstituted purines to the adenosine triphosphate-binding site of the human cyclin-dependent kinase 2 (CDK2). By iterating chemical library synthesis and biological screening, potent inhibitors

  6. A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

    Science.gov (United States)

    Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

    1996-01-01

    Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

  7. A Duo of Potassium-Responsive Histidine Kinases Govern the Multicellular Destiny of Bacillus subtilis

    Science.gov (United States)

    de Oña, Paula; Kunert, Maritta; Leñini, Cecilia; Gallegos-Monterrosa, Ramses; Mhatre, Eisha; Vileta, Darío; Hölscher, Theresa; Kuipers, Oscar P.

    2015-01-01

    ABSTRACT Multicellular biofilm formation and surface motility are bacterial behaviors considered mutually exclusive. However, the basic decision to move over or stay attached to a surface is poorly understood. Here, we discover that in Bacillus subtilis, the key root biofilm-controlling transcription factor Spo0A~Pi (phosphorylated Spo0A) governs the flagellum-independent mechanism of social sliding motility. A Spo0A-deficient strain was totally unable to slide and colonize plant roots, evidencing the important role that sliding might play in natural settings. Microarray experiments plus subsequent genetic characterization showed that the machineries of sliding and biofilm formation share the same main components (i.e., surfactin, the hydrophobin BslA, exopolysaccharide, and de novo-formed fatty acids). Sliding proficiency was transduced by the Spo0A-phosphorelay histidine kinases KinB and KinC. We discovered that potassium, a previously known inhibitor of KinC-dependent biofilm formation, is the specific sliding-activating signal through a thus-far-unnoticed cytosolic domain of KinB, which resembles the selectivity filter sequence of potassium channels. The differential expression of the Spo0A~Pi reporter abrB gene and the different levels of the constitutively active form of Spo0A, Sad67, in Δspo0A cells grown in optimized media that simultaneously stimulate motile and sessile behaviors uncover the spatiotemporal response of KinB and KinC to potassium and the gradual increase in Spo0A~Pi that orchestrates the sequential activation of sliding, followed by sessile biofilm formation and finally sporulation in the same population. Overall, these results provide insights into how multicellular behaviors formerly believed to be antagonistic are coordinately activated in benefit of the bacterium and its interaction with the host. PMID:26152584

  8. The Azaindole Framework in the Design of Kinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Jean-Yves Mérour

    2014-11-01

    Full Text Available This review article illustrates the growing use of azaindole derivatives as kinase inhibitors and their contribution to drug discovery and innovation. The different protein kinases which have served as targets and the known molecules which have emerged from medicinal chemistry and Fragment-Based Drug Discovery (FBDD programs are presented. The various synthetic routes used to access these compounds and the chemical pathways leading to their synthesis are also discussed. An analysis of their mode of binding based on X-ray crystallography data gives structural insights for the design of more potent and selective inhibitors.

  9. Small-molecule kinase inhibitors: an analysis of FDA-approved drugs

    DEFF Research Database (Denmark)

    Wu, Peng; Nielsen, Thomas Eiland; Clausen, Mads Hartvig

    2016-01-01

    Small-molecule kinase inhibitors (SMKIs), 28 of which are approved by the US Food and Drug Administration (FDA), have been actively pursued as promising targeted therapeutics. Here, we assess the key structural and physicochemical properties, target selectivity and mechanism of function, and ther......Small-molecule kinase inhibitors (SMKIs), 28 of which are approved by the US Food and Drug Administration (FDA), have been actively pursued as promising targeted therapeutics. Here, we assess the key structural and physicochemical properties, target selectivity and mechanism of function...

  10. Mini Screening of Kinase Inhibitors Affecting Period-length of Mammalian Cellular Circadian Clock

    International Nuclear Information System (INIS)

    Yagita, Kazuhiro; Yamanaka, Iori; Koinuma, Satoshi; Shigeyoshi, Yasufumi; Uchiyama, Yasuo

    2009-01-01

    In mammalian circadian rhythms, the transcriptional-translational feedback loop (TTFL) consisting of a set of clock genes is believed to elicit the circadian clock oscillation. The TTFL model explains that the accumulation and degradation of mPER and mCRY proteins control the period-length (tau) of the circadian clock. Although recent studies revealed that the Casein Kinase Iεδ (CKIεδ) regurates the phosphorylation of mPER proteins and the circadian period-length, other kinases are also likely to contribute the phosphorylation of mPER. Here, we performed small scale screening using 84 chemical compounds known as kinase inhibitors to identify candidates possibly affecting the circadian period-length in mammalian cells. Screening by this high-throughput real-time bioluminescence monitoring system revealed that the several chemical compounds apparently lengthened the cellular circadian clock oscillation. These compounds are known as inhibitors against kinases such as Casein Kinase II (CKII), PI3-kinase (PI3K) and c-Jun N-terminal Kinase (JNK) in addition to CKIεδ. Although these kinase inhibitors may have some non-specific effects on other factors, our mini screening identified new candidates contributing to period-length control in mammalian cells

  11. Molecular Mechanism of Selectivity among G Protein-Coupled Receptor Kinase 2 Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Thal, David M.; Yeow, Raymond Y.; Schoenau, Christian; Huber, Jochen; Tesmer, John J.G. (Sanofi); (Michigan)

    2012-07-11

    G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-G{beta}{gamma} complex in the presence of two of these inhibitors. Comparison with the apoGRK2-G{beta}{gamma} structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.

  12. Methods Of Using Chemical Libraries To Search For New Kinase Inhibitors

    Science.gov (United States)

    Gray, Nathanael S. , Schultz, Peter , Wodicka, Lisa , Meijer, Laurent , Lockhart, David J.

    2003-06-03

    The generation of selective inhibitors for specific protein kinases would provide new tools for analyzing signal transduction pathways and possibly new therapeutic agents. We have invented an approach to the development of selective protein kinase inhibitors based on the unexpected binding mode of 2,6,9-trisubstituted purines to the ATP binding site of human CDK2. The most potent inhibitor, purvalanol B (IC.sub.50 =6 nM), binds with a 30-fold greater affinity than the known CDK2 inhibitor, flavopiridol. The cellular effects of this class of compounds were examined and compared to those of flavopiridol by monitoring changes in mRNA expression levels for all genes in treated cells of Saccharomyces cerevisiae using high-density oligonucleotide probe arrays.

  13. Fluorescent Inhibitors as Tools To Characterize Enzymes: Case Study of the Lipid Kinase Phosphatidylinositol 4-Kinase IIIβ (PI4KB).

    Science.gov (United States)

    Humpolickova, Jana; Mejdrová, Ivana; Matousova, Marika; Nencka, Radim; Boura, Evzen

    2017-01-12

    The lipid kinase phosphatidylinositol 4-kinase IIIβ (PI4KB) is an essential host factor for many positive-sense single-stranded RNA (+RNA) viruses including human pathogens hepatitis C virus (HCV), Severe acute respiratory syndrome (SARS), coxsackie viruses, and rhinoviruses. Inhibitors of PI4KB are considered to be potential broad-spectrum virostatics, and it is therefore critical to develop a biochemical understanding of the kinase. Here, we present highly potent and selective fluorescent inhibitors that we show to be useful chemical biology tools especially in determination of dissociation constants. Moreover, we show that the coumarin-labeled inhibitor can be used to image PI4KB in cells using fluorescence-lifetime imaging microscopy (FLIM) microscopy.

  14. The two parallel photocycles of the Chlamydomonas sensory photoreceptor histidine kinase rhodopsin 1.

    Science.gov (United States)

    Luck, Meike; Hegemann, Peter

    2017-10-01

    Histidine kinase rhodopsins (HKRs) belong to a class of unexplored sensory photoreceptors that share a similar modular architecture. The light sensing rhodopsin domain is covalently linked to signal-transducing modules and in some cases to a C-terminal guanylyl-cyclase effector. In spite of their wide distribution in unicellular organisms, very little is known about their physiological role and mechanistic functioning. We investigated the photochemical properties of the recombinant rhodopsin-fragment of Cr-HKR1 originating from Chlamydomonas reinhardtii. Our spectroscopic studies revealed an unusual thermal stability of the photoproducts with the deprotonated retinal Schiff base (RSB). Upon UV-irradiation these Rh-UV states with maximal absorbance in the UVA-region (Rh-UV) photochemically convert to stable blue light absorbing rhodopsin (Rh-Bl) with protonated chromophore. The heterogeneity of the sample is based on two parallel photocycles with the chromophore in C 15 =N-syn- or -anti-configuration. This report represents an attempt to decipher the underlying reaction schemes and interconversions of the two coexisting photocycles. Copyright © 2017 Elsevier GmbH. All rights reserved.

  15. Promiscuity and selectivity of small-molecule inhibitors across TAM receptor tyrosine kinases in pediatric leukemia.

    Science.gov (United States)

    Liu, Mao-Hua; Chen, Shi-Bing; Yu, Juan; Liu, Cheng-Jun; Zhang, Xiao-Jing

    2017-08-01

    The TAM receptor tyrosine kinase family member Mer has been recognized as an attractive therapeutic target for pediatric leukemia. Beside Mer the family contains other two kinases, namely, Tyro3 and Axl, which are highly homologues with Mer and thus most existing small-molecule inhibitors show moderate or high promiscuity across the three kinases. Here, the structural basis and energetic property of selective binding of small-molecule inhibitors to the three kinases were investigated at molecular level. It is found that the selectivity is primarily determined by the size, shape and configuration of kinase's ATP-binding site; the Mer and Axl possess a small, closed active pocket as compared to the bulky, open pocket of Tyro3. The location and conformation of active-site residues of Mer and Axl are highly consistent, suggesting that small-molecule inhibitors generally have a low Mer-over-Axl selectivity and a high Mer-over-Tyro3 selectivity. We demonstrated that the difference in ATP binding potency to the three kinases is also responsible for inhibitor selectivity. We also found that the long-range interactions and allosteric effect arising from rest of the kinase's active site can indirectly influence inhibitor binding and selectivity. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Screening of protein kinase inhibitors identifies PKC inhibitors as inhibitors of osteoclastic acid secretion and bone resorption

    Directory of Open Access Journals (Sweden)

    Boutin Jean A

    2010-10-01

    Full Text Available Abstract Background Bone resorption is initiated by osteoclastic acidification of the resorption lacunae. This process is mediated by secretion of protons through the V-ATPase and chloride through the chloride antiporter ClC-7. To shed light on the intracellular signalling controlling extracellular acidification, we screened a protein kinase inhibitor library in human osteoclasts. Methods Human osteoclasts were generated from CD14+ monocytes. The effect of different kinase inhibitors on lysosomal acidification in human osteoclasts was investigated using acridine orange for different incubation times (45 minutes, 4 and 24 hours. The inhibitors were tested in an acid influx assay using microsomes isolated from human osteoclasts. Bone resorption by human osteoclasts on bone slices was measured by calcium release. Cell viability was measured using AlamarBlue. Results Of the 51 compounds investigated only few inhibitors were positive in both acidification and resorption assays. Rottlerin, GF109203X, Hypericin and Ro31-8220 inhibited acid influx in microsomes and bone resorption, while Sphingosine and Palmitoyl-DL-carnitine-Cl showed low levels of inhibition. Rottlerin inhibited lysosomal acidification in human osteoclasts potently. Conclusions In conclusion, a group of inhibitors all indicated to inhibit PKC reduced acidification in human osteoclasts, and thereby bone resorption, indicating that acid secretion by osteoclasts may be specifically regulated by PKC in osteoclasts.

  17. The Axl kinase domain in complex with a macrocyclic inhibitor offers first structural insights into an active TAM receptor kinase.

    Science.gov (United States)

    Gajiwala, Ketan S; Grodsky, Neil; Bolaños, Ben; Feng, Junli; Ferre, RoseAnn; Timofeevski, Sergei; Xu, Meirong; Murray, Brion W; Johnson, Ted W; Stewart, Al

    2017-09-22

    The receptor tyrosine kinase family consisting of Tyro3, Axl, and Mer (TAM) is one of the most recently identified receptor tyrosine kinase families. TAM receptors are up-regulated postnatally and maintained at high levels in adults. They all play an important role in immunity, but Axl has also been implicated in cancer and therefore is a target in the discovery and development of novel therapeutics. However, of the three members of the TAM family, the Axl kinase domain is the only one that has so far eluded structure determination. To this end, using differential scanning fluorimetry and hydrogen-deuterium exchange mass spectrometry, we show here that a lower stability and greater dynamic nature of the Axl kinase domain may account for its poor crystallizability. We present the first structural characterization of the Axl kinase domain in complex with a small-molecule macrocyclic inhibitor. The Axl crystal structure revealed two distinct conformational states of the enzyme, providing a first glimpse of what an active TAM receptor kinase may look like and suggesting a potential role for the juxtamembrane region in enzyme activity. We noted that the ATP/inhibitor-binding sites of the TAM members closely resemble each other, posing a challenge for the design of a selective inhibitor. We propose that the differences in the conformational dynamics among the TAM family members could potentially be exploited to achieve inhibitor selectivity for targeted receptors. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. A dual-specificity isoform of the protein kinase inhibitor PKI produced by alternate gene splicing.

    Science.gov (United States)

    Kumar, Priyadarsini; Walsh, Donal A

    2002-03-15

    We have previously shown that the protein kinase inhibitor beta (PKIbeta) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011-20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.

  19. Tyrosine kinase, aurora kinase and leucine aminopeptidase as attractive drug targets in anticancer therapy - characterisation of their inhibitors.

    Science.gov (United States)

    Ziemska, Joanna; Solecka, Jolanta

    Cancers are the leading cause of deaths all over the world. Available anticancer agents used in clinics exhibit low therapeutic index and usually high toxicity. Wide spreading drug resistance of cancer cells induce a demanding need to search for new drug targets. Currently, many on-going studies on novel compounds with potent anticancer activity, high selectivity as well as new modes of action are conducted. In this work, we describe in details three enzyme groups, which are at present of extensive interest to medical researchers and pharmaceutical companies. These include receptor tyrosine kinases (e.g. EGFR enzymes) and non-receptor tyrosine kinases (Src enzymes), type A, B and C Aurora kinases and aminopeptidases, especially leucine aminopeptidase. We discuss classification of these enzymes, biochemistry as well as their role in the cell cycle under normal conditions and during cancerogenesis. Further on, the work describes enzyme inhibitors that are under in vitro, preclinical, clinical studies as well as drugs available on the market. Both, chemical structures of discovered inhibitors and the role of chemical moieties in novel drug design are discussed. Described enzymes play essential role in cell cycle, especially in mitosis (Aurora kinases), cell differentiation, growth and apoptosis (tyrosine kinases) as well as G1/S transition (leucine aminopeptidase). In cancer cells, they are overexpressed and only their inhibition may stop tumor progression. This review presents the clinical outcomes of selected inhibitors and argues the safety of drug usage in human volunteers. Clinical studies of EGFR and Src kinase inhibitors in different tumors clearly show the need for molecular selection of patients (to those with mutations in genes coding EGFR and Src) to achieve positive clinical response. Current data indicates the great necessity for new anticancer treatment and actions to limit off-target activity.

  20. Recent advances in the discovery of small molecule c-Met Kinase inhibitors.

    Science.gov (United States)

    Parikh, Palak K; Ghate, Manjunath D

    2018-01-01

    c-Met is a prototype member of a subfamily of heterodimeric receptor tyrosine kinases (RTKs) and is the receptor for hepatocyte growth factor (HGF). Binding of HGF to its receptor c-Met, initiates a wide range of cellular signalling, including those involved in proliferation, motility, migration and invasion. Importantly, dysregulated HGF/c-Met signalling is a driving factor for numerous malignancies and promotes tumour growth, invasion, dissemination and/or angiogenesis. Dysregulated HGF/c-Met signalling has also been associated with poor clinical outcomes and resistance acquisition to some approved targeted therapies. Thus, c-Met kinase has emerged as a promising target for cancer drug development. Different therapeutic approaches targeting the HGF/c-Met signalling pathway are under development for targeted cancer therapy, among which small molecule inhibitors of c-Met kinase constitute the largest effort within the pharmaceutical industry. The review is an effort to summarize recent advancements in medicinal chemistry development of small molecule c-Met kinase inhibitors as potential anti-cancer agents which would certainly help future researchers to bring further developments in the discovery of small molecule c-Met kinase inhibitors. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  1. Structural Mechanism of the Pan-BCR-ABL Inhibitor Ponatinib (AP24534): Lessons for Overcoming Kinase Inhibitor Resistance

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Tianjun; Commodore, Lois; Huang, Wei-Sheng; Wang, Yihan; Thomas, Mathew; Keats, Jeff; Xu, Qihong; Rivera, Victor M.; Shakespeare, William C.; Clackson, Tim; Dalgarno, David C.; Zhu, Xiaotian (ARIAD)

    2012-01-20

    The BCR-ABL inhibitor imatinib has revolutionized the treatment of chronic myeloid leukemia. However, drug resistance caused by kinase domain mutations has necessitated the development of new mutation-resistant inhibitors, most recently against the T315I gatekeeper residue mutation. Ponatinib (AP24534) inhibits both native and mutant BCR-ABL, including T315I, acting as a pan-BCR-ABL inhibitor. Here, we undertook a combined crystallographic and structure-activity relationship analysis on ponatinib to understand this unique profile. While the ethynyl linker is a key inhibitor functionality that interacts with the gatekeeper, virtually all other components of ponatinib play an essential role in its T315I inhibitory activity. The extensive network of optimized molecular contacts found in the DFG-out binding mode leads to high potency and renders binding less susceptible to disruption by single point mutations. The inhibitory mechanism exemplified by ponatinib may have broad relevance to designing inhibitors against other kinases with mutated gatekeeper residues.

  2. Activation loop targeting strategy for design of receptor-interacting protein kinase 2 (RIPK2) inhibitors.

    Science.gov (United States)

    Suebsuwong, Chalada; Pinkas, Daniel M; Ray, Soumya S; Bufton, Joshua C; Dai, Bing; Bullock, Alex N; Degterev, Alexei; Cuny, Gregory D

    2018-02-15

    Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  3. Aurora kinase inhibitors: Progress towards the clinic

    Czech Academy of Sciences Publication Activity Database

    Kollareddy, M.; Zheleva, D.; Dzubak, P.; Brahmkshatriya, Pathik; Lepšík, Martin; Hajduch, M.

    2012-01-01

    Roč. 30, č. 6 (2012), s. 2411-2432 ISSN 0167-6997 Grant - others:GA ČR(CZ) GA301/08/1649; GA ČR(CZ) GD303/09/H048 Program:GA; GD Institutional research plan: CEZ:AV0Z40550506 Keywords : Aurora kinases * cancer * inhibitors Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.498, year: 2012

  4. Cyclin-dependent kinase inhibitors as anticancer drugs

    Czech Academy of Sciences Publication Activity Database

    Kryštof, Vladimír; Uldrijan, S.

    2010-01-01

    Roč. 11, č. 3 (2010), s. 291-302 ISSN 1389-4501 R&D Projects: GA ČR GA204/08/0511; GA ČR GA301/08/1649; GA MŠk(CZ) LC06077 Institutional research plan: CEZ:AV0Z50380511 Keywords : CDK * protein kinase * inhibitor Subject RIV: CE - Biochemistry Impact factor: 3.061, year: 2010

  5. Novel arylazopyrazole inhibitors of cyclin-dependent kinases

    Czech Academy of Sciences Publication Activity Database

    Jorda, Radek; Schütznerová, E.; Cankař, P.; Brychtová, Veronika; Navrátilová, Jana; Kryštof, Vladimír

    2015-01-01

    Roč. 23, č. 9 (2015), s. 1975-1981 ISSN 0968-0896 R&D Projects: GA ČR GAP305/12/0783; GA ČR GA14-19590S; GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Cyclin-dependent kinases * Inhibitor * Cell cycle Subject RIV: CE - Biochemistry Impact factor: 2.923, year: 2015

  6. BCR-ABL1 tyrosine kinase inhibitors for the treatment of chronic myeloid leukemia.

    Science.gov (United States)

    Cuellar, Sandra; Vozniak, Michael; Rhodes, Jill; Forcello, Nicholas; Olszta, Daniel

    2017-01-01

    The management of chronic myeloid leukemia with BCR-ABL1 tyrosine kinase inhibitors has evolved chronic myeloid leukemia into a chronic, manageable disease. A patient-centered approach is important for the appropriate management of chronic myeloid leukemia and optimization of long-term treatment outcomes. The pharmacist plays a key role in treatment selection, monitoring drug-drug interactions, identification and management of adverse events, and educating patients on adherence. The combination of tyrosine kinase inhibitors with unique safety profiles and individual patients with unique medical histories can make managing treatment difficult. This review will provide up-to-date information regarding tyrosine kinase inhibitor-based treatment of patients with chronic myeloid leukemia. Management strategies for adverse events and considerations for drug-drug interactions will not only vary among patients but also across tyrosine kinase inhibitors. Drug-drug interactions can be mild to severe. In instances where co-administration of concomitant medications cannot be avoided, it is critical to understand how drug levels are impacted and how subsequent dose modifications ensure therapeutic drug levels are maintained. An important component of patient-centered management of chronic myeloid leukemia also includes educating patients on the significance of early and regular monitoring of therapeutic milestones, emphasizing the importance of adhering to treatment in achieving these targets, and appropriately modifying treatment if these clinical goals are not being met. Overall, staying apprised of current research, utilizing the close pharmacist-patient relationship, and having regular interactions with patients, will help achieve successful long-term treatment of chronic myeloid leukemia in the age of BCR-ABL1 tyrosine kinase inhibitors.

  7. Phosphatidylinositol 3-Kinase (PI3K) and phosphatidylinositol 3-kinase-related kinase (PIKK) inhibitors: importance of the morpholine ring

    Czech Academy of Sciences Publication Activity Database

    Andrs, M.; Kobarecny, J.; Jun, D.; Hodný, Zdeněk; Bartek, Jiří; Kuca, K.

    2015-01-01

    Roč. 58, č. 1 (2015), s. 41-71 ISSN 0022-2623 R&D Projects: GA MŠk(CZ) CZ.1.07/2.3.00/30.0044 Grant - others:University Hospital Hradec Kralove(CZ) 00179906; Faculty of Military Health Sciences, University of Defence(CZ) SV/FVZ201402 Institutional support: RVO:68378050 Keywords : DEPENDENT PROTEIN-KINASE * STRAND BREAK REPAIR * SELECTIVE PI3K-BETA INHIBITORS * TELANGIECTASIA MUTATED KINASE Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.589, year: 2015

  8. Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate.

    Science.gov (United States)

    Navrátilová, Jarmila; Karasová, Martina; Kohutková Lánová, Martina; Jiráková, Ludmila; Budková, Zuzana; Pacherník, Jiří; Šmarda, Jan; Beneš, Petr

    2017-09-01

    Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis-activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti-cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down-regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  9. Kinase inhibitors can produce off-target effects and activate linked pathways by retroactivity

    Directory of Open Access Journals (Sweden)

    Wynn Michelle L

    2011-10-01

    Full Text Available Abstract Background It has been shown in experimental and theoretical work that covalently modified signaling cascades naturally exhibit bidirectional signal propagation via a phenomenon known as retroactivity. An important consequence of retroactivity, which arises due to enzyme sequestration in covalently modified signaling cascades, is that a downstream perturbation can produce a response in a component upstream of the perturbation without the need for explicit feedback connections. Retroactivity may, therefore, play an important role in the cellular response to a targeted therapy. Kinase inhibitors are a class of targeted therapies designed to interfere with a specific kinase molecule in a dysregulated signaling pathway. While extremely promising as anti-cancer agents, kinase inhibitors may produce undesirable off-target effects by non-specific interactions or pathway cross-talk. We hypothesize that targeted therapies such as kinase inhibitors can produce off-target effects as a consequence of retroactivity alone. Results We used a computational model and a series of simple signaling motifs to test the hypothesis. Our results indicate that within physiologically and therapeutically relevant ranges for all parameters, a targeted inhibitor can naturally induce an off-target effect via retroactivity. The kinetics governing covalent modification cycles in a signaling network were more important for propagating an upstream off-target effect in our models than the kinetics governing the targeted therapy itself. Our results also reveal the surprising and crucial result that kinase inhibitors have the capacity to turn "on" an otherwise "off" parallel cascade when two cascades share an upstream activator. Conclusions A proper and detailed characterization of a pathway's structure is important for identifying the optimal protein to target as well as what concentration of the targeted therapy is required to modulate the pathway in a safe and effective

  10. Rho-associated kinase inhibitors: a novel glaucoma therapy.

    Science.gov (United States)

    Inoue, Toshihiro; Tanihara, Hidenobu

    2013-11-01

    The rho-associated kinase (ROCK) signaling pathway is activated via secreted bioactive molecules or via integrin activation after extracellular matrix binding. These lead to polymerization of actin stress fibers and formation of focal adhesions. Accumulating evidence suggests that actin cytoskeleton-modulating signals are involved in aqueous outflow regulation. Aqueous humor contains various biologically active factors, some of which are elevated in glaucomatous eyes. These factors affect aqueous outflow, in part, through ROCK signaling modulation. Various drugs acting on the cytoskeleton have also been shown to increase aqueous outflow by acting directly on outflow tissue. In vivo animal studies have shown that the trabecular meshwork (TM) actin cytoskeleton in glaucomatous eyes is more disorganized and more randomly oriented than in non-glaucomatous control eyes. In a previous study, we introduced ROCK inhibitors as a potential glaucoma therapy by showing that a selective ROCK inhibitor significantly lowered rabbit IOP. Rho-associated kinase inhibitors directly affect the TM and Schlemm's canal (SC), differing from the target sight of other glaucoma drugs. The TM is affected earlier and more strongly than ciliary muscle cells by ROCK inhibitors, largely because of pharmacological affinity differences stemming from regulatory mechanisms. Additionally, ROCK inhibitors disrupt tight junctions, result in F-actin depolymerization, and modulate intracellular calcium level, effectively increasing SC-cell monolayer permeability. Perfusion of an enucleated eye with a ROCK inhibitor resulted in wider empty spaces in the juxtacanalicular (JCT) area and more giant vacuoles in the endothelial cells of SC, while the endothelial lining of SC was intact. Interestingly, ROCK inhibitors also increase retinal blood flow by relaxing vascular smooth muscle cells, directly protecting neurons against various stresses, while promoting wound healing. These additional effects may help

  11. Partial contribution of Rho-kinase inhibition to the bioactivity of Ganoderma lingzhi and its isolated compounds: insights on discovery of natural Rho-kinase inhibitors.

    Science.gov (United States)

    Amen, Yhiya; Zhu, Qinchang; Tran, Hai-Bang; Afifi, Mohamed S; Halim, Ahmed F; Ashour, Ahmed; Shimizu, Kuniyoshi

    2017-04-01

    Recent studies identified Rho-kinase enzymes (ROCK-I and ROCK-II) as important targets that are involved in a variety of diseases. Synthetic Rho-kinase inhibitors have emerged as potential therapeutic agents to treat disorders such as hypertension, stroke, cancer, diabetes, glaucoma, etc. Our study is the first to screen the total ethanol extract of the medicinal mushroom Ganoderma lingzhi with thirty-five compounds for Rho-kinase inhibitory activity. Moreover, a molecular binding experiment was designed to investigate the binding affinity of the compounds at the active sites of Rho-kinase enzymes. The structure-activity relationship analysis was investigated. Our results suggest that the traditional uses of G. lingzhi might be in part due to the ROCK-I and ROCK-II inhibitory potential of this mushroom. Structure-activity relationship studies revealed some interesting features of the lanostane triterpenes that potentiate their Rho-kinase inhibition. These findings would be helpful for further studies on the design of Rho-kinase inhibitors from natural sources and open the door for contributions from other researchers for optimizing the development of natural Rho-kinase inhibitors.

  12. Discovery of aminofurazan-azabenzimidazoles as inhibitors of Rho-kinase with high kinase selectivity and antihypertensive activity.

    Science.gov (United States)

    Stavenger, Robert A; Cui, Haifeng; Dowdell, Sarah E; Franz, Robert G; Gaitanopoulos, Dimitri E; Goodman, Krista B; Hilfiker, Mark A; Ivy, Robert L; Leber, Jack D; Marino, Joseph P; Oh, Hye-Ja; Viet, Andrew Q; Xu, Weiwei; Ye, Guosen; Zhang, Daohua; Zhao, Yongdong; Jolivette, Larry J; Head, Martha S; Semus, Simon F; Elkins, Patricia A; Kirkpatrick, Robert B; Dul, Edward; Khandekar, Sanjay S; Yi, Tracey; Jung, David K; Wright, Lois L; Smith, Gary K; Behm, David J; Doe, Christopher P; Bentley, Ross; Chen, Zunxuan X; Hu, Erding; Lee, Dennis

    2007-01-11

    The discovery, proposed binding mode, and optimization of a novel class of Rho-kinase inhibitors are presented. Appropriate substitution on the 6-position of the azabenzimidazole core provided subnanomolar enzyme potency in vitro while dramatically improving selectivity over a panel of other kinases. Pharmacokinetic data was obtained for the most potent and selective examples and one (6n) has been shown to lower blood pressure in a rat model of hypertension.

  13. Discovery of novel EGFR tyrosine kinase inhibitors by structure-based virtual screening.

    Science.gov (United States)

    Li, Siyuan; Sun, Xianqiang; Zhao, Hongli; Tang, Yun; Lan, Minbo

    2012-06-15

    By using of structure-based virtual screening, 13 novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors were discovered from 197,116 compounds in the SPECS database here. Among them, 8 compounds significantly inhibited EGFR kinase activity with IC(50) values lower than 10 μM. 3-{[1-(3-Chloro-4-fluorophenyl)-3,5-dioxo-4-pyrazolidinylidene]methyl}phenyl 2-thiophenecarboxylate (13), particularly, was the most potent inhibitor possessing the IC(50) value of 3.5 μM. The docking studies also provide some useful information that the docking models of the 13 compounds are beneficial to find a new path for designing novel EGFR inhibitors. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Structural and Functional Analysis of the Escherichia coli Acid-Sensing Histidine Kinase EvgS.

    Science.gov (United States)

    Sen, Hrishiraj; Aggarwal, Nikhil; Ishionwu, Chibueze; Hussain, Nosheen; Parmar, Chandni; Jamshad, Mohammed; Bavro, Vassiliy N; Lund, Peter A

    2017-09-15

    The EvgS/EvgA two-component system of Escherichia coli is activated in response to low pH and alkali metals and regulates many genes, including those for the glutamate-dependent acid resistance system and a number of efflux pumps. EvgS, the sensor kinase, is one of five unconventional histidine kinases (HKs) in E. coli and has a large periplasmic domain and a cytoplasmic PAS domain in addition to phospho-acceptor, HK and dimerization, internal receiver, and phosphotransfer domains. Mutations that constitutively activate the protein at pH 7 map to the PAS domain. Here, we built a homology model of the periplasmic region of EvgS, based on the structure of the equivalent region of the BvgS homologue, to guide mutagenesis of potential key residues in this region. We show that histidine 226 is required for induction and that it is structurally colocated with a proline residue (P522) at the top of the predicted transmembrane helix that is expected to play a key role in passing information to the cytoplasmic domains. We also show that the constitutive mutations in the PAS domain can be further activated by low external pH. Expression of the cytoplasmic part of the protein alone also gives constitutive activation, which is lost if the constitutive PAS mutations are present. These findings are consistent with a model in which EvgS senses both external and internal pH and is activated by a shift from a tight inactive to a weak active dimer, and we present an analysis of the purified cytoplasmic portion of EvgS that supports this. IMPORTANCE One of the ways bacteria sense their environment is through two-component systems, which have one membrane-bound protein to do the sensing and another inside the cell to turn genes on or off in response to what the membrane-bound protein has detected. The membrane-bound protein must thus be able to detect the stress and signal this detection event to the protein inside the cell. To understand this process, we studied a protein that helps

  15. Preparation of kinase-biased compounds in the search for lead inhibitors of kinase targets.

    Science.gov (United States)

    Lai, Justine Y Q; Langston, Steven; Adams, Ruth; Beevers, Rebekah E; Boyce, Richard; Burckhardt, Svenja; Cobb, James; Ferguson, Yvonne; Figueroa, Eva; Grimster, Neil; Henry, Andrew H; Khan, Nawaz; Jenkins, Kerry; Jones, Mark W; Judkins, Robert; Major, Jeremy; Masood, Abid; Nally, James; Payne, Helen; Payne, Lloyd; Raphy, Gilles; Raynham, Tony; Reader, John; Reader, Valérie; Reid, Alison; Ruprah, Parminder; Shaw, Michael; Sore, Hannah; Stirling, Matthew; Talbot, Adam; Taylor, Jess; Thompson, Stephen; Wada, Hiroki; Walker, David

    2005-05-01

    This work describes the preparation of approximately 13,000 compounds for rapid identification of hits in high-throughput screening (HTS). These compounds were designed as potential serine/threonine or tyrosine kinase inhibitors. The library consists of various scaffolds, e.g., purines, oxindoles, and imidazoles, whereby each core scaffold generally includes the hydrogen bond acceptor/donor properties known to be important for kinase binding. Several of these are based upon literature kinase templates, or adaptations of them to provide novelty. The routes to their preparation are outlined. A variety of automation techniques were used to prepare >500 compounds per scaffold. Where applicable, scavenger resins were employed to remove excess reagents and when necessary, preparative high performance liquid chromatography (HPLC) was used for purification. These compounds were screened against an 'in-house' kinase panel. The success rate in HTS was significantly higher than the corporate compound collection. Copyright (c) 2004 Wiley Periodicals, Inc.

  16. Maintaining glycogen synthase kinase-3 activity is critical for mTOR kinase inhibitors to inhibit cancer cell growth.

    Science.gov (United States)

    Koo, Junghui; Yue, Ping; Gal, Anthony A; Khuri, Fadlo R; Sun, Shi-Yong

    2014-05-01

    mTOR kinase inhibitors that target both mTORC1 and mTORC2 are being evaluated in cancer clinical trials. Here, we report that glycogen synthase kinase-3 (GSK3) is a critical determinant for the therapeutic response to this class of experimental drugs. Pharmacologic inhibition of GSK3 antagonized their suppressive effects on the growth of cancer cells similarly to genetic attenuation of GSK3. Conversely, expression of a constitutively activated form of GSK3β sensitized cancer cells to mTOR inhibition. Consistent with these findings, higher basal levels of GSK3 activity in a panel of human lung cancer cell lines correlated with more efficacious responses. Mechanistic investigations showed that mTOR kinase inhibitors reduced cyclin D1 levels in a GSK3β-dependent manner, independent of their effects on suppressing mTORC1 signaling and cap binding. Notably, selective inhibition of mTORC2 triggered proteasome-mediated cyclin D1 degradation, suggesting that mTORC2 blockade is responsible for GSK3-dependent reduction of cyclin D1. Silencing expression of the ubiquitin E3 ligase FBX4 rescued this reduction, implicating FBX4 in mediating this effect of mTOR inhibition. Together, our findings define a novel mechanism by which mTORC2 promotes cell growth, with potential implications for understanding the clinical action of mTOR kinase inhibitors. ©2014 AACR.

  17. Emerging roles for protein histidine phosphorylation in cellular signal transduction: lessons from the islet ?-cell

    OpenAIRE

    Kowluru, Anjaneyulu

    2008-01-01

    Protein phosphorylation represents one of the key regulatory events in physiological insulin secretion from the islet ?-cell. In this context, several classes of protein kinases (e.g. calcium-, cyclic nucleotide- and phospholipid-dependent protein kinases and tyrosine kinases) have been characterized in the ?-cell. The majority of phosphorylated amino acids identified include phosphoserine, phosphothreonine and phosphotyrosine. Protein histidine phosphorylation has been implicated in the prok...

  18. Aldehyde Dehydrogenase 1 and Raf Kinase Inhibitor Protein ...

    African Journals Online (AJOL)

    Aldehyde Dehydrogenase 1 and Raf Kinase Inhibitor Protein Expression Defines the Proliferative Nature of Cervical Cancer Stem Cells. ... of cervical cancer stem cells and also to validate them in initial and advanced stages of cervical cancer. Keywords: Cervical cancer, ALDH1, BALB/c-nu/nu, HeLa cells, RKIP, Sox2 ...

  19. Virulence Effects and Signaling Partners Modulated by Brucella melitensis Light-sensing Histidine Kinase

    Science.gov (United States)

    Gourley, Christopher R.

    The facultative intracellular pathogen Brucella melitensis utilizes diverse virulence factors. A Brucella light sensing histidine kinase can influence in vitro virulence of the bacteria during intracellular infection. First, we demonstrated that the B. melitensis light sensing kinase (BM-LOV-HK) affects virulence in an IRF-1-/- mouse model of infection. Infection with a Δ BM-LOV-HK strain resulted in less bacterial colonization of IRF-1-/- spleens and extended survivorship compared to mice infected with wild type B. melitensis 16M. Second, using PCR arrays, we observed less expression of innate and adaptive immune system activation markers in ΔBM-LOV-HK infected mouse spleens than wild type B. melitensis 16M infected mouse spleens 6 days after infection. Third, we demonstrated by microarray analysis of B. melitensis that deletion of BM-LOV-HK alters bacterial gene expression. Downregulation of genes involved in control of the general stress response system included the alternative sigma factor RpoE1 and its anti-anti sigma factor PhyR. Conversely, genes involved in flagella production, quorum sensing, and the type IV secretion system (VirB operon) were upregulated in the Δ BM-LOV-HK strain compared to the wild type B. melitensis 16M. Analysis of genes differentially regulated in Δ BM-LOV-HK versus the wild type strain indicated an overlap of 110 genes with data from previous quorum sensing regulator studies of Δ vjbR and/ΔblxR(babR) strains. Also, several predicted RpoE1 binding sites located upstream of genes were differentially regulated in the ΔBM-LOV-HK strain. Our results suggest BM-LOV-HK is important for in vivo Brucella virulence, and reveals that BM-LOV-HK directly or indirect regulates members of the Brucella quorum sensing, type IV secretion, and general stress systems.

  20. High-throughput screening to identify inhibitors which stabilize inactive kinase conformations in p38 alpha

    NARCIS (Netherlands)

    Simard, J.R.; Grutter, C.; Pawar, V.; Aust, B.; Wolf, A.; Rabiller, M.; Wulfert, S.; Robubi, A.; Kluter, S.; Ottmann, C.; Rauh, D.

    2009-01-01

    Small molecule kinase inhibitors are an attractive means to modulate kinase activities in medicinal chemistry and chemical biology research. In the physiological setting of a cell, kinase function is orchestrated by a plethora of regulatory processes involving the structural transition of kinases

  1. Bosutinib induced pleural effusions: Case report and review of tyrosine kinase inhibitors induced pulmonary toxicity

    Directory of Open Access Journals (Sweden)

    Natalia I. Moguillansky, MD

    2017-01-01

    Full Text Available Tyrosine kinase inhibitors are known to cause pulmonary complications. We report a case of bosutinib related bilateral pleural effusions in a patient with chronic myeloid leukemia. Characteristics of the pleural fluid are presented. We also discuss other tyrosine kinase inhibitors induced pulmonary toxicities, including pulmonary hypertension and interstitial lung disease.

  2. Computational Analysis of Epidermal Growth Factor Receptor Mutations Predicts Differential Drug Sensitivity Profiles toward Kinase Inhibitors.

    Science.gov (United States)

    Akula, Sravani; Kamasani, Swapna; Sivan, Sree Kanth; Manga, Vijjulatha; Vudem, Dashavantha Reddy; Kancha, Rama Krishna

    2018-05-01

    A significant proportion of patients with lung cancer carry mutations in the EGFR kinase domain. The presence of a deletion mutation in exon 19 or L858R point mutation in the EGFR kinase domain has been shown to cause enhanced efficacy of inhibitor treatment in patients with NSCLC. Several less frequent (uncommon) mutations in the EGFR kinase domain with potential implications in treatment response have also been reported. The role of a limited number of uncommon mutations in drug sensitivity was experimentally verified. However, a huge number of these mutations remain uncharacterized for inhibitor sensitivity or resistance. A large-scale computational analysis of clinically reported 298 point mutants of EGFR kinase domain has been performed, and drug sensitivity profiles for each mutant toward seven kinase inhibitors has been determined by molecular docking. In addition, the relative inhibitor binding affinity toward each drug as compared with that of adenosine triphosphate was calculated for each mutant. The inhibitor sensitivity profiles predicted in this study for a set of previously characterized mutants correlated well with the published clinical, experimental, and computational data. Both the single and compound mutations displayed differential inhibitor sensitivity toward first- and next-generation kinase inhibitors. The present study provides predicted drug sensitivity profiles for a large panel of uncommon EGFR mutations toward multiple inhibitors, which may help clinicians in deciding mutant-specific treatment strategies. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

  3. The Discovery of Aurora Kinase Inhibitor by Multi-Docking-Based Virtual Screening

    Directory of Open Access Journals (Sweden)

    Jun-Tae Kim

    2014-11-01

    Full Text Available We report the discovery of aurora kinase inhibitor using the fragment-based virtual screening by multi-docking strategy. Among a number of fragments collected from eMololecules, we found four fragment molecules showing potent activity (>50% at 100 μM against aurora kinase. Based on the explored fragment scaffold, we selected two compounds in our synthesized library and validated the biological activity against Aurora kinase.

  4. Structure of Human G Protein-Coupled Receptor Kinase 2 in Complex with the Kinase Inhibitor Balanol

    Energy Technology Data Exchange (ETDEWEB)

    Tesmer, John J.G.; Tesmer, Valerie M.; Lodowski, David T.; Steinhagen, Henning; Huber, Jochen (Sanofi); (Michigan); (Texas)

    2010-07-19

    G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with G{beta}{gamma} in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.

  5. Determination of human serum alpha1-acid glycoprotein and albumin binding of various marketed and preclinical kinase inhibitors.

    Science.gov (United States)

    Zsila, Ferenc; Fitos, Ilona; Bencze, Gyula; Kéri, György; Orfi, László

    2009-01-01

    There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.

  6. Variability in bioavailability of small molecular tyrosine kinase inhibitors

    NARCIS (Netherlands)

    Herbrink, Maikel; Nuijen, Bastiaan; Schellens, Jan H M; Beijnen, Jos H.

    2015-01-01

    Small molecular tyrosine kinase inhibitors (smTKIs) are in the centre of the very quickly expanding area of personalized chemotherapy and oral applicability thereof. The number of drugs in this class is rapidly growing, with twenty current approvals by both the European Medicines Agency (EMA) and

  7. Identifying Inhibitors of Inflammation: A Novel High-Throughput MALDI-TOF Screening Assay for Salt-Inducible Kinases (SIKs).

    Science.gov (United States)

    Heap, Rachel E; Hope, Anthony G; Pearson, Lesley-Anne; Reyskens, Kathleen M S E; McElroy, Stuart P; Hastie, C James; Porter, David W; Arthur, J Simon C; Gray, David W; Trost, Matthias

    2017-12-01

    Matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry has become a promising alternative for high-throughput drug discovery as new instruments offer high speed, flexibility and sensitivity, and the ability to measure physiological substrates label free. Here we developed and applied high-throughput MALDI TOF mass spectrometry to identify inhibitors of the salt-inducible kinase (SIK) family, which are interesting drug targets in the field of inflammatory disease as they control production of the anti-inflammatory cytokine interleukin-10 (IL-10) in macrophages. Using peptide substrates in in vitro kinase assays, we can show that hit identification of the MALDI TOF kinase assay correlates with indirect ADP-Hunter kinase assays. Moreover, we can show that both techniques generate comparable IC 50 data for a number of hit compounds and known inhibitors of SIK kinases. We further take these inhibitors to a fluorescence-based cellular assay using the SIK activity-dependent translocation of CRTC3 into the nucleus, thereby providing a complete assay pipeline for the identification of SIK kinase inhibitors in vitro and in cells. Our data demonstrate that MALDI TOF mass spectrometry is fully applicable to high-throughput kinase screening, providing label-free data comparable to that of current high-throughput fluorescence assays.

  8. Fragment-based approaches to the discovery of kinase inhibitors.

    Science.gov (United States)

    Mortenson, Paul N; Berdini, Valerio; O'Reilly, Marc

    2014-01-01

    Protein kinases are one of the most important families of drug targets, and aberrant kinase activity has been linked to a large number of disease areas. Although eminently targetable using small molecules, kinases present a number of challenges as drug targets, not least obtaining selectivity across such a large and relatively closely related target family. Fragment-based drug discovery involves screening simple, low-molecular weight compounds to generate initial hits against a target. These hits are then optimized to more potent compounds via medicinal chemistry, usually facilitated by structural biology. Here, we will present a number of recent examples of fragment-based approaches to the discovery of kinase inhibitors, detailing the construction of fragment-screening libraries, the identification and validation of fragment hits, and their optimization into potent and selective lead compounds. The advantages of fragment-based methodologies will be discussed, along with some of the challenges associated with using this route. Finally, we will present a number of key lessons derived both from our own experience running fragment screens against kinases and from a large number of published studies.

  9. Identification of anti-proliferative kinase inhibitors as potential therapeutic agents to treat canine osteosarcoma.

    Science.gov (United States)

    Mauchle, Ulrike; Selvarajah, Gayathri T; Mol, Jan A; Kirpensteijn, Jolle; Verheije, Monique H

    2015-08-01

    Osteosarcoma is the most common primary bone tumour in dogs but various forms of therapy have not significantly improved clinical outcomes. As dysregulation of kinase activity is often present in tumours, kinases represent attractive molecular targets for cancer therapy. The purpose of this study was to identify novel compounds targeting kinases with the potential to induce cell death in a panel of canine osteosarcoma cell lines. The ability of 80 well-characterized kinase inhibitor compounds to inhibit the proliferation of four canine osteosarcoma cell lines was investigated in vitro. For those compounds with activity, the mechanism of action and capability to potentiate the activity of doxorubicin was further evaluated. The screening showed 22 different kinase inhibitors that induced significant anti-proliferative effects across the four canine osteosarcoma cell lines investigated. Four of these compounds (RO 31-8220, 5-iodotubercidin, BAY 11-7082 and an erbstatin analog) showed significant cell growth inhibitory effects across all cell lines in association with variable induction of apoptosis. RO 31-8220 and 5-iodotubercidin showed the highest ability to potentiate the effects of doxorubicin on cell viability. In conclusion, the present study identified several potent kinase inhibitors targeting the PKC, CK1, PKA, ErbB2, mTOR and NF-κB pathways, which may warrant further investigations for the treatment of osteosarcoma in dogs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. [Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells].

    Science.gov (United States)

    Sheremet, Ia A; Emets, A I; Azmi, A; Vissenberg, K; Verbelen, J-P; Blium, Ia B

    2012-01-01

    In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

  11. Fragment-Based Drug Discovery of Potent Protein Kinase C Iota Inhibitors.

    Science.gov (United States)

    Kwiatkowski, Jacek; Liu, Boping; Tee, Doris Hui Ying; Chen, Guoying; Ahmad, Nur Huda Binte; Wong, Yun Xuan; Poh, Zhi Ying; Ang, Shi Hua; Tan, Eldwin Sum Wai; Ong, Esther Hq; Nurul Dinie; Poulsen, Anders; Pendharkar, Vishal; Sangthongpitag, Kanda; Lee, May Ann; Sepramaniam, Sugunavathi; Ho, Soo Yei; Cherian, Joseph; Hill, Jeffrey; Keller, Thomas H; Hung, Alvin W

    2018-05-24

    Protein kinase C iota (PKC-ι) is an atypical kinase implicated in the promotion of different cancer types. A biochemical screen of a fragment library has identified several hits from which an azaindole-based scaffold was chosen for optimization. Driven by a structure-activity relationship and supported by molecular modeling, a weakly bound fragment was systematically grown into a potent and selective inhibitor against PKC-ι.

  12. Review of current classification, molecular alterations, and tyrosine kinase inhibitor therapies in myeloproliferative disorders with hypereosinophilia

    Directory of Open Access Journals (Sweden)

    Havelange V

    2013-08-01

    Full Text Available Violaine Havelange,1,2 Jean-Baptiste Demoulin1 1de Duve Institute, Université catholique de Louvain, Brussels, Belgium; 2Department of Hematology, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium Abstract: Recent advances in our understanding of the molecular mechanisms underlying hypereosinophilia have led to the development of a 'molecular' classification of myeloproliferative disorders with eosinophilia. The revised 2008 World Health Organization classification of myeloid neoplasms included a new category called “myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB or FGFR1.” Despite the molecular heterogeneity of PDGFR (platelet-derived growth factor receptor rearrangements, tyrosine kinase inhibitors at low dose induce rapid and complete hematological remission in the majority of these patients. Other kinase inhibitors are promising. Further discoveries of new molecular alterations will direct the development of new specific inhibitors. In this review, an update of the classifications of myeloproliferative disorders associated with hypereosinophilia is discussed together with open and controversial questions. Molecular mechanisms and promising results of tyrosine kinase inhibitor treatments are reviewed. Keywords: hypereosinophilia, classification, myeloproliferative disorders, molecular alterations, tyrosine kinase inhibitor

  13. Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

    International Nuclear Information System (INIS)

    Okabe, Seiichi; Tauchi, Tetsuzo; Tanaka, Yuko; Ohyashiki, Kazuma

    2013-01-01

    Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzed the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance

  14. Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Okabe, Seiichi, E-mail: okabe@tokyo-med.ac.jp; Tauchi, Tetsuzo; Tanaka, Yuko; Ohyashiki, Kazuma

    2013-06-07

    Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzed the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance.

  15. Opportunities to Target Specific Contractile Abnormalities with Smooth Muscle Protein Kinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Annegret Ulke-Lemée

    2010-05-01

    Full Text Available Smooth muscle is a major component of most hollow organ systems (e.g., airways, vasculature, bladder and gut/gastrointestine; therefore, the coordinated regulation of contraction is a key property of smooth muscle. When smooth muscle functions normally, it contributes to general health and wellness, but its dysfunction is associated with morbidity and mortality. Rho-associated protein kinase (ROCK is central to calcium-independent, actomyosin-mediated contractile force generation in the vasculature, thereby playing a role in smooth muscle contraction, cell motility and adhesion. Recent evidence supports an important role for ROCK in the increased vasoconstriction and remodeling observed in various models of hypertension. This review will provide a commentary on the development of specific ROCK inhibitors and their clinical application. Fasudil will be discussed as an example of bench-to-bedside development of a clinical therapeutic that is used to treat conditions of vascular hypercontractility. Due to the wide spectrum of biological processes regulated by ROCK, many additional clinical indications might also benefit from ROCK inhibition. Apart from the importance of ROCK in smooth muscle contraction, a variety of other protein kinases are known to play similar roles in regulating contractile force. The zipper-interacting protein kinase (ZIPK and integrin-linked kinase (ILK are two well-described regulators of contraction. The relative contribution of each kinase to contraction depends on the muscle bed as well as hormonal and neuronal stimulation. Unfortunately, specific inhibitors for ZIPK and ILK are still in the development phase, but the success of fasudil suggests that inhibitors for these other kinases may also have valuable clinical applications. Notably, the directed inhibition of ZIPK with a pseudosubstrate molecule shows unexpected effects on the contractility of gastrointestinal smooth muscle.

  16. Functional and structural comparison of pyrrolnitrin- and iprodione-induced modifications in the class III histidine-kinase Bos1 of Botrytis cinerea.

    Directory of Open Access Journals (Sweden)

    Sabine Fillinger

    Full Text Available Dicarboximides and phenylpyrroles are commonly used fungicides against plant pathogenic ascomycetes. Although their effect on fungal osmosensing systems has been shown in many studies, their modes-of-action still remain unclear. Laboratory- or field-mutants of fungi resistant to either or both fungicide categories generally harbour point mutations in the sensor histidine kinase of the osmotic signal transduction cascade.In the present study we compared the mechanisms of resistance to the dicarboximide iprodione and to pyrrolnitrin, a structural analogue of phenylpyrrole fungicides, in Botrytis cinerea. Pyrrolnitrin-induced mutants and iprodione-induced mutants of B. cinerea were produced in vitro. For the pyrrolnitrin-induced mutants, a high level of resistance to pyrrolnitrin was associated with a high level of resistance to iprodione. For the iprodione-induced mutants, the high level of resistance to iprodione generated variable levels of resistance to pyrrolnitrin and phenylpyrroles. All selected mutants showed hypersensitivity to high osmolarity and regardless of their resistance levels to phenylpyrroles, they showed strongly reduced fitness parameters (sporulation, mycelial growth, aggressiveness on plants compared to the parental phenotypes. Most of the mutants presented modifications in the osmosensing class III histidine kinase affecting the HAMP domains. Site directed mutagenesis of the bos1 gene was applied to validate eight of the identified mutations. Structure modelling of the HAMP domains revealed that the replacements of hydrophobic residues within the HAMP domains generally affected their helical structure, probably abolishing signal transduction. Comparing mutant phenotypes to the HAMP structures, our study suggests that mutations perturbing helical structures of HAMP2-4 abolish signal-transduction leading to loss-of-function phenotype. The mutation of residues E529, M427, and T581, without consequences on HAMP structure

  17. The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes.

    Science.gov (United States)

    Standaert, M L; Avignon, A; Yamada, K; Bandyopadhyay, G; Farese, R V

    1996-02-01

    We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC-beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulated, phospholipase D-dependent PC hydrolysis and subsequent translocation of PKC-alpha and PKC-beta to the plasma membrane. Wortmannin did not inhibit PKC directly in vitro, or the PKC-dependent effects of phorbol esters on glucose transport in intact adipocytes. The PKC inhibitor RO 31-8220 did not inhibit PI 3-kinase directly or its activation in situ by insulin, but inhibited both insulin-stimulated and phorbol ester-stimulated glucose transport. Our findings suggest that insulin acts through PI 3-kinase to activate a PC-specific phospholipase D and causes the translocative activation of PKC-alpha and PKC-beta in plasma membranes of rat adipocytes.

  18. The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes.

    OpenAIRE

    Standaert, M L; Avignon, A; Yamada, K; Bandyopadhyay, G; Farese, R V

    1996-01-01

    We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC-beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulat...

  19. Prostatic-Like Syndrome in a Woman with Chronic Lymphocytic Leukemia: Sequential Kinase Inhibitor Therapy

    Directory of Open Access Journals (Sweden)

    Diego Velasco-Rodríguez

    2017-01-01

    Full Text Available Chronic lymphocytic leukemia (CLL is an incurable lymphoproliferative disorder with a heterogeneous genetic and clinical course. Two kinase inhibitors, ibrutinib and idelalisib, have demonstrated achievement of complete and durable remissions in relapse/refractory genetically unselected CLL patients. We present a case of relapsed CLL with extensive disease and hourglass deformity of urinary bladder as a result of the compression of two extraperitoneal paravesical soft tissue bulky masses, with excellent response to sequential kinase inhibitor therapy.

  20. Identification of p38α MAP kinase inhibitors by pharmacophore based virtual screening

    DEFF Research Database (Denmark)

    Gangwal, Rahul P; Das, Nihar R; Thanki, Kaushik

    2014-01-01

    The p38α mitogen-activated protein (MAP) kinase plays a vital role in treating many inflammatory diseases. In the present study, a combined ligand and structure based pharmacophore model was developed to identify potential DFG-in selective p38 MAP kinase inhibitors. Conformations of co...

  1. Combining RNA interference and kinase inhibitors against cell signalling components involved in cancer

    International Nuclear Information System (INIS)

    O'Grady, Michael; Raha, Debasish; Hanson, Bonnie J; Bunting, Michaeline; Hanson, George T

    2005-01-01

    The transcription factor activator protein-1 (AP-1) has been implicated in a large variety of biological processes including oncogenic transformation. The tyrosine kinases of the epidermal growth factor receptor (EGFR) constitute the beginning of one signal transduction cascade leading to AP-1 activation and are known to control cell proliferation and differentiation. Drug discovery efforts targeting this receptor and other pathway components have centred on monoclonal antibodies and small molecule inhibitors. Resistance to such inhibitors has already been observed, guiding the prediction of their use in combination therapies with other targeted agents such as RNA interference (RNAi). This study examines the use of RNAi and kinase inhibitors for qualification of components involved in the EGFR/AP-1 pathway of ME180 cells, and their inhibitory effects when evaluated individually or in tandem against multiple components of this important disease-related pathway. AP-1 activation was assessed using an ME180 cell line stably transfected with a beta-lactamase reporter gene under the control of AP-1 response element following epidermal growth factor (EGF) stimulation. Immunocytochemistry allowed for further quantification of small molecule inhibition on a cellular protein level. RNAi and RT-qPCR experiments were performed to assess the amount of knockdown on an mRNA level, and immunocytochemistry was used to reveal cellular protein levels for the targeted pathway components. Increased potency of kinase inhibitors was shown by combining RNAi directed towards EGFR and small molecule inhibitors acting at proximal or distal points in the pathway. After cellular stimulation with EGF and analysis at the level of AP-1 activation using a β-lactamase reporter gene, a 10–12 fold shift or 2.5–3 fold shift toward greater potency in the IC 50 was observed for EGFR and MEK-1 inhibitors, respectively, in the presence of RNAi targeting EGFR. EGFR pathway components were qualified as

  2. Mesenchymal stem cells are sensitive to treatment with kinase inhibitors and ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Nicolay, Nils H.; Debus, Juergen; Huber, Peter E. [Heidelberg University Hospital, Department of Radiation Oncology, Heidelberg (Germany); German Cancer Research Center (dkfz), Department of Molecular and Radiation Oncology, Heidelberg (Germany); Sommer, Eva; Lopez Perez, Ramon; Wirkner, Ute [German Cancer Research Center (dkfz), Department of Molecular and Radiation Oncology, Heidelberg (Germany); Bostel, Tilman [Heidelberg University Hospital, Department of Radiation Oncology, Heidelberg (Germany); Ho, Anthony D.; Saffrich, Rainer [Heidelberg University Hospital, Department of Hematology, Oncology and Rheumatology, Heidelberg (Germany); Lahn, Michael [Lilly Research Laboratories, Oncology Early Clinical Investigation, Indianapolis, IN (United States)

    2014-11-15

    Mesenchymal stem cells (MSCs) can regenerate damaged tissues and may therefore be of importance for normal tissue repair after cancer treatment. Small molecule receptor kinase inhibitors (RKIs) have recently been introduced into cancer treatment. However, the influence of these drugs - particularly in combination with radiotherapy - on the survival of MSCs is largely unknown. The sensitivity of human primary MSCs from healthy volunteers and primary human fibroblast cells to small molecule kinase inhibitors of the vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and transforming growth factor β (TGFβ) receptors, as well to inhibitors of c-Kit, was examined in combination with ionizing radiation (IR); cell survival and proliferation were assessed. Expression patterns of different kinase receptors and ligands were investigated using gene arrays. MSCs were highly sensitive to the tyrosine kinase inhibitors SU14816 (imatinib) and SU11657 (sunitinib), but showed only moderate sensitivity to the selective TGFβ receptor 1 inhibitor LY2109761. Primary adult human fibroblasts were comparably resistant to all three inhibitors. The addition of IR had an additive or supra-additive effect in the MSCs, but this was not the case for differentiated fibroblasts. Proliferation was markedly reduced in MSCs following kinase inhibition, both with and without IR. Gene expression analysis revealed high levels of the PDGF α and β receptors, and lower levels of the TGFβ receptor 2 and Abl kinase. IR did not alter the expression of kinase receptors or their respective ligands in either MSCs or adult fibroblasts. These data show that MSCs are highly sensitive to RKIs and combination treatments incorporating IR. Expression analyses suggest that high levels of PDGF receptors may contribute to this effect. (orig.) [German] Mesenchymale Stammzellen (MSCs) koennen die Geweberegeneration unterstuetzen und haben daher moeglicherweise eine Rolle bei der Reparatur

  3. Signal Sensing and Transduction by Histidine Kinases as Unveiled through Studies on a Temperature Sensor.

    Science.gov (United States)

    Abriata, Luciano A; Albanesi, Daniela; Dal Peraro, Matteo; de Mendoza, Diego

    2017-06-20

    Histidine kinases (HK) are the sensory proteins of two-component systems, responsible for a large fraction of bacterial responses to stimuli and environmental changes. Prototypical HKs are membrane-bound proteins that phosphorylate cognate response regulator proteins in the cytoplasm upon signal detection in the membrane or periplasm. HKs stand as potential drug targets but also constitute fascinating systems for studying proteins at work, specifically regarding the chemistry and mechanics of signal detection, transduction through the membrane, and regulation of catalytic outputs. In this Account, we focus on Bacillus subtilis DesK, a membrane-bound HK part of a two-component system that maintains appropriate membrane fluidity at low growth temperatures. Unlike most HKs, DesK has no extracytoplasmic signal-sensing domains; instead, sensing is carried out by 10 transmembrane helices (coming from two protomers) arranged in an unknown structure. The fifth transmembrane helix from each protomer connects, without any of the intermediate domains found in other HKs, into the dimerization and histidine phosphotransfer (DHp) domain located in the cytoplasm, which is followed by the ATP-binding domains (ABD). Throughout the years, genetic, biochemical, structural, and computational studies on wild-type, mutant, and truncated versions of DesK allowed us to dissect several aspects of DesK's functioning, pushing forward a more general understanding of its own structure/function relationships as well as those of other HKs. We have shown that the sensing mechanism is rooted in temperature-dependent membrane properties, most likely a combination of thickness, fluidity, and water permeability, and we have proposed possible mechanisms by which DesK senses these properties and transduces the signals. X-ray structures and computational models have revealed structural features of TM and cytoplasmic regions in DesK's kinase- and phosphatase-competent states. Biochemical and genetic

  4. Epigenetic Mechanisms Regulating Adaptive Responses to Targeted Kinase Inhibitors in Cancer.

    Science.gov (United States)

    Angus, Steven P; Zawistowski, Jon S; Johnson, Gary L

    2018-01-06

    Although targeted inhibition of oncogenic kinase drivers has achieved remarkable patient responses in many cancers, the development of resistance has remained a significant challenge. Numerous mechanisms have been identified, including the acquisition of gatekeeper mutations, activating pathway mutations, and copy number loss or gain of the driver or alternate nodes. These changes have prompted the development of kinase inhibitors with increased selectivity, use of second-line therapeutics to overcome primary resistance, and combination treatment to forestall resistance. In addition to genomic resistance mechanisms, adaptive transcriptional and signaling responses seen in tumors are gaining appreciation as alterations that lead to a phenotypic state change-often observed as an epithelial-to-mesenchymal shift or reversion to a cancer stem cell-like phenotype underpinned by remodeling of the epigenetic landscape. This epigenomic modulation driving cell state change is multifaceted and includes modulation of repressive and activating histone modifications, DNA methylation, enhancer remodeling, and noncoding RNA species. Consequently, the combination of kinase inhibitors with drugs targeting components of the transcriptional machinery and histone-modifying enzymes has shown promise in preclinical and clinical studies. Here, we review mechanisms of resistance to kinase inhibition in cancer, with special emphasis on the rewired kinome and transcriptional signaling networks and the potential vulnerabilities that may be exploited to overcome these adaptive signaling changes.

  5. cGMP-Dependent Protein Kinase Inhibitors in Health and Disease

    Directory of Open Access Journals (Sweden)

    Jens Schlossmann

    2013-02-01

    Full Text Available cGMP-dependent protein kinases (PKG exhibit diverse physiological functions in the mammalian system e.g., in vascular and gastrointestinal smooth muscles, in platelets, in kidney, in bone growth, nociception and in the central nervous system. Furthermore, PKG were found in insects and in the malaria parasite Plasmodium falciparum. Two different genes of PKG exist: a the PKG-I gene that is expressed as cytosolic PKG-Iα or PKG-Iβ isoform, and b the PKG-II gene, which expresses the membrane associated PKG-II protein. The enzyme kinetics, the localization and the substrates of these PKG enzymes differ utilizing different physiological functions. Various inhibitors of PKG were developed directed against diverse functional regions of the kinase. These inhibitors of PKG have been used to analyse the specific functions of these enzymes. The review article will summarize these different inhibitors regarding their specificity and their present applications in vitro and in vivo. Furthermore, it will be discussed that the distinct inhibition of the PKG enzymes could be used as a valuable pharmacological target e.g., in the treatment of cardiovascular diseases, diarrhea, cancer or malaria.

  6. Erkitinib, a novel EGFR tyrosine kinase inhibitor screened using a ProteoChip system from a phytochemical library

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Eung-Yoon; Choi, Young-Jin [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Innopharmascreen, Inc., Asan 336-795 (Korea, Republic of); Park, Chan-Won [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Dept. of Biological Science, Hoseo University, Asan 336-795 (Korea, Republic of); Kang, In-Cheol, E-mail: ickang@hoseo.edu [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Dept. of Biological Science, Hoseo University, Asan 336-795 (Korea, Republic of); Innopharmascreen, Inc., Asan 336-795 (Korea, Republic of)

    2009-11-20

    Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer. Therefore PTK inhibitors are currently under intensive investigation as potential drug candidates. Herein, we report on a ProteoChip-based screening of an epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitor, Erkitinibs, from phytochemical libraries. PLC-{gamma}-1 was used as a substrate immobilized on a ProteoChip and incubated with an EGFR kinase to phosphorylate tyrosine residues of the substrate, followed by a fluorescence detection of the substrate recognized by a phospho-specific monoclonal antibody. Erkitinibs inhibited HeLa cell proliferation in a dose-dependent manner. In conclusion, these data suggest that Erkitinibs can be a specific inhibitor of an EGFR kinase and can be further developed as a potent anti-tumor agent.

  7. Erkitinib, a novel EGFR tyrosine kinase inhibitor screened using a ProteoChip system from a phytochemical library

    International Nuclear Information System (INIS)

    Kim, Eung-Yoon; Choi, Young-Jin; Park, Chan-Won; Kang, In-Cheol

    2009-01-01

    Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer. Therefore PTK inhibitors are currently under intensive investigation as potential drug candidates. Herein, we report on a ProteoChip-based screening of an epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitor, Erkitinibs, from phytochemical libraries. PLC-γ-1 was used as a substrate immobilized on a ProteoChip and incubated with an EGFR kinase to phosphorylate tyrosine residues of the substrate, followed by a fluorescence detection of the substrate recognized by a phospho-specific monoclonal antibody. Erkitinibs inhibited HeLa cell proliferation in a dose-dependent manner. In conclusion, these data suggest that Erkitinibs can be a specific inhibitor of an EGFR kinase and can be further developed as a potent anti-tumor agent.

  8. Activity of dual SRC-ABL inhibitors highlights the role of BCR/ABL kinase dynamics in drug resistance

    Science.gov (United States)

    Azam, Mohammad; Nardi, Valentina; Shakespeare, William C.; Metcalf, Chester A.; Bohacek, Regine S.; Wang, Yihan; Sundaramoorthi, Raji; Sliz, Piotr; Veach, Darren R.; Bornmann, William G.; Clarkson, Bayard; Dalgarno, David C.; Sawyer, Tomi K.; Daley, George Q.

    2006-01-01

    Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance in patients with chronic myelogenous leukemia. Many mutations favor active kinase conformations that preclude imatinib binding. Because the active forms of ABL and SRC resemble one another, we tested two dual SRC-ABL kinase inhibitors, AP23464 and PD166326, against 58 imatinib-resistant (IMR) BCR/ABL kinase variants. Both compounds potently inhibit most IMR variants, and in vitro drug selection demonstrates that active (AP23464) and open (PD166326) conformation-specific compounds are less susceptible to resistance than imatinib. Combinations of inhibitors suppressed essentially all resistance mutations, with the notable exception of T315I. Guided by mutagenesis studies and molecular modeling, we designed a series of AP23464 analogues to target T315I. The analogue AP23846 inhibited both native and T315I variants of BCR/ABL with submicromolar potency but showed nonspecific cellular toxicity. Our data illustrate how conformational dynamics of the ABL kinase accounts for the activity of dual SRC-ABL inhibitors against IMR-mutants and provides a rationale for combining conformation specific inhibitors to suppress resistance. PMID:16754879

  9. Structural Bioinformatics-Based Prediction of Exceptional Selectivity of p38 MAP Kinase Inhibitor PH-797804

    Energy Technology Data Exchange (ETDEWEB)

    Xing, Li; Shieh, Huey S.; Selness, Shaun R.; Devraj, Rajesh V.; Walker, John K.; Devadas, Balekudru; Hope, Heidi R.; Compton, Robert P.; Schindler, John F.; Hirsch, Jeffrey L.; Benson, Alan G.; Kurumbail, Ravi G.; Stegeman, Roderick A.; Williams, Jennifer M.; Broadus, Richard M.; Walden, Zara; Monahan, Joseph B.; Pfizer

    2009-07-24

    PH-797804 is a diarylpyridinone inhibitor of p38{alpha} mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. On the basis of structural comparison with a different biaryl pyrazole template and supported by dozens of high-resolution crystal structures of p38{alpha} inhibitor complexes, PH-797804 is predicted to possess a high level of specificity across the broad human kinase genome. We used a structural bioinformatics approach to identify two selectivity elements encoded by the TXXXG sequence motif on the p38{alpha} kinase hinge: (i) Thr106 that serves as the gatekeeper to the buried hydrophobic pocket occupied by 2,4-difluorophenyl of PH-797804 and (ii) the bidentate hydrogen bonds formed by the pyridinone moiety with the kinase hinge requiring an induced 180{sup o} rotation of the Met109-Gly110 peptide bond. The peptide flip occurs in p38{alpha} kinase due to the critical glycine residue marked by its conformational flexibility. Kinome-wide sequence mining revealed rare presentation of the selectivity motif. Corroboratively, PH-797804 exhibited exceptionally high specificity against MAP kinases and the related kinases. No cross-reactivity was observed in large panels of kinase screens (selectivity ratio of >500-fold). In cellular assays, PH-797804 demonstrated superior potency and selectivity consistent with the biochemical measurements. PH-797804 has met safety criteria in human phase I studies and is under clinical development for several inflammatory conditions. Understanding the rationale for selectivity at the molecular level helps elucidate the biological function and design of specific p38{alpha} kinase inhibitors.

  10. Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors.

    Science.gov (United States)

    Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa; Xu, Jun; Haddock, Christopher J; Li, Chen; Lee, Brian J; Loredan, Denis G; Jiang, Wenxia; Vindigni, Alessandro; Wang, Dong; Rabadan, Raul; Zha, Shan

    2016-06-15

    Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (Atm(KD/-)) is more oncogenic than loss of ATM (Atm(-/-)) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate Atm(KD/-), but not Atm-proficientor Atm(-/-) leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy.

  11. Discovery of potent 1H-imidazo[4,5-b]pyridine-based c-Met kinase inhibitors via mechanism-directed structural optimization.

    Science.gov (United States)

    An, Xiao-De; Liu, Hongyan; Xu, Zhong-Liang; Jin, Yi; Peng, Xia; Yao, Ying-Ming; Geng, Meiyu; Long, Ya-Qiu

    2015-02-01

    Starting from our previously identified novel c-Met kinase inhibitors bearing 1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)-one scaffold, a global structural exploration was conducted to furnish an optimal binding motif for further development, directed by the enzyme inhibitory mechanism. First round SAR study picked two imidazonaphthyridinone frameworks with 1,8- and 3,5-disubstitution pattern as class I and class II c-Met kinase inhibitors, respectively. Further structural optimization on type II inhibitors by truncation of the imidazonaphthyridinone core and incorporation of an N-phenyl cyclopropane-1,1-dicarboxamide pharmacophore led to the discovery of novel imidazopyridine-based c-Met kinase inhibitors, displaying nanomolar enzyme inhibitory activity and improved Met kinase selectivity. More significantly, the new chemotype c-Met kinase inhibitors effectively inhibited Met phosphorylation and its downstream signaling as well as the proliferation of Met-dependent EBC-1 human lung cancer cells at submicromolar concentrations. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Oncogenic Kinase Bcr-Abl and Its Resistance to Pharmacological Inhibitors

    Czech Academy of Sciences Publication Activity Database

    Kryštof, Vladimír

    2008-01-01

    Roč. 102, č. 9 (2008), s. 795-800 E-ISSN 1213-7103 Institutional research plan: CEZ:AV0Z50380511 Keywords : imatinib * inhibitor * chronic myeloid leukaemia * kinase * cancer Subject RIV: CE - Biochemistry www.chemicke-listy.cz/docs/full/2008_09_795-800.pdf

  13. Identification of hematein as a novel inhibitor of protein kinase CK2 from a natural product library

    International Nuclear Information System (INIS)

    Hung, Ming-Szu; Xu, Zhidong; Lin, Yu-Ching; Mao, Jian-Hua; Yang, Cheng-Ta; Chang, Pey-Jium; Jablons, David M; You, Liang

    2009-01-01

    Casein kinase 2 (CK2) is dysregulated in various human cancers and is a promising target for cancer therapy. To date, there is no small molecular CK2 inhibitor in clinical trial yet. With the aim to identify novel CK2 inhibitors, we screened a natural product library. We adopted cell-based proliferation and CK2 kinase assays to screen CK2 inhibitors from a natural compound library. Dose-dependent response of CK2 inhibitors in vitro was determined by a radioisotope kinase assay. Western blot analysis was used to evaluate down stream Akt phosphorylation and apoptosis. Apoptosis was also evaluated by annexin-V/propidium iodide (PI) labeling method using flow cytometry. Inhibition effects of CK2 inhibitors on the growth of cancer and normal cells were evaluated by cell proliferation and viability assays. Hematein was identified as a novel CK2 inhibitor that is highly selective among a panel of kinases. It appears to be an ATP non-competitive and partially reversible CK2 inhibitor with an IC 50 value of 0.55 μM. In addition, hematein inhibited cancer cell growth partially through down-regulation of Akt phosphorylation and induced apoptosis in these cells. Furthermore, hematein exerted stronger inhibition effects on the growth of cancer cells than in normal cells. In this study, we showed that hematein is a novel selective and cell permeable small molecule CK2 inhibitor. Hematein showed stronger growth inhibition effects to cancer cells when compared to normal cells. This compound may represent a promising class of CK2 inhibitors

  14. Identification and Structure-Function Analysis of Subfamily Selective G Protein-Coupled Receptor Kinase Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Homan, Kristoff T.; Larimore, Kelly M.; Elkins, Jonathan M.; Szklarz, Marta; Knapp, Stefan; Tesmer, John J.G. [Michigan; (Oxford)

    2015-02-13

    Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.

  15. Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region.

    Science.gov (United States)

    Ahn, Yu Mi; Clare, Michael; Ensinger, Carol L; Hood, Molly M; Lord, John W; Lu, Wei-Ping; Miller, David F; Patt, William C; Smith, Bryan D; Vogeti, Lakshminarayana; Kaufman, Michael D; Petillo, Peter A; Wise, Scott C; Abendroth, Jan; Chun, Lawrence; Clark, Robin; Feese, Michael; Kim, Hidong; Stewart, Lance; Flynn, Daniel L

    2010-10-01

    Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase. Copyright © 2010 Elsevier Ltd. All rights reserved.

  16. Small Molecule Tyrosine Kinase Inhibitors of ErbB2/HER2/Neu in the Treatment of Aggressive Breast Cancer

    Directory of Open Access Journals (Sweden)

    Richard L. Schroeder

    2014-09-01

    Full Text Available The human epidermal growth factor receptor 2 (HER2 is a member of the erbB class of tyrosine kinase receptors. These proteins are normally expressed at the surface of healthy cells and play critical roles in the signal transduction cascade in a myriad of biochemical pathways responsible for cell growth and differentiation. However, it is widely known that amplification and subsequent overexpression of the HER2 encoding oncogene results in unregulated cell proliferation in an aggressive form of breast cancer known as HER2-positive breast cancer. Existing therapies such as trastuzumab (Herceptin® and lapatinib (Tyverb/Tykerb®, a monoclonal antibody inhibitor and a dual EGFR/HER2 kinase inhibitor, respectively, are currently used in the treatment of HER2-positive cancers, although issues with high recurrence and acquired resistance still remain. Small molecule tyrosine kinase inhibitors provide attractive therapeutic targets, as they are able to block cell signaling associated with many of the proposed mechanisms for HER2 resistance. In this regard we aim to present a review on the available HER2 tyrosine kinase inhibitors, as well as those currently in development. The use of tyrosine kinase inhibitors as sequential or combinatorial therapeutic strategies with other HER family inhibitors is also discussed.

  17. The structure and dynamic properties of the complete histidine phosphotransfer domain of the chemotaxis specific histidine autokinase CheA from Thermotoga maritima

    International Nuclear Information System (INIS)

    Vu, Anh; Hamel, Damon J.; Zhou Hongjun; Dahlquist, Frederick W.

    2011-01-01

    The bacterial histidine autokinase CheA contains a histidine phosphotransfer (Hpt) domain that accepts a phosphate from the catalytic domain and donates the phosphate to either target response regulator protein, CheY or CheB. The Hpt domain forms a helix-bundle structure with a conserved four-helix bundle motif and a variable fifth helix. Observation of two nearly equally populated conformations in the crystal structure of a Hpt domain fragment of CheA from Thermotoga maritima containing only the first four helices suggests more mobility in a tightly packed helix bundle structure than previously thought. In order to examine how the structures of Hpt domain homologs may differ from each other particularly in the conformation of the last helix, and whether an alternative conformation exists in the intact Hpt domain in solution, we have solved a high-resolution, solution structure of the CheA Hpt from T. maritima and characterized the backbone dynamics of this protein. The structure contains a four-helix bundle characteristic of histidine phosphotransfer domains. The position and orientation of the fifth helix resembles those in known Hpt domain crystal and solution structures in other histidine kinases. The alternative conformation that was reported in the crystal structure of the CheA Hpt from T. maritima missing the fifth helix is not detected in the solution structure, suggesting a role for the fifth helix in providing stabilizing forces to the overall structure.

  18. Photoactivatable Caged Prodrugs of VEGFR-2 Kinase Inhibitors

    OpenAIRE

    Boris Pinchuk; Rebecca Horbert; Alexander Döbber; Lydia Kuhl; Christian Peifer

    2016-01-01

    In this study, we report on the design, synthesis, photokinetic properties and in vitro evaluation of photoactivatable caged prodrugs for the receptor tyrosine kinase VEGFR-2. Highly potent VEGFR-2 inhibitors 1 and 3 were caged by introduction of a photoremovable protecting group (PPG) to yield the caged prodrugs 4 and 5. As expected, enzymatic and cellular proliferation assays showed dramatically diminished efficacy of caged prodrugs in vitro. Upon ultraviolet (UV) irradiation of the prodrug...

  19. QSAR, molecular docking studies of thiophene and imidazopyridine derivatives as polo-like kinase 1 inhibitors

    Science.gov (United States)

    Cao, Shandong

    2012-08-01

    The purpose of the present study was to develop in silico models allowing for a reliable prediction of polo-like kinase inhibitors based on a large diverse dataset of 136 compounds. As an effective method, quantitative structure activity relationship (QSAR) was applied using the comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). The proposed QSAR models showed reasonable predictivity of thiophene analogs (Rcv2=0.533, Rpred2=0.845) and included four molecular descriptors, namely IC3, RDF075m, Mor02m and R4e+. The optimal model for imidazopyridine derivatives (Rcv2=0.776, Rpred2=0.876) was shown to perform good in prediction accuracy, using GATS2m and BEHe1 descriptors. Analysis of the contour maps helped to identify structural requirements for the inhibitors and served as a basis for the design of the next generation of the inhibitor analogues. Docking studies were also employed to position the inhibitors into the polo-like kinase active site to determine the most probable binding mode. These studies may help to understand the factors influencing the binding affinity of chemicals and to develop alternative methods for prescreening and designing of polo-like kinase inhibitors.

  20. Virtual screening filters for the design of type II p38 MAP kinase inhibitors: a fragment based library generation approach.

    Science.gov (United States)

    Badrinarayan, Preethi; Sastry, G Narahari

    2012-04-01

    In this work, we introduce the development and application of a three-step scoring and filtering procedure for the design of type II p38 MAP kinase leads using allosteric fragments extracted from virtual screening hits. The design of the virtual screening filters is based on a thorough evaluation of docking methods, DFG-loop conformation, binding interactions and chemotype specificity of the 138 p38 MAP kinase inhibitors from Protein Data Bank bound to DFG-in and DFG-out conformations using Glide, GOLD and CDOCKER. A 40 ns molecular dynamics simulation with the apo, type I with DFG-in and type II with DFG-out forms was carried out to delineate the effects of structural variations on inhibitor binding. The designed docking-score and sub-structure filters were first tested on a dataset of 249 potent p38 MAP kinase inhibitors from seven diverse series and 18,842 kinase inhibitors from PDB, to gauge their capacity to discriminate between kinase and non-kinase inhibitors and likewise to selectively filter-in target-specific inhibitors. The designed filters were then applied in the virtual screening of a database of ten million (10⁷) compounds resulting in the identification of 100 hits. Based on their binding modes, 98 allosteric fragments were extracted from the hits and a fragment library was generated. New type II p38 MAP kinase leads were designed by tailoring the existing type I ATP site binders with allosteric fragments using a common urea linker. Target specific virtual screening filters can thus be easily developed for other kinases based on this strategy to retrieve target selective compounds. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. [Recent Advances and Prospect of Advanced Non-small Cell Lung Cancer Targeted 
Therapy: Focus on Small Molecular Tyrosine Kinase Inhibitors].

    Science.gov (United States)

    Zhang, Guowei; Wang, Huijuan; Ma, Zhiyong

    2017-04-20

    At present the treatment of advanced non-small cell lung cancer enters a targeted era and develops rapidly. New drugs appear constantly. Small molecular tyrosine kinase inhibitors have occupied the biggest piece of the territory, which commonly have a clear biomarker as predictor, and show remarkable effect in specific molecular classification of patients. The epidermal growth factor tyrosine kinase inhibitors such as gefitinib, erlotinib, icotinib and anaplastic lymphoma kinase tyrosine kinase inhibitors crizotinib have brought a milestone advance. In recent years new generations of tyrosine kinase inhibitors have achieved a great success in patients with acquired resistance to the above two kinds of drugs. At the same time new therapeutic targets are constantly emerging. So in this paper, we reviewed and summarized the important drugs and clinical trails on this topic, and made a prospect of the future development.

  2. Recent Advances and Prospect of Advanced Non-small Cell Lung Cancer Targeted 
Therapy: Focus on Small Molecular Tyrosine Kinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Guowei ZHANG

    2017-04-01

    Full Text Available At present the treatment of advanced non-small cell lung cancer enters a targeted era and develops rapidly. New drugs appear constantly. Small molecular tyrosine kinase inhibitors have occupied the biggest piece of the territory, which commonly have a clear biomarker as predictor, and show remarkable effect in specific molecular classification of patients. The epidermal growth factor tyrosine kinase inhibitors such as gefitinib, erlotinib, icotinib and anaplastic lymphoma kinase tyrosine kinase inhibitors crizotinib have brought a milestone advance. In recent years new generations of tyrosine kinase inhibitors have achieved a great success in patients with acquired resistance to the above two kinds of drugs. At the same time new therapeutic targets are constantly emerging. So in this paper, we reviewed and summarized the important drugs and clinical trails on this topic, and made a prospect of the future development.

  3. Alanine rich peptide from Populus trichocarpa inhibit growth of Staphylococcus aureus via targetting its extracellular domain of Sensor Histidine Kinase YycGex protein.

    Science.gov (United States)

    Al Akeel, Raid; Mateen, Ayesha; Syed, Rabbani; Al-Qahtani, Mohammed S; Alqahtani, A

    2018-05-11

    Due to growing concern towards microbial resistance, ongoing search for developing novel bioactive compounds such as peptides is on rise. The aim of this study was to evaluate antimicrobial effect of Populus trichocarpa extract, chemically identify the active peptide fraction and finds its target in Staphylococcus aureus. In this study the active fraction of P. trichocarpa crude extract was purified and characterized using MS/MS. This peptide PT13 antimicrobial activity was confirmed by in-vitro agar based disk diffusion and in-vivo infection model of G. mellonella. The proteomic expression analysis of S. aureus under influence of PT13 was studied using LTQ-Orbitrap-MS in-solution digestion and identity of target protein was acquired with their quantified expression using label-free approach of Progenesis QI software. Docking study was performed with peptide PT13 and its target YycG protein using CABS-dock. The active fraction PT13 sequence was identified as KVPVAAAAAAAAAVVASSMVVAAAK, with 25 amino acid including 13 alanine having M/Z 2194.2469. PT13 was uniformly inhibited growth S. aureus SA91 and MIC was determined 16 μg/mL for SA91 S. aureus strain. Sensor histidine kinase (YycG) was most significant target found differentially expressed under influence of PT13. G. mellonella larvae were killed rapidly due to S aureus infection, whereas death in protected group was insignificant in compare to control. The docking models showed ten docking models with RMSD value 1.89 for cluster 1 and RMSD value 3.95 for cluster 2 which is predicted to be high quality model. Alanine rich peptide could be useful in constructing as antimicrobial peptide for targeting extracellular Domain of Sensor Histidine Kinase YycG from S. aureus used in the study. Copyright © 2018. Published by Elsevier Ltd.

  4. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J. (Abbott)

    2012-03-02

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

  5. STK33 kinase inhibitor BRD-8899 has no effect on KRAS-dependent cancer cell viability.

    Science.gov (United States)

    Luo, Tuoping; Masson, Kristina; Jaffe, Jacob D; Silkworth, Whitney; Ross, Nathan T; Scherer, Christina A; Scholl, Claudia; Fröhling, Stefan; Carr, Steven A; Stern, Andrew M; Schreiber, Stuart L; Golub, Todd R

    2012-02-21

    Approximately 30% of human cancers harbor oncogenic gain-of-function mutations in KRAS. Despite interest in KRAS as a therapeutic target, direct blockade of KRAS function with small molecules has yet to be demonstrated. Based on experiments that lower mRNA levels of protein kinases, KRAS-dependent cancer cells were proposed to have a unique requirement for the serine/threonine kinase STK33. Thus, it was suggested that small-molecule inhibitors of STK33 might have therapeutic benefit in these cancers. Here, we describe the development of selective, low nanomolar inhibitors of STK33's kinase activity. The most potent and selective of these, BRD8899, failed to kill KRAS-dependent cells. While several explanations for this result exist, our data are most consistent with the view that inhibition of STK33's kinase activity does not represent a promising anti-KRAS therapeutic strategy.

  6. Tyrosine kinase inhibitors and mesenchymal stromal cells: effects on self-renewal, commitment and functions

    Science.gov (United States)

    Borriello, Adriana; Caldarelli, Ilaria; Bencivenga, Debora; Stampone, Emanuela; Perrotta, Silverio; Oliva, Adriana; Ragione, Fulvio Della

    2017-01-01

    The hope of selectively targeting cancer cells by therapy and eradicating definitively malignancies is based on the identification of pathways or metabolisms that clearly distinguish “normal” from “transformed” phenotypes. Some tyrosine kinase activities, specifically unregulated and potently activated in malignant cells, might represent important targets of therapy. Consequently, tyrosine kinase inhibitors (TKIs) might be thought as the “vanguard” of molecularly targeted therapy for human neoplasias. Imatinib and the successive generations of inhibitors of Bcr-Abl1 kinase, represent the major successful examples of TKI use in cancer treatment. Other tyrosine kinases have been selected as targets of therapy, but the efficacy of their inhibition, although evident, is less definite. Two major negative effects exist in this therapeutic strategy and are linked to the specificity of the drugs and to the role of the targeted kinase in non-malignant cells. In this review, we will discuss the data available on the TKIs effects on the metabolism and functions of mesenchymal stromal cells (MSCs). MSCs are widely distributed in human tissues and play key physiological roles; nevertheless, they might be responsible for important pathologies. At present, bone marrow (BM) MSCs have been studied in greater detail, for both embryological origins and functions. The available data are evocative of an unexpected degree of complexity and heterogeneity of BM-MSCs. It is conceivable that this grade of intricacy occurs also in MSCs of other organs. Therefore, in perspective, the negative effects of TKIs on MSCs might represent a critical problem in long-term cancer therapies based on such inhibitors. PMID:27750212

  7. Structure-Based Design of a Novel Series of Potent, Selective Inhibitors of the Class I Phosphatidylinositol 3-Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Adrian L.; D’Angelo, Noel D.; Bo, Yunxin Y.; Booker, Shon K.; Cee, Victor J.; Herberich, Brad; Hong, Fang-Tsao; Jackson, Claire L.M.; Lanman, Brian A.; Liu, Longbin; Nishimura, Nobuko; Pettus, Liping H.; Reed, Anthony B.; Tadesse, Seifu; Tamayo, Nuria A.; Wurz, Ryan P.; Yang, Kevin; Andrews, Kristin L.; Whittington, Douglas A.; McCarter, John D.; Miguel, Tisha San; Zalameda, Leeanne; Jiang, Jian; Subramanian, Raju; Mullady, Erin L.; Caenepeel, Sean; Freeman, Daniel J.; Wang, Ling; Zhang, Nancy; Wu, Tian; Hughes, Paul E.; Norman, Mark H. (Amgen)

    2012-09-17

    A highly selective series of inhibitors of the class I phosphatidylinositol 3-kinases (PI3Ks) has been designed and synthesized. Starting from the dual PI3K/mTOR inhibitor 5, a structure-based approach was used to improve potency and selectivity, resulting in the identification of 54 as a potent inhibitor of the class I PI3Ks with excellent selectivity over mTOR, related phosphatidylinositol kinases, and a broad panel of protein kinases. Compound 54 demonstrated a robust PD-PK relationship inhibiting the PI3K/Akt pathway in vivo in a mouse model, and it potently inhibited tumor growth in a U-87 MG xenograft model with an activated PI3K/Akt pathway.

  8. The inhibitors of cyclin-dependent kinases and GSK-3β enhance osteoclastogenesis

    Directory of Open Access Journals (Sweden)

    Yosuke Akiba

    2016-03-01

    Full Text Available Osteoclasts are multinucleated cells with bone resorption activity that is crucial for bone remodeling. RANK‐RANKL (receptor activator of nuclear factor κB ligand signaling has been shown as a main signal pathway for osteoclast differentiation. However, the molecular mechanism and the factors regulating osteoclastogenesis remain to be fully understood. In this study, we performed a chemical genetic screen, and identified a Cdks/GSK-3β (cyclin-dependent kinases/glycogen synthase kinaseinhibitor, kenpaullone, and two Cdks inhibitors, olomoucine and roscovitine, all of which significantly enhance osteoclastogenesis of RAW264.7 cells by upregulating NFATc1 (nuclear factor of activated T cells, cytoplasmic 1 levels. We also determined that the all three compounds increase the number of osteoclast differentiated from murine bone marrow cells. Furthermore, the three inhibitors, especially kenpaullone, promoted maturation of cathepsin K, suggesting that the resorption activity of the resultant osteoclasts is also activated. Our findings indicate that inhibition of GSK-3β and/or Cdks enhance osteoclastogenesis by modulating the RANK–RANKL signaling pathway.

  9. Reduction of Fibrogenesis by Selective Delivery of a Rho Kinase Inhibitor to Hepatic Stellate Cells in Mice

    NARCIS (Netherlands)

    van Beuge, M. M.; Prakash, J.; Lacombe, M.; Gosens, R.; Post, E.; Reker-Smit, C.; Beljaars, L.; Poelstra, K.

    One of the pathways activated during liver fibrosis is the Rho kinase pathway, which regulates activation, migration, and contraction of hepatic stellate cells (HSC). Inhibition of this kinase by the Rho kinase inhibitor Y27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide

  10. A historical overview of protein kinases and their targeted small molecule inhibitors.

    Science.gov (United States)

    Roskoski, Robert

    2015-10-01

    catalytic subunits. PKA and all other protein kinase domains have a small amino-terminal lobe and large carboxyterminal lobe as determined by X-ray crystallography. The N-lobe and C-lobe form a cleft that serves as a docking site for MgATP. Nearly all active protein kinases contain a K/E/D/D signature sequence that plays important structural and catalytic roles. Protein kinases contain hydrophobic catalytic and regulatory spines and collateral shell residues that are required to assemble the active enzyme. There are two general kinds of conformational changes associated with most protein kinases. The first conformational change involves the formation of an intact regulatory spine to form an active enzyme. The second conformational change occurs in active kinases as they toggle between open and closed conformations during their catalytic cycles. Because mutations and dysregulation of protein kinases play causal roles in human disease, this family of enzymes has become one of the most important drug targets over the past two decades. Imatinib was approved by the United States FDA for the treatment of chronic myelogenous leukemia in 2001; this small molecule inhibits the BCR-Abl protein kinase oncoprotein that results from the formation of the Philadelphia chromosome. More than two dozen other orally effective mechanism-based small molecule protein kinase inhibitors have been subsequently approved by the FDA. These drugs bind to the ATP-binding site of their target enzymes and extend into nearby hydrophobic pockets. Most of these protein kinase inhibitors prolong survival in cancer patients only weeks or months longer than standard cytotoxic therapies. In contrast, the clinical effectiveness of imatinib against chronic myelogenous leukemia is vastly superior to that of any other targeted protein kinase inhibitor with overall survival lasting a decade or more. However, the near universal and expected development of drug resistance in the treatment of neoplastic disorders

  11. An Aminopyridazine Inhibitor of Death Associated Protein Kinase Attenuates Hypoxia-Ischemia Induced Brain Damage

    Energy Technology Data Exchange (ETDEWEB)

    Velentza, A.V.; Wainwright, M.S.; Zasadzki, M.; Mirzoeva, S.; Haiech, J.; Focia, P.J.; Egli, M.; Watterson, D.M.

    2010-03-08

    Death associated protein kinase (DAPK) is a calcium and calmodulin regulated enzyme that functions early in eukaryotic programmed cell death, or apoptosis. To validate DAPK as a potential drug discovery target for acute brain injury, the first small molecule DAPK inhibitor was synthesized and tested in vivo. A single injection of the aminopyridazine-based inhibitor administered 6 h after injury attenuated brain tissue or neuronal biomarker loss measured, respectively, 1 week and 3 days later. Because aminopyridazine is a privileged structure in neuropharmacology, we determined the high-resolution crystal structure of a binary complex between the kinase domain and a molecular fragment of the DAPK inhibitor. The co-crystal structure describes a structural basis for interaction and provides a firm foundation for structure-assisted design of lead compounds with appropriate molecular properties for future drug development.

  12. Photoactivatable Caged Prodrugs of VEGFR-2 Kinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Boris Pinchuk

    2016-04-01

    Full Text Available In this study, we report on the design, synthesis, photokinetic properties and in vitro evaluation of photoactivatable caged prodrugs for the receptor tyrosine kinase VEGFR-2. Highly potent VEGFR-2 inhibitors 1 and 3 were caged by introduction of a photoremovable protecting group (PPG to yield the caged prodrugs 4 and 5. As expected, enzymatic and cellular proliferation assays showed dramatically diminished efficacy of caged prodrugs in vitro. Upon ultraviolet (UV irradiation of the prodrugs original inhibitory activity was completely restored and even distinctly reinforced, as was the case for the prodrug 4. The presented results are a further evidence for caging technique being an interesting approach in the protein kinase field. It could enable spatial and temporal control for the inhibition of VEGFR-2. The described photoactivatable prodrugs might be highly useful as biological probes for studying the VEGFR-2 signal transduction.

  13. Combined effects of EGFR tyrosine kinase inhibitors and vATPase inhibitors in NSCLC cells

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hyeon-Ok [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Hong, Sung-Eun [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Kim, Chang Soon [Department of Microbiological Engineering, Kon-Kuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 143–701 (Korea, Republic of); Park, Jin-Ah; Kim, Jin-Hee; Kim, Ji-Young; Kim, Bora [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Chang, Yoon Hwan; Hong, Seok-Il; Hong, Young Jun [Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Park, In-Chul, E-mail: parkic@kirams.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Lee, Jin Kyung, E-mail: jklee@kirams.re.kr [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of)

    2015-08-15

    Despite excellent initial clinical responses of non-small cell lung cancer (NSCLC) patients to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), many patients eventually develop resistance. According to a recent report, vacuolar H + ATPase (vATPase) is overexpressed and is associated with chemotherapy drug resistance in NSCLC. We investigated the combined effects of EGFR TKIs and vATPase inhibitors and their underlying mechanisms in the regulation of NSCLC cell death. We found that combined treatment with EGFR TKIs (erlotinib, gefitinib, or lapatinib) and vATPase inhibitors (bafilomycin A1 or concanamycin A) enhanced synergistic cell death compared to treatments with each drug alone. Treatment with bafilomycin A1 or concanamycin A led to the induction of Bnip3 expression in an Hif-1α dependent manner. Knock-down of Hif-1α or Bnip3 by siRNA further enhanced cell death induced by bafilomycin A1, suggesting that Hif-1α/Bnip3 induction promoted resistance to cell death induced by the vATPase inhibitors. EGFR TKIs suppressed Hif-1α and Bnip3 expression induced by the vATPase inhibitors, suggesting that they enhanced the sensitivity of the cells to these inhibitors by decreasing Hif-1α/Bnip3 expression. Taken together, we conclude that EGFR TKIs enhance the sensitivity of NSCLC cells to vATPase inhibitors by decreasing Hif-1α/Bnip3 expression. We suggest that combined treatment with EGFR TKIs and vATPase inhibitors is potentially effective for the treatment of NSCLC. - Highlights: • Co-treatment with EGFR TKIs and vATPase inhibitors induces synergistic cell death • EGFR TKIs enhance cell sensitivity to vATPase inhibitors via Hif-1α downregulation • Co-treatment of these inhibitors is potentially effective for the treatment of NSCLC.

  14. Comparisons of actin filament disruptors and Rho kinase inhibitors as potential antiglaucoma medications

    OpenAIRE

    Tian, Baohe; Kaufman, Paul L

    2012-01-01

    Dynamics of the actin cytoskeleton in the trabecular meshwork play a crucial role in the regulation of trabecular outflow resistance. The actin filament disruptors and Rho kinase inhibitors affect the dynamics of the actomyosin system by either disrupting the actin filaments or inhibiting the Rho kinase-activated cellular contractility. Both approaches induce similar morphological changes and resistance decreases in the trabecular outflow pathway, and thus both have potential as antiglaucoma ...

  15. TYROSINE KINASE INHIBITORS AND PREGNANCY

    Directory of Open Access Journals (Sweden)

    Elisabetta Abruzzese

    2014-04-01

    Full Text Available The management of patients with chronic myeloid leukemia (CML during pregnancy has became recently a matter of continuous debate.  The introduction of the Tyrosine Kinase Inhibitors (TKIs in clinical practice has dramatically changed the prognosis of CML patients.  Patients diagnosed in chronic phase can reasonably expect many years of excellent disease control and good quality of life, as well as a normal life expectancy.  This fact has come the necessity to address issues relating to fertility and pregnancy. Physicians are not infrequently being asked for advice regarding the need for, and or the appropriateness of, stopping treatment in order to conceive. In this report we will review the data published in terms of fertility, conception, pregnancy, pregnancy outcome and illness control for all the approved TKIs, as well as suggest how to manage a planned and/or unplanned pregnancy.

  16. An integrative study to identify novel scaffolds for sphingosine kinase 1 inhibitors

    Czech Academy of Sciences Publication Activity Database

    Vettorazzi, M.; Angelina, E.; Lima, S.; Goněc, T.; Otevřel, J.; Marvanová, P.; Padrtová, T.; Mokrý, P.; Bobáľ, P.; Acosta, L. M.; Palma, A.; Cobo, J.; Bobálová, Janette; Csollei, J.; Malik, I.; Alvarez, S.; Spiegel, S.; Jampílek, J.; Enriz, R. D.

    2017-01-01

    Roč. 139, OCT (2017), s. 461-481 ISSN 0223-5234 Institutional support: RVO:68081715 Keywords : sphingosine kinase 1 inhibitors * bioassays * molecular modelling * virtual screening Subject RIV: CB - Analytical Chemistry, Separation OBOR OECD: Analytical chemistry Impact factor: 4.519, year: 2016

  17. Modulation of neutrophil superoxide generation by inhibitors of protein kinase C, calmodulin, diacylglycerol and myosin light chain kinases, and peptidyl prolyl cis-trans isomerase.

    Science.gov (United States)

    Bergstrand, H; Eriksson, T; Hallberg, A; Johansson, B; Karabelas, K; Michelsen, P; Nybom, A

    1992-12-01

    To assess the role of protein kinase C (PKC) in the respiratory burst of adherent human polymorphonuclear leukocytes (PMNL), reduction of ferricytochrome C by cells triggered with a phorbol ester (PMA), ionophore A23187, serum-treated zymosan (STZ) or three lipid derivatives, 3-decanoyl-sn-glycerol (G-3-OCOC9), (R,R)-1,4-diethyl-2-O-decyl-L-tartrate (Tt-2-OC10) and 3-decyloxy-5-hydroxymethylphenol (DHP) was examined in a microtiter plate procedure in the presence of inhibitors of PKC and, for comparison, inhibitors of calmodulin, diacylglycerol and myosin light chain kinases and the peptidyl-prolyl cis-trans isomerase activity of fujiphilin. 1) Of the protein kinase inhibitors examined, Ro 31-7549 and staurosporine reduced responses to all stimuli except possibly STZ; in contrast, K252a and the myosin light chain kinase inhibitors ML-7 and ML-9 blocked responses to A23187 and STZ better than those triggered by PMA. H-7 reduced responses to A23187, DHP and G-3-OCOC9, and calphostin, palmitoyl carnitine, sphingosine and the multifunctional drugs TMB-8 and W-7 reduced A23187; they also, when examined, reduced decane derivative-induced O2- production more effectively than PMA- and STZ-triggered responses. Polymyxin B, 4 alpha-PMA and retinal displayed no inhibitory capacity. 2) Of the selective calmodulin antagonists, CGS 9343B, Ro 22-4839 and calmidazolium did not inhibit the oxidative response irrespective of the stimulus used, whereas metofenazate reduced those evoked by A23187, DHP, G-3-OCOC9 and STZ.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. A comprehensive target selectivity survey of the BCR-ABL kinase inhibitor INNO-406 by kinase profiling and chemical proteomics in chronic myeloid leukemia cells.

    Science.gov (United States)

    Rix, U; Remsing Rix, L L; Terker, A S; Fernbach, N V; Hantschel, O; Planyavsky, M; Breitwieser, F P; Herrmann, H; Colinge, J; Bennett, K L; Augustin, M; Till, J H; Heinrich, M C; Valent, P; Superti-Furga, G

    2010-01-01

    Resistance to the BCR-ABL tyrosine kinase inhibitor imatinib poses a pressing challenge in treating chronic myeloid leukemia (CML). This resistance is often caused by point mutations in the ABL kinase domain or by overexpression of LYN. The second-generation BCR-ABL inhibitor INNO-406 is known to inhibit most BCR-ABL mutants and LYN efficiently. Knowledge of its full target spectrum would provide the molecular basis for potential side effects or suggest novel therapeutic applications and possible combination therapies. We have performed an unbiased chemical proteomics native target profile of INNO-406 in CML cells combined with functional assays using 272 recombinant kinases thereby identifying several new INNO-406 targets. These include the kinases ZAK, DDR1/2 and various ephrin receptors. The oxidoreductase NQO2, inhibited by both imatinib and nilotinib, is not a relevant target of INNO-406. Overall, INNO-406 has an improved activity over imatinib but a slightly broader target profile than both imatinib and nilotinib. In contrast to dasatinib and bosutinib, INNO-406 does not inhibit all SRC kinases and most TEC family kinases and is therefore expected to elicit fewer side effects. Altogether, these properties may make INNO-406 a valuable component in the drug arsenal against CML.

  19. Structure-Based Design of Potent and Selective 3-Phosphoinositide-Dependent Kinase-1 (PDK1) Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Medina, Jesus R.; Becker, Christopher J.; Blackledge, Charles W.; Duquenne, Celine; Feng, Yanhong; Grant, Seth W.; Heerding, Dirk; Li, William H.; Miller, William H.; Romeril, Stuart P.; Scherzer, Daryl; Shu, Arthur; Bobko, Mark A.; Chadderton, Antony R.; Dumble, Melissa; Gardiner, Christine M.; Gilbert, Seth; Liu, Qi; Rabindran, Sridhar K.; Sudakin, Valery; Xiang, Hong; Brady, Pat G.; Campobasso, Nino; Ward, Paris; Axten, Jeffrey M. (GSKPA)

    2014-10-02

    Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.

  20. Pharmacogenetics of telatinib, a VEGFR-2 and VEGFR-3 tyrosine kinase inhibitor, used in patients with solid tumors

    NARCIS (Netherlands)

    N. Steeghs (Neeltje); A.J. Gelderblom (Hans); J.A.M. Wessels (Judith); F.A.L.M. Eskens (Ferry); N. de Bont (Natasja); J.W. Nortier (Johan); H.J. Guchelaar (Henk Jan)

    2011-01-01

    textabstractSummary: Purpose Telatinib is an orally active small-molecule tyrosine kinase inhibitor of kinase insert domain receptor (KDR; VEGFR-2) and fms-related tyrosine kinase 4 (FLT4; VEGFR-3). This study aims at the identification of relationships between single nucleotide polymorphisms (SNPs)

  1. Differential expression of mRNAs for protein kinase inhibitor isoforms in mouse brain.

    OpenAIRE

    Seasholtz, A F; Gamm, D M; Ballestero, R P; Scarpetta, M A; Uhler, M D

    1995-01-01

    Many neurotransmitters are known to regulate neuronal cell function by means of activation of cAMP-dependent protein kinase (PKA) and phosphorylation of neuronal substrate proteins, including transcription factors and ion channels. Here, we have characterized the gene expression of two isoforms of a protein kinase inhibitor (PKI) specific for PKA in mouse brain by RNase protection and in situ hybridization histochemistry. The studies demonstrate that the PKI alpha isoform is abundant in many ...

  2. Generation of a proton motive force by histidine decarboxylation and electrogenic histidine/histamine antiport in Lactobacillus buchneri.

    Science.gov (United States)

    Molenaar, D; Bosscher, J S; ten Brink, B; Driessen, A J; Konings, W N

    1993-05-01

    Lactobacillus buchneri ST2A vigorously decarboxylates histidine to the biogenic amine histamine, which is excreted into the medium. Cells grown in the presence of histidine generate both a transmembrane pH gradient, inside alkaline, and an electrical potential (delta psi), inside negative, upon addition of histidine. Studies of the mechanism of histidine uptake and histamine excretion in membrane vesicles and proteoliposomes devoid of cytosolic histidine decarboxylase activity demonstrate that histidine uptake, histamine efflux, and histidine/histamine exchange are electrogenic processes. Histidine/histamine exchange is much faster than the unidirectional fluxes of these substrates, is inhibited by an inside-negative delta psi and is stimulated by an inside positive delta psi. These data suggest that the generation of metabolic energy from histidine decarboxylation results from an electrogenic histidine/histamine exchange and indirect proton extrusion due to the combined action of the decarboxylase and carrier-mediated exchange. The abundance of amino acid decarboxylation reactions among bacteria suggests that this mechanism of metabolic energy generation and/or pH regulation is widespread.

  3. Preferential Selectivity of Inhibitors with Human Tau Protein Kinase Gsk3 Elucidates Their Potential Roles for Off-Target Alzheimer’s Therapy

    Directory of Open Access Journals (Sweden)

    Jagadeesh Kumar Dasappa

    2013-01-01

    Full Text Available Alzheimer’s disease (AD is a neurodegenerative disorder characterized by the accumulation of amyloid beta peptides (A and neurofibrillary tangles (NFTs. The abnormal phosphorylation of tau leads to the formation of NFTs produced by the action of tau kinases, resulting in the loss of neurons and synapse, leading to dementia. Hence, tau kinases have become potential drug target candidates for small molecule inhibitors. With an aim to explore the identification of a common inhibitor, this investigation was undertaken towards analyzing all 10 tau kinases which are implicated in phosphorylation of AD. A set of 7 inhibitors with varied scaffolds were collected from the Protein Data Bank (PDB. The analysis, involving multiple sequence alignment, 3D structural alignment, catalytic active site overlap, and docking studies, has enabled elucidation of the pharmacophoric patterns for the class of 7 inhibitors. Our results divulge that tau protein kinases share a specific set of conserved structural elements for the binding of inhibitors and ATP, respectively. The scaffold of 3-aminopyrrolidine (inhibitor 6 exhibits high preferential affinity with GSK3. Surprisingly, the PDB does not contain the structural details of GSK3 with this specific inhibitor. Thus, our investigations provide vital clues towards design of novel off-target drugs for Alzheimer’s.

  4. The effect of MRN complex and ATM kinase inhibitors on Zebrafish embryonic development

    Science.gov (United States)

    Kumaran, Malina; Fazry, Shazrul

    2018-04-01

    Zebrafish is an ideal animal model to study developmental biology due to its transparent embryos and rapid development stages of embryogenesis. Here we investigate the role of DNA damage proteins, specifically Mre11/Rad50/NBN (MRN) complex and ataxia-telangiectasia mutated (ATM) kinase during embryogenesis by inhibiting its function using specific MRN complex (Mirin) and ATM Kinase inhibitors (Ku60019 and Ku55933). Zebrafish embryos at midblastula transition (MBT) stage are treated with Mirin, Ku60019 and Ku55933. The embryonic development of the embryos was monitored at 24 hours-post fertilisation (hpf), 48 hpf and 72 hpf. We observed that at the lowest concentrations (3 µM of Mirin, 1.5 nM of Ku60019 and 3 nM of Ku55933), the inhibitors treated embryos have 100% survivability. However, with increasing inhibitor concentration, the survivability drops. Control or mock treatment of all embryos shows 100 % survivability rate. This study suggests that DNA damage repair proteins may be crucial for normal zebrafish embryo development and survival.

  5. Identification of novel inhibitors for Pim-1 kinase using pharmacophore modeling based on a novel method for selecting pharmacophore generation subsets

    Science.gov (United States)

    Shahin, Rand; Swellmeen, Lubna; Shaheen, Omar; Aboalhaija, Nour; Habash, Maha

    2016-01-01

    Targeting Proviral integration-site of murine Moloney leukemia virus 1 kinase, hereafter called Pim-1 kinase, is a promising strategy for treating different kinds of human cancer. Headed for this a total list of 328 formerly reported Pim-1 kinase inhibitors has been explored and divided based on the pharmacophoric features of the most active molecules into 10 subsets projected to represent potential active binding manners accessible to ligands within the binding pocket of Pim-1 kinase. Discovery Studio 4.1 (DS 4.1) was employed to detect potential pharmacophoric active binding manners anticipated by Pim-1 Kinase inhibitors. The pharmacophoric models were then allowed to compete within Quantitative Structure Activity Relationship (QSAR) framework with other 2D descriptors. Accordingly Genetic algorithm and multiple linear regression investigation were engaged to find the finest QSAR equation that has the best predictive power r 262 2 = 0.70, F = 119.14, r LOO 2 = 0.693, r PRESS 2 against 66 external test inhibitors = 0.71 q2 = 0.55. Three different pharmacophores appeared in the successful QSAR equation this represents three different binding modes for inhibitors within the Pim-1 kinase binding pocket. Pharmacophoric models were later used to screen compounds within the National Cancer Institute database. Several low micromolar Pim-1 Kinase inhibitors were captured. The most potent hits show IC50 values of 0.77 and 1.03 µM. Also, upon analyzing the successful QSAR Equation we found that some polycyclic aromatic electron-rich structures namely 6-Chloro-2-methoxy-acridine can be considered as putative hits for Pim-1 kinase inhibition.

  6. Sibiriline, a new small chemical inhibitor of receptor-interacting protein kinase 1, prevents immune-dependent hepatitis.

    Science.gov (United States)

    Le Cann, Fabienne; Delehouzé, Claire; Leverrier-Penna, Sabrina; Filliol, Aveline; Comte, Arnaud; Delalande, Olivier; Desban, Nathalie; Baratte, Blandine; Gallais, Isabelle; Piquet-Pellorce, Claire; Faurez, Florence; Bonnet, Marion; Mettey, Yvette; Goekjian, Peter; Samson, Michel; Vandenabeele, Peter; Bach, Stéphane; Dimanche-Boitrel, Marie-Thérèse

    2017-09-01

    Necroptosis is a regulated form of cell death involved in several disease models including in particular liver diseases. Receptor-interacting protein kinases, RIPK1 and RIPK3, are the main serine/threonine kinases driving this cell death pathway. We screened a noncommercial, kinase-focused chemical library which allowed us to identify Sibiriline as a new inhibitor of necroptosis induced by tumor necrosis factor (TNF) in Fas-associated protein with death domain (FADD)-deficient Jurkat cells. Moreover, Sib inhibits necroptotic cell death induced by various death ligands in human or mouse cells while not protecting from caspase-dependent apoptosis. By using competition binding assay and recombinant kinase assays, we demonstrated that Sib is a rather specific competitive RIPK1 inhibitor. Molecular docking analysis shows that Sib is trapped closed to human RIPK1 adenosine triphosphate-binding site in a relatively hydrophobic pocket locking RIPK1 in an inactive conformation. In agreement with its RIPK1 inhibitory property, Sib inhibits both TNF-induced RIPK1-dependent necroptosis and RIPK1-dependent apoptosis. Finally, Sib protects mice from concanavalin A-induced hepatitis. These results reveal the small-molecule Sib as a new RIPK1 inhibitor potentially of interest for the treatment of immune-dependent hepatitis. © 2017 Federation of European Biochemical Societies.

  7. Leveraging the Pre-DFG Residue Thr-406 To Obtain High Kinase Selectivity in an Aminopyrazole-Type PAK1 Inhibitor Series.

    Science.gov (United States)

    Rudolph, Joachim; Aliagas, Ignacio; Crawford, James J; Mathieu, Simon; Lee, Wendy; Chao, Qi; Dong, Ping; Rouge, Lionel; Wang, Weiru; Heise, Christopher; Murray, Lesley J; La, Hank; Liu, Yanzhou; Manning, Gerard; Diederich, François; Hoeflich, Klaus P

    2015-06-11

    To increase kinase selectivity in an aminopyrazole-based PAK1 inhibitor series, analogues were designed to interact with the PAK1 deep-front pocket pre-DFG residue Thr-406, a residue that is hydrophobic in most kinases. This goal was achieved by installing lactam head groups to the aminopyrazole hinge binding moiety. The corresponding analogues represent the most kinase selective ATP-competitive Group I PAK inhibitors described to date. Hydrogen bonding with the Thr-406 side chain was demonstrated by X-ray crystallography, and inhibitory activities, particularly against kinases with hydrophobic pre-DFG residues, were mitigated. Leveraging hydrogen bonding side chain interactions with polar pre-DFG residues is unprecedented, and similar strategies should be applicable to other appropriate kinases.

  8. Tyrosine Kinase Inhibitors Induced Thyroid Dysfunction: A Review of Its Incidence, Pathophysiology, Clinical Relevance, and Treatment

    Directory of Open Access Journals (Sweden)

    Hala Ahmadieh

    2013-01-01

    Full Text Available Tyrosine kinase inhibitors (TKI belong to a new class of molecular multitargeted anticancer therapy which targets different growth factor receptors and hence attenuates cancer cell survival and growth. Since their introduction as adjunct treatment for renal cell carcinoma and gastrointestinal stromal tumors (GIST, a number of reports have demonstrated that TKI can induce thyroid dysfunction which was especially more common with sunitinib maleate. Many mechanisms with respect to this adverse effect of tyrosine kinase inhibitors have been proposed including their induction of thyroiditis, capillary regression in the thyroid gland, antithyroid peroxidase antibody production, and their ability to decrease iodine uptake by the thyroid gland. Of interest is the observation that TKI-induced thyroid dysfunction may actually be protective as it was shown to improve overall survival, and it was suggested that it may have a prognostic value. Followup on thyroid function tests while patients are maintained on tyrosine kinase inhibitor is strongly recommended. When thyroid dysfunction occurs, appropriate treatment should be individualized depending on patients symptoms and thyroid stimulating hormone level.

  9. An Investigation of Molecular Docking and Molecular Dynamic Simulation on Imidazopyridines as B-Raf Kinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Huiding Xie

    2015-11-01

    Full Text Available In the recent cancer treatment, B-Raf kinase is one of key targets. Nowadays, a group of imidazopyridines as B-Raf kinase inhibitors have been reported. In order to investigate the interaction between this group of inhibitors and B-Raf kinase, molecular docking, molecular dynamic (MD simulation and binding free energy (ΔGbind calculation were performed in this work. Molecular docking was carried out to identify the key residues in the binding site, and MD simulations were performed to determine the detail binding mode. The results obtained from MD simulation reveal that the binding site is stable during the MD simulations, and some hydrogen bonds (H-bonds in MD simulations are different from H-bonds in the docking mode. Based on the obtained MD trajectories, ΔGbind was computed by using Molecular Mechanics Generalized Born Surface Area (MM-GBSA, and the obtained energies are consistent with the activities. An energetic analysis reveals that both electrostatic and van der Waals contributions are important to ΔGbind, and the unfavorable polar solvation contribution results in the instability of the inhibitor with the lowest activity. These results are expected to understand the binding between B-Raf and imidazopyridines and provide some useful information to design potential B-Raf inhibitors.

  10. Inflammatory Signaling by NOD-RIPK2 Is Inhibited by Clinically Relevant Type II Kinase Inhibitors.

    Science.gov (United States)

    Canning, Peter; Ruan, Qui; Schwerd, Tobias; Hrdinka, Matous; Maki, Jenny L; Saleh, Danish; Suebsuwong, Chalada; Ray, Soumya; Brennan, Paul E; Cuny, Gregory D; Uhlig, Holm H; Gyrd-Hansen, Mads; Degterev, Alexei; Bullock, Alex N

    2015-09-17

    RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Overall survival after immunotherapy, tyrosine kinase inhibitors and surgery in treatment of metastatic renal cell cancer

    DEFF Research Database (Denmark)

    de Lichtenberg, Trine Honnens; Hermann, Gregers G.; Rorth, Mikael

    2014-01-01

    Abstract Objective. The aim of this study was to evaluate overall survival (OS) after treatment of metastatic renal cell carcinoma (mRCC) following the introduction of tyrosine kinase inhibitors (TKIs) and mammalian target of rapamycin (mTOR) inhibitors. Material and methods. One-hundred and forty...

  12. Tofacitinib and analogs as inhibitors of the histone kinase PRK1 (PKN1).

    Science.gov (United States)

    Ostrovskyi, Dmytro; Rumpf, Tobias; Eib, Julia; Lumbroso, Alexandre; Slynko, Inna; Klaeger, Susan; Heinzlmeir, Stephanie; Forster, Michael; Gehringer, Matthias; Pfaffenrot, Ellen; Bauer, Silke Mona; Schmidtkunz, Karin; Wenzler, Sandra; Metzger, Eric; Kuster, Bernhard; Laufer, Stefan; Schüle, Roland; Sippl, Wolfgang; Breit, Bernhard; Jung, Manfred

    2016-09-01

    The histone kinase PRK1 has been identified as a potential target to combat prostate cancer but selective PRK1 inhibitors are lacking. The US FDA -approved JAK1-3 inhibitor tofacitinib also potently inhibits PRK1 in vitro. We show that tofacitinib also inhibits PRK1 in a cellular setting. Using tofacitinib as a starting point for structure-activity relationship studies, we identified a more potent and another more selective PRK1 inhibitor compared with tofacitinib. Furthermore, we found two potential PRK1/JAK3-selectivity hotspots. The identified inhibitors and the selectivity hotspots lay the basis for the development of selective PRK1 inhibitors. The identification of PRK1, but also of other cellular tofacitinib targets, has implications on its clinical use and on future development of tofacitinib-like JAK inhibitors. [Formula: see text].

  13. Generation of a Proton Motive Force by Histidine Decarboxylation and Electrogenic Histidine/Histamine Antiport in Lactobacillus buchneri

    OpenAIRE

    Molenaar, Douwe; Bosscher, Jaap S.; Brink, Bart ten; Driessen, Arnold J.M.; Konings, Wil N.

    1993-01-01

    Lactobacillus buchneri ST2A vigorously decarboxylates histidine to the biogenic amine histamine, which is excreted into the medium. Cells grown in the presence of histidine generate both a transmembrane pH gradient, inside alkaline, and an electrical potential (delta psi), inside negative, upon addition of histidine. Studies of the mechanism of histidine uptake and histamine excretion in membrane vesicles and proteoliposomes devoid of cytosolic histidine decarboxylase activity demonstrate tha...

  14. Development of Pharmacophore Model for Indeno[1,2-b]indoles as Human Protein Kinase CK2 Inhibitors and Database Mining

    Directory of Open Access Journals (Sweden)

    Samer Haidar

    2017-01-01

    Full Text Available Protein kinase CK2, initially designated as casein kinase 2, is an ubiquitously expressed serine/threonine kinase. This enzyme, implicated in many cellular processes, is highly expressed and active in many tumor cells. A large number of compounds has been developed as inhibitors comprising different backbones. Beside others, structures with an indeno[1,2-b]indole scaffold turned out to be potent new leads. With the aim of developing new inhibitors of human protein kinase CK2, we report here on the generation of common feature pharmacophore model to further explain the binding requirements for human CK2 inhibitors. Nine common chemical features of indeno[1,2-b]indole-type CK2 inhibitors were determined using MOE software (Chemical Computing Group, Montreal, Canada. This pharmacophore model was used for database mining with the aim to identify novel scaffolds for developing new potent and selective CK2 inhibitors. Using this strategy several structures were selected by searching inside the ZINC compound database. One of the selected compounds was bikaverin (6,11-dihydroxy-3,8-dimethoxy-1-methylbenzo[b]xanthene-7,10,12-trione, a natural compound which is produced by several kinds of fungi. This compound was tested on human recombinant CK2 and turned out to be an active inhibitor with an IC50 value of 1.24 µM.

  15. Selectivity analysis of protein kinase CK2 inhibitors DMAT, TBB and resorufin in cisplatin-induced stress responses

    DEFF Research Database (Denmark)

    Fritz, Gerhard; Issinger, Olaf-Georg; Olsen, Birgitte Brinkmann

    2009-01-01

    Targeting protein kinases as a therapeutic approach to treat various diseases, especially cancer is currently a fast growing business. Although many inhibitors are available, exhibiting remarkable potency, the major challenge is their selectivity. Here we show that the protein kinase CK2 inhibito...

  16. Systems biology analysis of mitogen activated protein kinase inhibitor resistance in malignant melanoma.

    Science.gov (United States)

    Zecena, Helma; Tveit, Daniel; Wang, Zi; Farhat, Ahmed; Panchal, Parvita; Liu, Jing; Singh, Simar J; Sanghera, Amandeep; Bainiwal, Ajay; Teo, Shuan Y; Meyskens, Frank L; Liu-Smith, Feng; Filipp, Fabian V

    2018-04-04

    Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma. The cellular model evolved in response to clinical dosage of the BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring was uncovered to be based on non-genomic adaptation and validated in two distinct melanoma models, SK-MEL-28 and A375. Both cell lines have activating BRAF mutations and display metastatic potential. Downregulation of dual specific phosphatases, tumor suppressors, and negative MAPK regulators reengages mitogenic signaling. Upregulation of growth factors, cytokines, and cognate receptors triggers signaling pathways circumventing BRAF blockage. Further, changes in amino acid and one-carbon metabolism support cellular proliferation despite MAPK inhibitor treatment. In addition, treatment-resistant cells upregulate pigmentation and melanogenesis, pathways which partially overlap with MAPK signaling. Upstream regulator analysis discovered significant perturbation in oncogenic forkhead box and hypoxia inducible factor family transcription factors. The established cellular models offer mechanistic insight into cellular changes and therapeutic targets under inhibitor resistance in malignant melanoma. At a systems biology level, the MAPK pathway undergoes major rewiring while acquiring inhibitor resistance

  17. Pim kinase inhibition sensitizes FLT3-ITD acute myeloid leukemia cells to topoisomerase 2 inhibitors through increased DNA damage and oxidative stress

    Science.gov (United States)

    Doshi, Kshama A.; Trotta, Rossana; Natarajan, Karthika; Rassool, Feyruz V.; Tron, Adriana E.; Huszar, Dennis; Perrotti, Danilo; Baer, Maria R.

    2016-01-01

    Internal tandem duplication of fms-like tyrosine kinase-3 (FLT3-ITD) is frequent (30 percent) in acute myeloid leukemia (AML), and is associated with short disease-free survival following chemotherapy. The serine threonine kinase Pim-1 is a pro-survival oncogene transcriptionally upregulated by FLT3-ITD that also promotes its signaling in a positive feedback loop. Thus inhibiting Pim-1 represents an attractive approach in targeting FLT3-ITD cells. Indeed, co-treatment with the pan-Pim kinase inhibitor AZD1208 or expression of a kinase-dead Pim-1 mutant sensitized FLT3-ITD cell lines to apoptosis triggered by chemotherapy drugs including the topoisomerase 2 inhibitors daunorubicin, etoposide and mitoxantrone, but not the nucleoside analog cytarabine. AZD1208 sensitized primary AML cells with FLT3-ITD to topoisomerase 2 inhibitors, but did not sensitize AML cells with wild-type FLT3 or remission bone marrow cells, supporting a favorable therapeutic index. Mechanistically, the enhanced apoptosis observed with AZD1208 and topoisomerase 2 inhibitor combination treatment was associated with increased DNA double-strand breaks and increased levels of reactive oxygen species (ROS), and co-treatment with the ROS scavenger N-acetyl cysteine rescued FLT3-ITD cells from AZD1208 sensitization to topoisomerase 2 inhibitors. Our data support testing of Pim kinase inhibitors with topoisomerase 2 inhibitors, but not with cytarabine, to improve treatment outcomes in AML with FLT3-ITD. PMID:27374090

  18. Combining the pan-aurora kinase inhibitor AMG 900 with histone deacetylase inhibitors enhances antitumor activity in prostate cancer

    International Nuclear Information System (INIS)

    Paller, Channing J; Wissing, Michel D; Mendonca, Janet; Sharma, Anup; Kim, Eugene; Kim, Hea-Soo; Kortenhorst, Madeleine S Q; Gerber, Stephanie; Rosen, Marc; Shaikh, Faraz; Zahurak, Marianna L; Rudek, Michelle A; Hammers, Hans; Rudin, Charles M; Carducci, Michael A; Kachhap, Sushant K

    2014-01-01

    Histone deacetylase inhibitors (HDACIs) are being tested in clinical trials for the treatment of solid tumors. While most studies have focused on the reexpression of silenced tumor suppressor genes, a number of genes/pathways are downregulated by HDACIs. This provides opportunities for combination therapy: agents that further disable these pathways through inhibition of residual gene function are speculated to enhance cell death in combination with HDACIs. A previous study from our group indicated that mitotic checkpoint kinases such as PLK1 and Aurora A are downregulated by HDACIs. We used in vitro and in vivo xenograft models of prostate cancer (PCA) to test whether combination of HDACIs with the pan-aurora kinase inhibitor AMG 900 can synergistically or additively kill PCA cells. AMG 900 and HDACIs synergistically decreased cell proliferation activity and clonogenic survival in DU-145, LNCaP, and PC3 PCA cell lines compared to single-agent treatment. Cellular senescence, polyploidy, and apoptosis was significantly increased in all cell lines after combination treatment. In vivo xenograft studies indicated decreased tumor growth and decreased aurora B kinase activity in mice treated with low-dose AMG 900 and vorinostat compared to either agent alone. Pharmacodynamics was assessed by scoring for phosphorylated histone H3 through immunofluorescence. Our results indicate that combination treatment with low doses of AMG 900 and HDACIs could be a promising therapy for future clinical trials against PCA

  19. Identification and Characterization of Amlexanox as a G Protein-Coupled Receptor Kinase 5 Inhibitor

    Directory of Open Access Journals (Sweden)

    Kristoff T. Homan

    2014-10-01

    Full Text Available G protein-coupled receptor kinases (GRKs have been implicated in human diseases ranging from heart failure to diabetes. Previous studies have identified several compounds that selectively inhibit GRK2, such as paroxetine and balanol. Far fewer selective inhibitors have been reported for GRK5, a target for the treatment of cardiac hypertrophy, and the mechanism of action of reported compounds is unknown. To identify novel scaffolds that selectively inhibit GRK5, a differential scanning fluorometry screen was used to probe a library of 4480 compounds. The best hit was amlexanox, an FDA-approved anti-inflammatory, anti-allergic immunomodulator. The crystal structure of amlexanox in complex with GRK1 demonstrates that its tricyclic aromatic ring system forms ATP-like interactions with the hinge of the kinase domain, which is likely similar to how this drug binds to IκB kinase ε (IKKε, another kinase known to be inhibited by this compound. Amlexanox was also able to inhibit myocyte enhancer factor 2 transcriptional activity in neonatal rat ventricular myocytes in a manner consistent with GRK5 inhibition. The GRK1 amlexanox structure thus serves as a springboard for the rational design of inhibitors with improved potency and selectivity for GRK5 and IKKε.

  20. Differential effects on cell motility, embryonic stem cell self-renewal and senescence by diverse Src kinase family inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Tamm, Christoffer, E-mail: christoffer.tamm@imbim.uu.se; Galito, Sara Pijuan, E-mail: sara.pijuan@imbim.uu.se; Anneren, Cecilia, E-mail: cecilia.anneren@imbim.uu.se

    2012-02-15

    The Src family of non-receptor tyrosine kinases (SFKs) has been shown to play an intricate role in embryonic stem (ES) cell maintenance. In the present study we have focused on the underlying molecular mechanisms responsible for the vastly different effects induced by various commonly used SFK inhibitors. We show that several diverse cell types, including fibroblasts completely lacking SFKs, cannot undergo mitosis in response to SU6656 and that this is caused by an unselective inhibition of Aurora kinases. In contrast, PP2 and PD173952 block motility immediately upon exposure and forces cells to grow in dense colonies. The subsequent halt in proliferation of fibroblast and epithelial cells in the center of the colonies approximately 24 h post-treatment appears to be caused by cell-to-cell contact inhibition rather than a direct effect of SFK kinase inhibition. Interestingly, in addition to generating more homogenous and dense ES cell cultures, without any diverse effect on proliferation, PP2 and PD173652 also promote ES cell self-renewal by reducing the small amount of spontaneous differentiation typically observed under standard ES cell culture conditions. These effects could not be mirrored by the use of Gleevec, a potent inhibitor of c-Abl and PDGFR kinases that are also inhibited by PP2. -- Highlights: Black-Right-Pointing-Pointer SFK inhibitor SU6656 induces senescence in mouse ES cells. Black-Right-Pointing-Pointer SU6656 inhibits mitosis in a SFK-independent manner via cross-selectivity for Aurora kinases. Black-Right-Pointing-Pointer SFK inhibitor PP2 impairs cell motility in various cell lines, including mouse ES cells. Black-Right-Pointing-Pointer Ensuing impeded motility, PP2 inhibits proliferation of various cells lines except for mouse ES cells. Black-Right-Pointing-Pointer SFK inhibitors PP2 and PD173952 impede spontaneous differentiation in standard mouse ES culture maintenance.

  1. Differential effects on cell motility, embryonic stem cell self-renewal and senescence by diverse Src kinase family inhibitors

    International Nuclear Information System (INIS)

    Tamm, Christoffer; Galitó, Sara Pijuan; Annerén, Cecilia

    2012-01-01

    The Src family of non-receptor tyrosine kinases (SFKs) has been shown to play an intricate role in embryonic stem (ES) cell maintenance. In the present study we have focused on the underlying molecular mechanisms responsible for the vastly different effects induced by various commonly used SFK inhibitors. We show that several diverse cell types, including fibroblasts completely lacking SFKs, cannot undergo mitosis in response to SU6656 and that this is caused by an unselective inhibition of Aurora kinases. In contrast, PP2 and PD173952 block motility immediately upon exposure and forces cells to grow in dense colonies. The subsequent halt in proliferation of fibroblast and epithelial cells in the center of the colonies approximately 24 h post-treatment appears to be caused by cell-to-cell contact inhibition rather than a direct effect of SFK kinase inhibition. Interestingly, in addition to generating more homogenous and dense ES cell cultures, without any diverse effect on proliferation, PP2 and PD173652 also promote ES cell self-renewal by reducing the small amount of spontaneous differentiation typically observed under standard ES cell culture conditions. These effects could not be mirrored by the use of Gleevec, a potent inhibitor of c-Abl and PDGFR kinases that are also inhibited by PP2. -- Highlights: ► SFK inhibitor SU6656 induces senescence in mouse ES cells. ► SU6656 inhibits mitosis in a SFK-independent manner via cross-selectivity for Aurora kinases. ► SFK inhibitor PP2 impairs cell motility in various cell lines, including mouse ES cells. ► Ensuing impeded motility, PP2 inhibits proliferation of various cells lines except for mouse ES cells. ► SFK inhibitors PP2 and PD173952 impede spontaneous differentiation in standard mouse ES culture maintenance.

  2. Recent Advances of Colony-Stimulating Factor-1 Receptor (CSF-1R) Kinase and Its Inhibitors.

    Science.gov (United States)

    El-Gamal, Mohammed I; Al-Ameen, Shahad K; Al-Koumi, Dania M; Hamad, Mawadda G; Jalal, Nouran A; Oh, Chang-Hyun

    2018-01-17

    Colony stimulation factor-1 receptor (CSF-1R), which is also known as FMS kinase, plays an important role in initiating inflammatory, cancer, and bone disorders when it is overstimulated by its ligand, CSF-1. Innate immunity, as well as macrophage differentiation and survival, are regulated by the stimulation of the CSF-1R. Another ligand, interlukin-34 (IL-34), was recently reported to activate the CSF-1R receptor in a different manner. The relationship between CSF-1R and microglia has been reviewed. Both CSF-1 antibodies and small molecule CSF-1R kinase inhibitors have now been tested in animal models and in humans. In this Perspective, we discuss the role of CSF-1 and IL-34 in producing cancer, bone disorders, and inflammation. We also review the newly discovered and improved small molecule kinase inhibitors and monoclonal antibodies that have shown potent activity toward CSF-1R, reported from 2012 until 2017.

  3. HSP90 inhibitors potentiate PGF2α-induced IL-6 synthesis via p38 MAP kinase in osteoblasts.

    Directory of Open Access Journals (Sweden)

    Kazuhiko Fujita

    Full Text Available Heat shock protein 90 (HSP90 that is ubiquitously expressed in various tissues, is recognized to be a major molecular chaperone. We have previously reported that prostaglandin F2α (PGF2α, a potent bone remodeling mediator, stimulates the synthesis of interleukin-6 (IL-6 through p44/p42 mitogen-activated protein (MAP kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that Rho-kinase acts at a point upstream of p38 MAP kinase. In the present study, we investigated the involvement of HSP90 in the PGF2α-stimulated IL-6 synthesis and the underlying mechanism in MC3T3-E1 cells. Geldanamycin, an inhibitor of HSP90, significantly amplified both the PGF2α-stimulated IL-6 release and the mRNA expression levels. In addition, other HSP90 inhibitors, 17-allylamino-17demethoxy-geldanamycin (17-AAG and 17-dimethylamino-ethylamino-17-demethoxy-geldanamycin (17-DMAG and onalespib, enhanced the PGF2α-stimulated IL-6 release. Geldanamycin, 17-AAG and onalespib markedly strengthened the PGF2α-induced phosphorylation of p38 MAP kinase. Geldanamycin and 17-AAG did not affect the PGF2α-induced phosphorylation of p44/p42 MAP kinase and myosin phosphatase targeting subunit (MYPT-1, a substrate of Rho-kinase, and the protein levels of RhoA and Rho-kinase. In addition, HSP90-siRNA enhanced the PGF2α-induced phosphorylation of p38 MAP kinase. Furthermore, SB203580, an inhibitor of p38 MAP kinase, significantly suppressed the amplification by geldanamycin, 17-AAG or 17-DMAG of the PGF2α-stimulated IL-6 release. Our results strongly suggest that HSP90 negatively regulates the PGF2α-stimulated IL-6 synthesis in osteoblasts, and that the effect of HSP90 is exerted through regulating p38 MAP kinase activation.

  4. In silico design and biological evaluation of a dual specificity kinase inhibitor targeting cell cycle progression and angiogenesis.

    Science.gov (United States)

    Latham, Antony M; Kankanala, Jayakanth; Fearnley, Gareth W; Gage, Matthew C; Kearney, Mark T; Homer-Vanniasinkam, Shervanthi; Wheatcroft, Stephen B; Fishwick, Colin W G; Ponnambalam, Sreenivasan

    2014-01-01

    Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues. We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2) and cyclin-dependent kinase 1 (CDK1). This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A)-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis. We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery.

  5. Inhibitors of JAK-family kinases: an update on the patent literature 2013-2015, part 2.

    Science.gov (United States)

    Kettle, Jason G; Åstrand, Annika; Catley, Matthew; Grimster, Neil P; Nilsson, Magnus; Su, Qibin; Woessner, Richard

    2017-02-01

    Janus kinases (JAKs) are a family of four enzymes; JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) that are critical in cytokine signalling and are strongly linked to both cancer and inflammatory diseases. There are currently two launched JAK inhibitors for the treatment of human conditions: tofacitinib for Rheumatoid arthritis (RA) and ruxolitinib for myeloproliferative neoplasms including intermediate or high risk myelofibrosis and polycythemia vera. Areas covered: This review covers patents claiming activity against one or more JAK family members in the period 2013-2015 inclusive, and covers 95 patents from 42 applicants, split over two parts. The authors have ordered recent patents according to the primary applicant's name, with part 2 covering J through Z. Expert opinion: Inhibition of JAK-family kinases is an area of growing interest, catalysed by the maturity of data on marketed inhibitors ruxolitinib and tofacitinib in late stage clinical trials. Many applicants are pursuing traditional fast-follower strategies around these inhibitors, with a range of chemical strategies adopted. The challenge will be to show sufficient differentiation to the originator compounds, since dose limiting toxicities with such agents appear to be on target and mechanism-related and also considering that such agents may be available as generic compounds by the time follower agents reach market.

  6. Inhibitors of JAK-family kinases: an update on the patent literature 2013-2015, part 1.

    Science.gov (United States)

    Kettle, Jason G; Åstrand, Annika; Catley, Matthew; Grimster, Neil P; Nilsson, Magnus; Su, Qibin; Woessner, Richard

    2017-02-01

    Janus kinases (JAKs) are a family of four enzymes; JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) that are critical in cytokine signalling and are strongly linked to both cancer and inflammatory diseases. There are currently two launched JAK inhibitors for the treatment of human conditions: tofacitinib for Rheumatoid arthritis (RA) and ruxolitinib for myeloproliferative neoplasms including intermediate or high risk myelofibrosis and polycythemia vera. Areas covered: This review covers patents claiming activity against one or more JAK family members in the period 2013-2015 inclusive, and covers 95 patents from 42 applicants, split over two parts. The authors have ordered recent patents according to the primary applicant's name, with part 1 covering A through to I. Expert opinion: Inhibition of JAK-family kinases is an area of growing interest, catalysed by the maturity of data on marketed inhibitors ruxolitinib and tofacitinib in late stage clinical trials. Many applicants are pursuing traditional fast-follower strategies around these inhibitors, with a range of chemical strategies adopted. The challenge will be to show sufficient differentiation to the originator compounds, since dose limiting toxicities with such agents appear to be on target and mechanism-related and also considering that such agents may be available as generic compounds by the time follower agents reach market.

  7. Structural characterization of nonactive site, TrkA-selective kinase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Su, Hua-Poo; Rickert, Keith; Burlein, Christine; Narayan, Kartik; Bukhtiyarova, Marina; Hurzy, Danielle M.; Stump, Craig A.; Zhang, Xufang; Reid, John; Krasowska-Zoladek, Alicja; Tummala, Srivanya; Shipman, Jennifer M.; Kornienko, Maria; Lemaire, Peter A.; Krosky, Daniel; Heller, Amanda; Achab, Abdelghani; Chamberlin, Chad; Saradjian, Peter; Sauvagnat, Berengere; Yang, Xianshu; Ziebell, Michael R.; Nickbarg, Elliott; Sanders, John M.; Bilodeau, Mark T.; Carroll, Steven S.; Lumb, Kevin J.; Soisson, Stephen M.; Henze, Darrell A.; Cooke, Andrew J. (Merck)

    2016-12-30

    Current therapies for chronic pain can have insufficient efficacy and lead to side effects, necessitating research of novel targets against pain. Although originally identified as an oncogene, Tropomyosin-related kinase A (TrkA) is linked to pain and elevated levels of NGF (the ligand for TrkA) are associated with chronic pain. Antibodies that block TrkA interaction with its ligand, NGF, are in clinical trials for pain relief. Here, we describe the identification of TrkA-specific inhibitors and the structural basis for their selectivity over other Trk family kinases. The X-ray structures reveal a binding site outside the kinase active site that uses residues from the kinase domain and the juxtamembrane region. Three modes of binding with the juxtamembrane region are characterized through a series of ligand-bound complexes. The structures indicate a critical pharmacophore on the compounds that leads to the distinct binding modes. The mode of interaction can allow TrkA selectivity over TrkB and TrkC or promiscuous, pan-Trk inhibition. This finding highlights the difficulty in characterizing the structure-activity relationship of a chemical series in the absence of structural information because of substantial differences in the interacting residues. These structures illustrate the flexibility of binding to sequences outside of—but adjacent to—the kinase domain of TrkA. This knowledge allows development of compounds with specificity for TrkA or the family of Trk proteins.

  8. Exploring the Hypersensitivity of PTEN Deleted Prostate Cancer Stem Cells to WEE1 Tyrosine Kinase Inhibitors

    Science.gov (United States)

    2015-12-01

    AWARD NUMBER: W81XWH-14-1-0251 TITLE: Exploring the Hypersensitivity of PTEN Deleted Prostate Cancer Stem Cells to WEE1 Tyrosine Kinase... Tyrosine Kinase Inhibitors 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0251 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Kiran Mahajan 5d...ABSTRACT Central to all cycling cells-including prostate cancer stem cells- is the expression of WEE1 tyrosine kinase. WEE1 monitors duplication of

  9. Ibrutinib (PCI-32765), the first BTK (Bruton's tyrosine kinase) inhibitor in clinical trials.

    Science.gov (United States)

    Brown, Jennifer R

    2013-03-01

    Ibrutinib is a potent covalent kinase inhibitor that targets BTK. BTK, or Bruton's tyrosine kinase, is an obvious target for therapy of B cell diseases because inactivating mutations lead to B cell aplasia in humans and the disease X-linked agammaglobulinemia. Ibrutinib has modest cytotoxicity against CLL cells in vitro but also blocks trophic stimuli from the microenvironment. As with other inhibitors of the BCR pathway, ibrutinib causes rapid nodal reduction and response associated with rapid increase in lymphocytosis, which then returns to baseline over time. The ORR of ibrutinib in relapsed refractory CLL is 67 % with PFS 88 % at 15 months. In a cohort of untreated patients 65 years and over, the estimated 15 month PFS is 96 %. Registration trials have been initiated, and the difficult task that remains is to determine where in the course of CLL therapy this drug will have the greatest impact and benefit for patients.

  10. Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase Pathway

    Science.gov (United States)

    2017-01-01

    v e V ia b il it y Figure 8. PC3-LN4 cells in normoxia or hypoxia were treated with Pim inhibitors. Left panel shows a Western blot and the...3728-36, PMID 25241892 4. Warfel, NA, Kraft, AS. Pim kinase (and Akt) biology and signaling in tumors. Pharmacol Ther. 2015 Jul; 151: 41 - 9. doi: 10.1016...Associated Fibroblast Biology in Prostate Cancer These studies will accelerate and significantly advance the rational development of targeted agents

  11. The evolving field of kinase inhibitors in thyroid cancer.

    Science.gov (United States)

    Marotta, V; Sciammarella, C; Vitale, M; Colao, A; Faggiano, A

    2015-01-01

    Most of the genetic events implicated in the pathogenesis of thyroid cancer (TC) involve genes with kinase activity. Thus, kinase inhibitors (KIs) are very relevant in this field. KIs are considered the most suitable treatment for patients with iodine-refractory differentiated TC; these patients comprise the subgroup with the poorer prognosis. To date, only sorafenib has been approved for this indication, but promising results have been reported with several other KIs. In particular, lenvatinib has demonstrated excellent efficacy, with both progression-free survival and objective tumour response being better than with sorafenib. Despite being considered to be well tolerated, both sorafenib and lenvatinib have shown a remarkable toxicity, which has led to dose reductions in the majority of patients and to treatment discontinuation in a significant proportion of cases. The role of KIs in differentiated TC may be revolutionised by the finding that selumetinib may restore a clinical response to radioactive iodine (RAI). Vandetanib and cabozantinib have been approved for the treatment of advanced, progressive medullary TC (MTC). Nevertheless, the toxicity of both compounds suggests their selective use in those patients with strong disease progression. Treatment with the mTOR-inhibitor everolimus, alone or in combination with somatostatin analogues, should be studied in metastatic MTC patients with slow progression of disease, these representing the vast majority of patients. KIs did not significantly impact on the clinical features of anaplastic TC (ATC). Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  12. How to Achieve Better Results Using PASS-Based Virtual Screening: Case Study for Kinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Pavel V. Pogodin

    2018-04-01

    Full Text Available Discovery of new pharmaceutical substances is currently boosted by the possibility of utilization of the Synthetically Accessible Virtual Inventory (SAVI library, which includes about 283 million molecules, each annotated with a proposed synthetic one-step route from commercially available starting materials. The SAVI database is well-suited for ligand-based methods of virtual screening to select molecules for experimental testing. In this study, we compare the performance of three approaches for the analysis of structure-activity relationships that differ in their criteria for selecting of “active” and “inactive” compounds included in the training sets. PASS (Prediction of Activity Spectra for Substances, which is based on a modified Naïve Bayes algorithm, was applied since it had been shown to be robust and to provide good predictions of many biological activities based on just the structural formula of a compound even if the information in the training set is incomplete. We used different subsets of kinase inhibitors for this case study because many data are currently available on this important class of drug-like molecules. Based on the subsets of kinase inhibitors extracted from the ChEMBL 20 database we performed the PASS training, and then applied the model to ChEMBL 23 compounds not yet present in ChEMBL 20 to identify novel kinase inhibitors. As one may expect, the best prediction accuracy was obtained if only the experimentally confirmed active and inactive compounds for distinct kinases in the training procedure were used. However, for some kinases, reasonable results were obtained even if we used merged training sets, in which we designated as inactives the compounds not tested against the particular kinase. Thus, depending on the availability of data for a particular biological activity, one may choose the first or the second approach for creating ligand-based computational tools to achieve the best possible results in

  13. Call for Action: Invasive Fungal Infections Associated With Ibrutinib and Other Small Molecule Kinase Inhibitors Targeting Immune Signaling Pathways.

    Science.gov (United States)

    Chamilos, Georgios; Lionakis, Michail S; Kontoyiannis, Dimitrios P

    2018-01-06

    Opportunistic infections caused by Pneumocystis jirovecii, Cryptococcus neoformans, and ubiquitous airborne filamentous fungi have been recently reported in patients with hematological cancers historically considered at low risk for invasive fungal infections (IFIs), after receipt of the Bruton tyrosine kinase inhibitor ibrutinib. The spectrum and severity of IFIs often observed in these patients implies the presence of a complex immunodeficiency that may not be solely attributed to mere inhibition of Bruton tyrosine kinase. In view of the surge in development of small molecule kinase inhibitors for treatment of malignant and autoimmune diseases, it is possible that there would be an emergence of IFIs associated with the effects of these molecules on the immune system. Preclinical assessment of the immunosuppressive effects of kinase inhibitors and human studies aimed at improving patient risk stratification for development of IFIs could lead to prevention, earlier diagnosis, and better outcomes in affected patients. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  14. Ibrutinib: a first in class covalent inhibitor of Bruton's tyrosine kinase.

    Science.gov (United States)

    Davids, Matthew S; Brown, Jennifer R

    2014-05-01

    Ibrutinib (formerly PCI-32765) is a potent, covalent inhibitor of Bruton's tyrosine kinase, a kinase downstream of the B-cell receptor that is critical for B-cell survival and proliferation. In preclinical studies, ibrutinib bound to Bruton's tyrosine kinase with high affinity, leading to inhibition of B-cell receptor signaling, decreased B-cell activation and induction of apoptosis. In clinical studies, ibrutinib has been well-tolerated and has demonstrated profound anti-tumor activity in a variety of hematologic malignancies, most notably chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), leading to US FDA approval for relapsed CLL and MCL. Ongoing studies are evaluating ibrutinib in other types of non-Hodgkin's lymphoma, such as diffuse large B-cell lymphoma and Waldenström's macrogobulinemia, in larger Phase III studies in CLL and MCL, and in combination studies with monoclonal antibodies and chemotherapy. Future studies will combine ibrutinib with other promising novel agents currently in development in hematologic malignancies.

  15. All-trans-retinoic acid enhances apoptosis induction by tyrosine kinase inhibitors in the eosinophilic leukemia-derived EoL-1 cell line.

    Science.gov (United States)

    Robert, Carine; Apàti, Agota; Chomienne, Christine; Papp, Béla

    2008-02-01

    Imatinib and retinoids induce apoptosis in FIP1L1/PDGFRalpha-positive EoL-1 leukemia cells. Although imatinib induces complete remission in most FIP1L1/PDGFRalpha-positive patients, response to imatinib is sometimes suboptimal. In order to enhance the potency of the molecularly targeted therapy of eosinophilic leukemia, we investigated the effect of retinoids combined with tyrosine kinase inhibitors on EoL-1 cells. We demonstrate that retinoids combined with tyrosine kinase inhibitors lead to enhanced apoptosis induction in EoL-1 cells. Our results suggest that tyrosine kinase inhibitors combined with retinoids may constitute a valuable therapeutic approach for sensitive neoplasias that may display enhanced anti-leukemic potency when compared to single drug treatments.

  16. Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells.

    Science.gov (United States)

    Sheremet, Ya A; Yemets, A I; Azmi, A; Vissenberg, K; Verbelen, J P; Blume, Ya B

    2012-01-01

    To test whether reversible tubulin phosphorylation plays any role in the process of plant mitosis the effects of inhibitors of tyrosine kinases, herbimycin A, genistein and tyrphostin AG 18, and of an inhibitor of tyrosine phosphatases, sodium orthovanadate, on microtubule organization and mitosis progression in a synchronized BY-2 culture has been investigated. It was found that treatment with inhibitors of tyrosine kinases of BY-2 cells at the G2/M transition did not lead to visible disturbances of mitotic microtubule structures, while it did reduce the frequency of their appearance. We assume that a decreased tyrosine phosphorylation level could alter the microtubule dynamic instability parameters during interphase/prophase transition. All types of tyrosine kinase inhibitors used caused a prophase delay: herbimycin A and genistein for 2 h, and tyrphostin AG18 for 1 h. Thereafter the peak of mitosis was displaced for 1 h by herbimycin A or genistein exposure, but after tyrphostin AG18 treatment the timing of the mitosis-peak was comparable to that in control cells. Enhancement of tyrosine phosphorylation induced by the tyrosine phosphatase inhibitor resulted in the opposite effect on BY-2 mitosis transition. Culture treatment with sodium orthovanadate during 1 h resulted in an accelerated start of the prophase and did not lead to the alteration in time of the mitotic index peak formation, as compared to control cells. We suppose that the reversible tyrosine phosphorylation can be involved in the regulation of interphase to M phase transition possibly through regulation of microtubule dynamics in plant cells.

  17. Akt Inhibitor A-443654 Interferes with Mitotic Progression by Regulating Aurora A Kinase Expression

    Directory of Open Access Journals (Sweden)

    Xuesong Liu

    2008-08-01

    Full Text Available Both Akt and Aurora A kinase have been shown to be important targets for intervention for cancer therapy. We report here that Compound A (A-443654, a specific Akt inhibitor, interferes with mitotic progression and bipolar spindle formation. Compound A induces G2/M accumulation, defects in centrosome separation, and formation of either monopolar arrays or disorganized spindles. On the basis of gene expression array studies, we identified Aurora A as one of the genes regulated transcriptionally by Akt inhibitors including Compound A. Inhibition of the phosphatidylinositol 3-kinase (PI3K/Akt pathway, either by PI3K inhibitor LY294002 or by Compound A, dramatically inhibits the promoter activity of Aurora A, whereas the mammalian target of rapamycin inhibitor has little effect, suggesting that Akt might be responsible for up-regulating Aurora A for mitotic progression. Further analysis of the Aurora A promoter region indicates that the Ets element but not the Sp1 element is required for Compound A-sensitive transcriptional control of Aurora A. Overexpression of Aurora A in cells treated with Compound A attenuates the mitotic arrest and the defects in bipolar spindle formation induced by Akt inhibition. Our studies suggest that that Akt may promote mitotic progression through the transcriptional regulation of Aurora A.

  18. HER2-targeted therapy in breast cancer. Monoclonal antibodies and tyrosine kinase inhibitors

    DEFF Research Database (Denmark)

    Nielsen, Dorte Lisbet; Andersson, Michael; Kamby, Claus

    2008-01-01

    There is strong clinical evidence that trastuzumab, a monoclonal antibody targeting the human epidermal growth factor receptor (HER) two tyrosine kinase receptor, is an important component of first-line treatment of patients with HER2-positive metastatic breast cancer. In particular the combination...... of trastuzumab to chemotherapy improves disease-free and overall survival. The use of lapatinib, a dual tyrosine kinase inhibitor of both HER1 and HER2, in combination with capecitabine in the second-line treatment of HER2-positive patients with metastatic breast cancer previously treated with trastuzumab has...

  19. Development of dihydropyridone indazole amides as selective Rho-kinase inhibitors.

    Science.gov (United States)

    Goodman, Krista B; Cui, Haifeng; Dowdell, Sarah E; Gaitanopoulos, Dimitri E; Ivy, Robert L; Sehon, Clark A; Stavenger, Robert A; Wang, Gren Z; Viet, Andrew Q; Xu, Weiwei; Ye, Guosen; Semus, Simon F; Evans, Christopher; Fries, Harvey E; Jolivette, Larry J; Kirkpatrick, Robert B; Dul, Edward; Khandekar, Sanjay S; Yi, Tracey; Jung, David K; Wright, Lois L; Smith, Gary K; Behm, David J; Bentley, Ross; Doe, Christopher P; Hu, Erding; Lee, Dennis

    2007-01-11

    Rho kinase (ROCK1) mediates vascular smooth muscle contraction and is a potential target for the treatment of hypertension and related disorders. Indazole amide 3 was identified as a potent and selective ROCK1 inhibitor but possessed poor oral bioavailability. Optimization of this lead resulted in the discovery of a series of dihydropyridones, exemplified by 13, with improved pharmacokinetic parameters relative to the initial lead. Indazole substitution played a critical role in decreasing clearance and improving oral bioavailability.

  20. Optimization of Allosteric With-No-Lysine (WNK) Kinase Inhibitors and Efficacy in Rodent Hypertension Models

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Ken; Levell, Julian; Yoon, Taeyong; Kohls, Darcy; Yowe, David; Rigel, Dean F.; Imase, Hidetomo; Yuan, Jun; Yasoshima, Kayo; DiPetrillo, Keith; Monovich, Lauren; Xu, Lingfei; Zhu, Meicheng; Kato, Mitsunori; Jain, Monish; Idamakanti, Neeraja; Taslimi, Paul; Kawanami, Toshio; Argikar, Upendra A.; Kunjathoor, Vidya; Xie, Xiaoling; Yagi, Yukiko I.; Iwaki, Yuki; Robinson, Zachary; Park, Hyi-Man (Novartis)

    2017-08-03

    The observed structure–activity relationship of three distinct ATP noncompetitive With-No-Lysine (WNK) kinase inhibitor series, together with a crystal structure of a previously disclosed allosteric inhibitor bound to WNK1, led to an overlay hypothesis defining core and side-chain relationships across the different series. This in turn enabled an efficient optimization through scaffold morphing, resulting in compounds with a good balance of selectivity, cellular potency, and pharmacokinetic profile, which were suitable for in vivo proof-of-concept studies. When dosed orally, the optimized compound reduced blood pressure in mice overexpressing human WNK1, and induced diuresis, natriuresis and kaliuresis in spontaneously hypertensive rats (SHR), confirming that this mechanism of inhibition of WNK kinase activity is effective at regulating cardiovascular homeostasis.

  1. In silico design and biological evaluation of a dual specificity kinase inhibitor targeting cell cycle progression and angiogenesis.

    Directory of Open Access Journals (Sweden)

    Antony M Latham

    Full Text Available Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues.We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2 and cyclin-dependent kinase 1 (CDK1. This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis.We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery.

  2. A kinase inhibitor screen identifies Mcl-1 and Aurora kinase A as novel treatment targets in antiestrogen-resistant breast cancer cells

    DEFF Research Database (Denmark)

    Thrane, S; Pedersen, A M; Thomsen, M B H

    2015-01-01

    Antiestrogen resistance is a major problem in breast cancer treatment. Therefore, the search for new therapeutic targets and biomarkers for antiestrogen resistance is crucial. In this study, we performed a kinase inhibitor screen on antiestrogen responsive MCF-7 cells and a panel of MCF-7-derived...

  3. Protein kinase inhibitor peptide (PKI): a family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function.

    Science.gov (United States)

    Dalton, George D; Dewey, William L

    2006-02-01

    Signal transduction cascades involving cAMP-dependent protein kinase are highly conserved among a wide variety of organisms. Given the universal nature of this enzyme it is not surprising that cAMP-dependent protein kinase plays a critical role in numerous cellular processes. This is particularly evident in the nervous system where cAMP-dependent protein kinase is involved in neurotransmitter release, gene transcription, and synaptic plasticity. Protein kinase inhibitor peptide (PKI) is an endogenous thermostable peptide that modulates cAMP-dependent protein kinase function. PKI contains two distinct functional domains within its amino acid sequence that allow it to: (1) potently and specifically inhibit the activity of the free catalytic subunit of cAMP-dependent protein kinase and (2) export the free catalytic subunit of cAMP-dependent protein kinase from the nucleus. Three distinct PKI isoforms (PKIalpha, PKIbeta, PKIgamma) have been identified and each isoform is expressed in the brain. PKI modulates neuronal synaptic activity, while PKI also is involved in morphogenesis and symmetrical left-right axis formation. In addition, PKI also plays a role in regulating gene expression induced by cAMP-dependent protein kinase. Future studies should identify novel physiological functions for endogenous PKI both in the nervous system and throughout the body. Most interesting will be the determination whether functional differences exist between individual PKI isoforms which is an intriguing possibility since these isoforms exhibit: (1) cell-type specific tissue expression patterns, (2) different potencies for the inhibition of cAMP-dependent protein kinase activity, and (3) expression patterns that are hormonally, developmentally and cell-cycle regulated. Finally, synthetic peptide analogs of endogenous PKI will continue to be invaluable tools that are used to elucidate the role of cAMP-dependent protein kinase in a variety of cellular processes throughout the nervous

  4. Ret function in muscle stem cells points to tyrosine kinase inhibitor therapy for facioscapulohumeral muscular dystrophy.

    Science.gov (United States)

    Moyle, Louise A; Blanc, Eric; Jaka, Oihane; Prueller, Johanna; Banerji, Christopher Rs; Tedesco, Francesco Saverio; Harridge, Stephen Dr; Knight, Robert D; Zammit, Peter S

    2016-11-14

    Facioscapulohumeral muscular dystrophy (FSHD) involves sporadic expression of DUX4, which inhibits myogenesis and is pro-apoptotic. To identify target genes, we over-expressed DUX4 in myoblasts and found that the receptor tyrosine kinase Ret was significantly up-regulated, suggesting a role in FSHD. RET is dynamically expressed during myogenic progression in mouse and human myoblasts. Constitutive expression of either RET9 or RET51 increased myoblast proliferation, whereas siRNA-mediated knockdown of Ret induced myogenic differentiation. Suppressing RET activity using Sunitinib, a clinically-approved tyrosine kinase inhibitor, rescued differentiation in both DUX4-expressing murine myoblasts and in FSHD patient-derived myoblasts. Importantly, Sunitinib also increased engraftment and differentiation of FSHD myoblasts in regenerating mouse muscle. Thus, DUX4-mediated activation of Ret prevents myogenic differentiation and could contribute to FSHD pathology by preventing satellite cell-mediated repair. Rescue of DUX4-induced pathology by Sunitinib highlights the therapeutic potential of tyrosine kinase inhibitors for treatment of FSHD.

  5. The selective Aurora B kinase inhibitor AZD1152 is a potential new treatment for multiple myeloma.

    Science.gov (United States)

    Evans, Robert P; Naber, Claudia; Steffler, Tara; Checkland, Tamara; Maxwell, Christopher A; Keats, Jonathan J; Belch, Andrew R; Pilarski, Linda M; Lai, Raymond; Reiman, Tony

    2008-02-01

    Aurora kinases are potential targets for cancer therapy. Previous studies have validated Aurora kinase A as a therapeutic target in multiple myeloma (MM), and have demonstrated in vitro anti-myeloma effects of small molecule Aurora kinase inhibitors that inhibit both Aurora A and B. This study demonstrated that Aurora B kinase was strongly expressed in myeloma cell lines and primary plasma cells. The selective Aurora B inhibitor AZD1152-induced apoptotic death in myeloma cell lines at nanomolar concentrations, with a cell cycle phenotype consistent with that reported previously for Aurora B inhibition. In some cases, AZD1152 in combination with dexamethasone showed increased anti-myeloma activity compared with the use of either agent alone. AZD1152 was active against sorted CD138(+) BM plasma cells from myeloma patients but also, as expected, was toxic to CD138(-) marrow cells from the same patients. In a murine myeloma xenograft model, AZD1152-inhibited tumour growth at well-tolerated doses and induced cell death in established tumours, with associated mild, transient leucopenia. AZD1152 shows promise in these preclinical studies as a novel treatment for MM.

  6. Identification of critical chemical features for Aurora kinase-B inhibitors using Hip-Hop, virtual screening and molecular docking

    Science.gov (United States)

    Sakkiah, Sugunadevi; Thangapandian, Sundarapandian; John, Shalini; Lee, Keun Woo

    2011-01-01

    This study was performed to find the selective chemical features for Aurora kinase-B inhibitors using the potent methods like Hip-Hop, virtual screening, homology modeling, molecular dynamics and docking. The best hypothesis, Hypo1 was validated toward a wide range of test set containing the selective inhibitors of Aurora kinase-B. Homology modeling and molecular dynamics studies were carried out to perform the molecular docking studies. The best hypothesis Hypo1 was used as a 3D query to screen the chemical databases. The screened molecules from the databases were sorted based on ADME and drug like properties. The selective hit compounds were docked and the hydrogen bond interactions with the critical amino acids present in Aurora kinase-B were compared with the chemical features present in the Hypo1. Finally, we suggest that the chemical features present in the Hypo1 are vital for a molecule to inhibit the Aurora kinase-B activity.

  7. A Comparative Study of the Aneugenic and Polyploidy-inducing Effects of Fisetin and Two Model Aurora Kinase Inhibitors

    Science.gov (United States)

    Gollapudi, P.; Hasegawa, L.S.; Eastmond, D.A.

    2014-01-01

    Fisetin, a plant flavonol commonly found in fruits, nuts and vegetables, is frequently added to nutritional supplements due to its reported cardioprotective, anti-carcinogenic and antioxidant properties. Earlier reports from our laboratory and others have indicated that fisetin has both aneugenic and clastogenic properties in cultured cells. More recently, fisetin has also been reported to target Aurora B kinase, a Ser/Thr kinase involved in ensuring proper microtubule attachment at the spindle assembly checkpoint, and an enzyme that is overexpressed in several types of cancer. Here we have further characterized the chromosome damage caused by fisetin and compared it with that induced by two known Aurora kinase inhibitors, VX-680 and ZM-447439, in cultured TK6 cells using the micronucleus assay with CREST staining as well as a flow cytometry-based assay that measures multiple types of numerical chromosomal aberrations. The three compounds were highly effective in inducing aneuploidy and polyploidy as evidenced by increases in kinetochore-positive micronuclei, hyperdiploidy, and polyploidy. With fisetin, however, the latter two effects were most significantly observed only after cells were allowed to overcome a cell cycle delay, and occurred at higher concentrations than those induced by the other Aurora kinase inhibitors. Modest increases in kinetochore-negative micronuclei were also seen with the model Aurora kinase inhibitors. These results indicate that fisetin induces multiple types of chromosome abnormalities in human cells, and indicate a need for a thorough investigation of fisetin-augmented dietary supplements. PMID:24680981

  8. 2-Aminopyridine-Based Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Inhibitors: Assessment of Mechanism-Based Safety.

    Science.gov (United States)

    Dow, Robert L; Ammirati, Mark; Bagley, Scott W; Bhattacharya, Samit K; Buckbinder, Leonard; Cortes, Christian; El-Kattan, Ayman F; Ford, Kristen; Freeman, Gary B; Guimarães, Cristiano R W; Liu, Shenping; Niosi, Mark; Skoura, Athanasia; Tess, David

    2018-04-12

    Studies have linked the serine-threonine kinase MAP4K4 to the regulation of a number of biological processes and/or diseases, including diabetes, cancer, inflammation, and angiogenesis. With a majority of the members of our lead series (e.g., 1) suffering from time-dependent inhibition (TDI) of CYP3A4, we sought design avenues that would eliminate this risk. One such approach arose from the observation that carboxylic acid-based intermediates employed in our discovery efforts retained high MAP4K4 inhibitory potency and were devoid of the TDI risk. The medicinal chemistry effort that led to the discovery of this central nervous system-impaired inhibitor together with its preclinical safety profile is described.

  9. 1-Methoxy-agroclavine from Penicillium sp. WC75209, a novel inhibitor of the Lck tyrosine kinase.

    Science.gov (United States)

    Padmanabha, R; Shu, Y Z; Cook, L S; Veitch, J A; Donovan, M; Lowe, S; Huang, S; Pirnik, D; Manly, S P

    1998-03-17

    A high-throughput screen was developed and implemented to identify inhibitors of the Lck tyrosine kinase. This report describes the identification of a specific inhibitor of this enzyme from the solid fermentation culture of the Penicillium sp., WC75209. The active compound was isolated and structurally characterized as 1-methoxy-5R, 10S-agroclavine, a new member of the ergot alkaloid family.

  10. Cancer Cell Resistance to Aurora Kinase Inhibitors: Identification of Novel Targets for Cancer Therapy

    Czech Academy of Sciences Publication Activity Database

    Hrabáková, Rita; Kollaredy, M.; Tylečková, Jiřina; Halada, Petr; Hajdúch, M.; Gadher, S. J.; Kovářová, Hana

    2013-01-01

    Roč. 12, č. 1 (2013), s. 455-469 ISSN 1535-3893 R&D Projects: GA MŠk LC07017 Institutional support: RVO:67985904 ; RVO:61388971 Keywords : Aurora kinase inhibitors * resistance * p53 * apoptosis Subject RIV: CE - Biochemistry Impact factor: 5.001, year: 2013

  11. Investigation of Unanticipated Alkylation at the N(π) Position of a Histidyl Residue Under Mitsunobu Conditions and Synthesis of Orthogonally Protected Histidine Analogues

    Science.gov (United States)

    Qian, Wenjian; Liu, Fa; Burke, Terrence R.

    2011-01-01

    We had previously reported that Mitsunobu-based introduction of alkyl substituents onto the imidazole N(π)-position of a key histidine residue in phosphothreonine-containing peptides can impart high binding affinity against the polo box domain of polo like kinase 1. Our current paper investigates the mechanism leading to this N(π)-alkylation and provides synthetic methodologies that permit the facile synthesis of histidine N(π)-modified peptides. These agents represent new and potentially important tools for biological studies. PMID:21950469

  12. LONG-TERM RESULTS OF TARGET THERAPY WITH FIRST AND * SECOND-LINE TYROSINE KINASE INHIBITORS IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA

    Directory of Open Access Journals (Sweden)

    L. L. Vysotskaya

    2015-01-01

    Full Text Available Aim: To assess long-term efficacy of firstand second-line tyrosine kinase inhibitors in non-selected patients with chronic myeloid leukemia in a real-life clinical setting.Materials and methods: The assessment is based on long-term results of a prospective single center comparative clinical trial that was based on non-selected groups of 116 patients with various stages of chronic myeloid leukemia being treated with a first generation tyrosine kinase inhibitor imatinib, and of 44 patients being treated with a second generation tyrosine kinase inhibitor nilotinib. We analyzed all-cause mortality, progression-free survival from April 2005 to April 2013, with a median of the follow-up of 128 months.Results: In 116 patients with chronic myeloid leukemia treated with imatinib, the Kaplan-Meier survival estimate was 120 months. In 44 patients at an early chronic phase, 5-year overall survival and progression-free survival was 93.2% and 8-year overall and progression-free survival was 79.5%. In 44 patients at a late chronic stage, 5-year overall and progression-free survival was 95.5%, 8-year overall and progression-free survival, 72.7%. In 28 patients at acceleration phase, 5-years overall survival was 78.6% and 8-year overall survival, 46%. Median of overall survival in patients treated with nilotinib was not reached. During 78.6 months of combination treatment with cytotoxic agents, tyrosine kinase inhibitors of the first (imatinib and second line (nilotinib, overall survival was 100%.Conclusion: In clinical practice, inclusion of patients with chronic myeloid leukemia and imatinib resistance (disease relapse or imatinib intolerance into the treatment program with frontline therapy with general cytotoxic agents and thereafter with firstand second-line tyrosine kinase inhibitors significantly improves overall survival.

  13. Ibrutinib: a first in class covalent inhibitor of Bruton’s tyrosine kinase

    Science.gov (United States)

    Davids, Matthew S; Brown, Jennifer R

    2015-01-01

    Ibrutinib (formerly PCI-32765) is a potent, covalent inhibitor of Bruton’s tyrosine kinase, a kinase downstream of the B-cell receptor that is critical for B-cell survival and proliferation. In preclinical studies, ibrutinib bound to Bruton’s tyrosine kinase with high affinity, leading to inhibition of B-cell receptor signaling, decreased B-cell activation and induction of apoptosis. In clinical studies, ibrutinib has been well-tolerated and has demonstrated profound anti-tumor activity in a variety of hematologic malignancies, most notably chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), leading to US FDA approval for relapsed CLL and MCL. Ongoing studies are evaluating ibrutinib in other types of non-Hodgkin’s lymphoma, such as diffuse large B-cell lymphoma and Waldenström’s macrogobulinemia, in larger Phase III studies in CLL and MCL, and in combination studies with monoclonal antibodies and chemotherapy. Future studies will combine ibrutinib with other promising novel agents currently in development in hematologic malignancies. PMID:24941982

  14. Multi-tyrosine kinase inhibitors in preclinical studies for pediatric CNS AT/RT: Evidence for synergy with Topoisomerase-I inhibition

    Directory of Open Access Journals (Sweden)

    Jayanthan Aarthi

    2011-12-01

    Full Text Available Abstract Background Currently, Atypical Teratoid Rhabdoid Tumor (AT/RT constitutes one of the most difficult to treat malignancies in pediatrics. Hence, new knowledge of potential targets for therapeutics and the development of novel treatment approaches are urgently needed. We have evaluated the presence of cytokine pathways and the effects of two clinically available multi-tyrosine kinase inhibitors for cytotoxicity, target modulation and drug combinability against AT/RT cell lines. Results AT/RT cell lines expressed measurable quantities of VEGF, FGF, PDGF and SDF-1, although the absolute amounts varied between the cell lines. The targeted receptor tyrosine kinase inhibitor sorafenib inhibited the key signaling molecule Erk, which was activated following the addition of own conditioned media, suggesting the existence of autocrine/paracrine growth stimulatory pathways. The multi-tyrosine kinase inhibitors sorafenib and sunitinib also showed significant growth inhibition of AT/RT cells and their activity was enhanced by combination with the topoisomerase inhibitor, irinotecan. The loss of cytoplasmic NF-kappa-B in response to irinotecan was diminished by sorafenib, providing evidence for a possible benefit for this drug combination. Conclusions In addition to previously described involvement of insulin like growth factor (IGF family of cytokines, a multitude of other growth factors may contribute to the growth and survival of AT/RT cells. However, consistent with the heterogeneous nature of this tumor, quantitative and qualitative differences may exist among different tumor samples. Multi-tyrosine kinase inhibitors appear to have effective antitumor activity against all cell lines studied. In addition, the target modulation studies and drug combinability data provide the groundwork for additional studies and support the evaluation of these agents in future treatment protocols.

  15. Navigating into the binding pockets of the HER family protein kinases: discovery of novel EGFR inhibitor as antitumor agent.

    Science.gov (United States)

    Liu, Wei; Ning, Jin-Feng; Meng, Qing-Wei; Hu, Jing; Zhao, Yan-Bin; Liu, Chao; Cai, Li

    2015-01-01

    The epidermal growth factor receptor (EGFR) family has been validated as a successful antitumor drug target for decades. Known EGFR inhibitors were exposed to distinct drug resistance against the various EGFR mutants within non-small-cell lung cancer (NSCLC), particularly the T790M mutation. Although so far a number of studies have been reported on the development of third-generation EGFR inhibitors for overcoming the resistance issue, the design procedure largely depends on the intuition of medicinal chemists. Here we retrospectively make a detailed analysis of the 42 EGFR family protein crystal complexes deposited in the Protein Data Bank (PDB). Based on the analysis of inhibitor binding modes in the kinase catalytic cleft, we identified a potent EGFR inhibitor (compound A-10) against drug-resistant EGFR through fragment-based drug design. This compound showed at least 30-fold more potency against EGFR T790M than the two control molecules erlotinib and gefitinib in vitro. Moreover, it could exhibit potent HER2 inhibitory activities as well as tumor growth inhibitory activity. Molecular docking studies revealed a structural basis for the increased potency and mutant selectivity of this compound. Compound A-10 may be selected as a promising candidate in further preclinical studies. In addition, our findings could provide a powerful strategy to identify novel selective kinase inhibitors on the basis of detailed kinase-ligand interaction space in the PDB.

  16. Design, Synthesis and Evaluation of Ribose-modified Anilinopyrimidine Derivatives as EGFR Tyrosine Kinase Inhibitors

    Science.gov (United States)

    Hu, Xiuqin; Wang, Disha; Tong, Yi; Tong, Linjiang; Wang, Xia; Zhu, Lili; Xie, Hua; Li, Shiliang; Yang, You; Xu, Yufang

    2017-11-01

    The synthesis of a series of ribose-modified anilinopyrimidine derivatives was efficiently achieved by utilizing DBU or tBuOLi-promoted coupling of ribosyl alcohols with 2,4,5-trichloropyrimidine as key step. Preliminary biological evaluation of this type of compounds as new EGFR tyrosine kinase inhibitors for combating EGFR L858R/T790M mutant associated with drug resistance in the treatment of non-small cell lung cancer revealed that 3-N-acryloyl-5-O-anilinopyrimidine ribose derivative 1a possessed potent and specific inhibitory activity against EGFR L858R/T790M over WT EGFR. Based upon molecular docking studies of the binding mode between compound 1a and EGFR, the distance between the Michael receptor and the pyrimidine scaffold is considered as an important factor for the inhibitory potency and future design of selective EGFR tyrosine kinase inhibitors against EGFR L858R/T790M mutants.

  17. Histidine-Containing Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2000-01-01

    Peptide nucleic acids containing histidine moieties are provided. These compounds have applications including diagnostics, research and potential therapeutics.......Peptide nucleic acids containing histidine moieties are provided. These compounds have applications including diagnostics, research and potential therapeutics....

  18. A high-throughput screening assay for eukaryotic elongation factor 2 kinase inhibitors

    Directory of Open Access Journals (Sweden)

    Ting Xiao

    2016-10-01

    Full Text Available Eukaryotic elongation factor 2 kinase (eEF2K inhibitors may aid in the development of new therapeutic agents to combat cancer. Purified human eEF2K was obtained from an Escherichia coli expression system and a luminescence-based high-throughput screening (HTS assay was developed using MH-1 peptide as the substrate. The luminescent readouts correlated with the amount of adenosine triphosphate remaining in the kinase reaction. This method was applied to a large-scale screening campaign against a diverse compound library and subsequent confirmation studies. Nine initial hits showing inhibitory activities on eEF2K were identified from 56,000 synthetic compounds during the HTS campaign, of which, five were chosen to test their effects in cancer cell lines.

  19. Activity of the novel BCR kinase inhibitor IQS019 in preclinical models of B-cell non-Hodgkin lymphoma

    Directory of Open Access Journals (Sweden)

    P. Balsas

    2017-03-01

    Full Text Available Abstract Background Pharmacological inhibition of B cell receptor (BCR signaling has recently emerged as an effective approach in a wide range of B lymphoid neoplasms. However, despite promising clinical activity of the first Bruton’s kinase (Btk and spleen tyrosine kinase (Syk inhibitors, a small fraction of patients tend to develop progressive disease after initial response to these agents. Methods We evaluated the antitumor activity of IQS019, a new BCR kinase inhibitor with increased affinity for Btk, Syk, and Lck/Yes novel tyrosine kinase (Lyn, in a set of 34 B lymphoid cell lines and primary cultures, including samples with acquired resistance to the first-in-class Btk inhibitor ibrutinib. Safety and efficacy of the compound were then evaluated in two xenograft mouse models of B cell lymphoma. Results IQS019 simultaneously engaged a rapid and dose-dependent de-phosphorylation of both constitutive and IgM-activated Syk, Lyn, and Btk, leading to impaired cell proliferation, reduced CXCL12-dependent cell migration, and induction of caspase-dependent apoptosis. Accordingly, B cell lymphoma-bearing mice receiving IQS019 presented a reduced tumor outgrowth characterized by a decreased mitotic index and a lower infiltration of malignant cells in the spleen, in tight correlation with downregulation of phospho-Syk, phospho-Lyn, and phospho-Btk. More interestingly, IQS019 showed improved efficacy in vitro and in vivo when compared to the first-in-class Btk inhibitor ibrutinib, and was active in cells with acquired resistance to this latest. Conclusions These results define IQS019 as a potential drug candidate for a variety of B lymphoid neoplasms, including cases with acquired resistance to current BCR-targeting therapies.

  20. Cytokinesis defect in BY-2 cells caused by ATP-competitive kinase inhibitors.

    Science.gov (United States)

    Kozgunova, Elena; Higashiyama, Tetsuya; Kurihara, Daisuke

    2016-10-02

    Cytokinesis is last but not least in cell division as it completes the formation of the two cells. The main role in cell plate orientation and expansion have been assigned to microtubules and kinesin proteins. However, recently we reported severe cytokinesis defect in BY-2 cells not accompanied by changes in microtubules dynamics. Here we also confirmed that distribution of kinesin NACK1 is not the cause of cytokinesis defect. We further explored inhibition of the cell plate expansion by ATP-competitive inhibitors. Two different inhibitors, 5-Iodotubercidin and ML-7 resulted in a very similar phenotype, which indicates that they target same protein cascade. Interestingly, in our previous study we showed that 5-Iodotubercidin treatment affects concentration of actin filaments on the cell plate, while ML-7 is inhibitor of myosin light chain kinase. Although not directly, it indicates importance of actomyosin complex in plant cytokinesis.

  1. Molecular docking and NMR binding studies to identify novel inhibitors of human phosphomevalonate kinase

    Energy Technology Data Exchange (ETDEWEB)

    Boonsri, Pornthip [Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI 53201 (United States); Department of Chemistry, NANOTEC Center of Nanotechnology, National Nanotechnology Center, Faculty of Science, Kasetsart University, Bangkok 10900 (Thailand); Neumann, Terrence S.; Olson, Andrew L.; Cai, Sheng [Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI 53201 (United States); Herdendorf, Timothy J.; Miziorko, Henry M. [Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110 (United States); Hannongbua, Supa [Department of Chemistry, NANOTEC Center of Nanotechnology, National Nanotechnology Center, Faculty of Science, Kasetsart University, Bangkok 10900 (Thailand); Sem, Daniel S., E-mail: daniel.sem@cuw.edu [Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, Milwaukee, WI 53201 (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Natural and synthetic inhibitors of human phosphomevalonate kinase identified. Black-Right-Pointing-Pointer Virtual screening yielded a hit rate of 15%, with inhibitor K{sub d}'s of 10-60 {mu}M. Black-Right-Pointing-Pointer NMR studies indicate significant protein conformational changes upon binding. -- Abstract: Phosphomevalonate kinase (PMK) phosphorylates mevalonate-5-phosphate (M5P) in the mevalonate pathway, which is the sole source of isoprenoids and steroids in humans. We have identified new PMK inhibitors with virtual screening, using autodock. Promising hits were verified and their affinity measured using NMR-based {sup 1}H-{sup 15}N heteronuclear single quantum coherence (HSQC) chemical shift perturbation and fluorescence titrations. Chemical shift changes were monitored, plotted, and fitted to obtain dissociation constants (K{sub d}). Tight binding compounds with K{sub d}'s ranging from 6-60 {mu}M were identified. These compounds tended to have significant polarity and negative charge, similar to the natural substrates (M5P and ATP). HSQC cross peak changes suggest that binding induces a global conformational change, such as domain closure. Compounds identified in this study serve as chemical genetic probes of human PMK, to explore pharmacology of the mevalonate pathway, as well as starting points for further drug development.

  2. Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution.

    Directory of Open Access Journals (Sweden)

    Amanda Tse

    Full Text Available Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib and promiscuous (Bosutinib, Dasatinib kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations

  3. Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution

    Science.gov (United States)

    Tse, Amanda; Verkhivker, Gennady M.

    2015-01-01

    Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib) and promiscuous (Bosutinib, Dasatinib) kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations of key mediating

  4. Computational-experimental approach to drug-target interaction mapping: A case study on kinase inhibitors.

    Directory of Open Access Journals (Sweden)

    Anna Cichonska

    2017-08-01

    Full Text Available Due to relatively high costs and labor required for experimental profiling of the full target space of chemical compounds, various machine learning models have been proposed as cost-effective means to advance this process in terms of predicting the most potent compound-target interactions for subsequent verification. However, most of the model predictions lack direct experimental validation in the laboratory, making their practical benefits for drug discovery or repurposing applications largely unknown. Here, we therefore introduce and carefully test a systematic computational-experimental framework for the prediction and pre-clinical verification of drug-target interactions using a well-established kernel-based regression algorithm as the prediction model. To evaluate its performance, we first predicted unmeasured binding affinities in a large-scale kinase inhibitor profiling study, and then experimentally tested 100 compound-kinase pairs. The relatively high correlation of 0.77 (p < 0.0001 between the predicted and measured bioactivities supports the potential of the model for filling the experimental gaps in existing compound-target interaction maps. Further, we subjected the model to a more challenging task of predicting target interactions for such a new candidate drug compound that lacks prior binding profile information. As a specific case study, we used tivozanib, an investigational VEGF receptor inhibitor with currently unknown off-target profile. Among 7 kinases with high predicted affinity, we experimentally validated 4 new off-targets of tivozanib, namely the Src-family kinases FRK and FYN A, the non-receptor tyrosine kinase ABL1, and the serine/threonine kinase SLK. Our sub-sequent experimental validation protocol effectively avoids any possible information leakage between the training and validation data, and therefore enables rigorous model validation for practical applications. These results demonstrate that the kernel

  5. The tyrosine kinase inhibitor imatinib mesylate suppresses uric acid crystal-induced acute gouty arthritis in mice.

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    Laurent L Reber

    Full Text Available Gouty arthritis is caused by the deposition of monosodium urate (MSU crystals in joints. Despite many treatment options for gout, there is a substantial need for alternative treatments for patients unresponsive to current therapies. Tyrosine kinase inhibitors have demonstrated therapeutic benefit in experimental models of antibody-dependent arthritis and in rheumatoid arthritis in humans, but to date, the potential effects of such inhibitors on gouty arthritis has not been evaluated. Here we demonstrate that treatment with the tyrosine kinase inhibitor imatinib mesylate (imatinib can suppress inflammation induced by injection of MSU crystals into subcutaneous air pouches or into the ankle joint of wild type mice. Moreover, imatinib treatment also largely abolished the lower levels of inflammation which developed in IL-1R1-/- or KitW-sh/W-sh mice, indicating that this drug can inhibit IL-1-independent pathways, as well as mast cell-independent pathways, contributing to pathology in this model. Imatinib treatment not only prevented ankle swelling and synovial inflammation when administered before MSU crystals but also diminished these features when administrated after the injection of MSU crystals, a therapeutic protocol more closely mimicking the clinical situation in which treatment occurs after the development of an acute gout flare. Finally, we also assessed the efficiency of local intra-articular injections of imatinib-loaded poly(lactic-co-glycolic acid (PLGA nanoparticles in this model of acute gout. Treatment with low doses of this long-acting imatinib:PLGA formulation was able to reduce ankle swelling in a therapeutic protocol. Altogether, these results raise the possibility that tyrosine kinase inhibitors might have utility in the treatment of acute gout in humans.

  6. Paroxetine Is a Direct Inhibitor of G Protein-Coupled Receptor Kinase 2 and Increases Myocardial Contractility

    Energy Technology Data Exchange (ETDEWEB)

    Thal, David M. [Univ. of Michigan, Ann Arbor, MI (United States); Homan, Kristoff T. [Univ. of Michigan, Ann Arbor, MI (United States); Chen, Jun [Univ. of New Mexico Health Sciences Center, Albuquerque, NM (United States); Wu, Emily K. [Univ. of Michigan, Ann Arbor, MI (United States); Hinkle, Patricia M. [Univ. of Rochester Medical Center, Rochester, NY (United States); Huang, Z. Maggie [Temple Univ. School of Medicine, Philadelphia, Pennsylvania (United States); Chuprun, J. Kurt [Temple Univ. School of Medicine, Philadelphia, Pennsylvania (United States); Song, Jianliang [Temple Univ. School of Medicine, Philadelphia, Pennsylvania (United States); Gao, Erhe [Temple Univ. School of Medicine, Philadelphia, Pennsylvania (United States); Cheung, Joseph Y. [Temple Univ. School of Medicine, Philadelphia, Pennsylvania (United States); Sklar, Larry A. [Univ. of New Mexico Health Sciences Center, Albuquerque, NM (United States); Koch, Walter J. [Temple Univ. School of Medicine, Philadelphia, Pennsylvania (United States); Tesmer, John J.G. [Univ. of Michigan, Ann Arbor, MI (United States)

    2012-08-10

    G protein-coupled receptor kinase 2 (GRK2) is a well-established therapeutic target for the treatment of heart failure. In this paper we identify the selective serotonin reuptake inhibitor (SSRI) paroxetine as a selective inhibitor of GRK2 activity both in vitro and in living cells. In the crystal structure of the GRK2·paroxetine–Gβγ complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Isolated cardiomyocytes show increased isoproterenol-induced shortening and contraction amplitude in the presence of paroxetine, and pretreatment of mice with paroxetine before isoproterenol significantly increases left ventricular inotropic reserve in vivo with no significant effect on heart rate. Neither is observed in the presence of the SSRI fluoxetine. Our structural and functional results validate a widely available drug as a selective chemical probe for GRK2 and represent a starting point for the rational design of more potent and specific GRK2 inhibitors.

  7. [Protein kinase A inhibitor H-89 blocks polyploidization of SP600125-induced CMK cells by regulating phosphorylation of ribosomal protein S6 kinase 1].

    Science.gov (United States)

    Zhao, Song; Yang, Jingang; Li, Changling; Xing, Sining; Yu, Ying; Liu, Shuo; Pu, Feifei; Ma, Dongchu

    2016-10-01

    Objective To investigate the regulatory effect of post-translation modification of ribosomal protein S6 kinase 1 (S6K1) on the polyploidization of megakaryocytes. Methods SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and H-89, a cAMP-dependent protein kinase (PKA) inhibitor, were used to treat CMK cells separately or in combination. With propidium iodide (PI) to dye DNA in the treated cells, the relative DNA content was detected by flow cytometry, and then the DNA polyploidy was analyzed. The change of expression and phosphorylation of ribosomal protein S6 kinase 1 (S6K1), an important mammalian target of rapamycin (mTOR) downstream target molecule, was analyzed by Western blotting. Molecular docking study and kinase activity assay were performed to analyze the combination of H-89 with S6K1 and the effect of H-89 on the activity of S6K1 kinase. Results SP600125 induced CMK cell polyploidization in a time-dependent and dose-dependent manner. At the same time, it increased the phosphorylation of S6K1 at Thr421/Ser424 and decreased the phosphorylation of S6K1 at Thr389. H-89 not only blocked polyploidization, but also decreased the phosphorylation of S6K1 at Thr421/Ser424 and increased the phosphorylation of S6K1 at Thr389. Molecular docking and kinase activity assay showed that H-89 occupied the ATP binding sites of S6K1 and inhibited its activity. Noticeably, both H-89 and SP600125 inhibited the activity of PKA. Moreover, the two drugs further inhibited the activity of PKA when used together. Therefore, these data indicated that H-89 blocked the SP600125-induced polyploidization of CMK cells mainly by changing S6K1 phosphorylation state, rather than its inhibitory effect on PKA. Conclusion H-89 can block the polyploidization of SP600125-induced CMK cells by regulating S6K1 phosphorylation state.

  8. Treatment of vitiligo with the topical Janus kinase inhibitor ruxolitinib.

    Science.gov (United States)

    Rothstein, Brooke; Joshipura, Deep; Saraiya, Ami; Abdat, Rana; Ashkar, Huda; Turkowski, Yana; Sheth, Vaneeta; Huang, Victor; Au, Shiu Chung; Kachuk, Courtney; Dumont, Nicole; Gottlieb, Alice B; Rosmarin, David

    2017-06-01

    Existing therapies for vitiligo are limited in efficacy and can be associated with undesirable side effects. Topical Janus kinase inhibitors may offer a new therapeutic option for vitiligo. We sought to assess the role of topical ruxolitinib 1.5% cream, a Janus kinase inhibitor, in vitiligo treatment. This 20-week, open-label, proof-of-concept trial of twice-daily topical ruxolitinib 1.5% cream was conducted in 12 patients with a minimum of 1% affected body surface area of vitiligo. The primary outcome was percent improvement in Vitiligo Area Scoring Index from baseline to week 20. Of 12 patients screened, 11 were enrolled and 9 completed the study (54.5% men; mean age, 52 years). Four patients with significant facial involvement at baseline had a 76% improvement in facial Vitiligo Area Scoring Index scores at week 20 (95% confidence interval, 53-99%; P = .001). A 23% improvement in overall Vitiligo Area Scoring Index scores was observed in all enrolled patients at week 20 (95% confidence interval, 4-43%; P = .02). Three of 8 patients responded on body surfaces and 1 of 8 patients responded on acral surfaces. Adverse events were minor, including erythema, hyperpigmentation, and transient acne. Limitations of the study include the small sample size and open-label study design. Topical ruxolitinib 1.5% cream provided significant repigmentation in facial vitiligo and may offer a valuable new treatment for vitiligo. Copyright © 2017 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

  9. Tyrosine kinase inhibitors for brain metastases in HER2-positive breast cancer.

    Science.gov (United States)

    Duchnowska, Renata; Loibl, Sibylle; Jassem, Jacek

    2018-06-01

    Approximately 30-50% of advanced HER2-positive breast cancer patients will develop central nervous system (CNS) metastases, with an annual risk of around 10%, and a half of them will die from brain progression. An increased risk of brain metastases is also seen in patients with early HER2-positive breast cancer administered curative therapy. Brain metastases in HER2-positive breast cancer patients usually constitute the first site of recurrence. The administration of anti-HER2 monoclonal antibodies, trastuzumab and pertuzumab, considerably delays the onset of symptomatic brain disease: however, the limited penetration of these compounds into the CNS hinders their efficacy. The small-molecule tyrosine kinase inhibitors of epidermal growth factor receptors family have established activity in HER2-positive breast cancer in both advanced disease and neoadjuvant setting. Favorable physico-chemical properties of these compounds allow them for a more efficient penetration through the blood-brain barrier, and hold the promise for more effective prevention and treatment of brain metastases. In this article we review the role of currently available or investigational HER2 tyrosine kinase inhibitors: lapatinib, neratinib, afatinib and tucatinib in the treatment of brain metastases in HER2-positive breast cancer patients. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Substituted aminopyrimidine protein kinase B (PknB) inhibitors show activity against Mycobacterium tuberculosis

    Science.gov (United States)

    Chapman, Timothy M.; Bouloc, Nathalie; Buxton, Roger S.; Chugh, Jasveen; Lougheed, Kathryn E.A.; Osborne, Simon A.; Saxty, Barbara; Smerdon, Stephen J.; Taylor, Debra L.; Whalley, David

    2012-01-01

    A high-throughput screen against PknB, an essential serine–threonine protein kinase present in Mycobacterium tuberculosis (M. tuberculosis), allowed the identification of an aminoquinazoline inhibitor which was used as a starting point for SAR investigations. Although a significant improvement in enzyme affinity was achieved, the aminoquinazolines showed little or no cellular activity against M. tuberculosis. However, switching to an aminopyrimidine core scaffold and the introduction of a basic amine side chain afforded compounds with nanomolar enzyme binding affinity and micromolar minimum inhibitory concentrations against M. tuberculosis. Replacement of the pyrazole head group with pyridine then allowed equipotent compounds with improved selectivity against a human kinase panel to be obtained. PMID:22469702

  11. Selectivity Profiling and Biological Activity of Novel β-Carbolines as Potent and Selective DYRK1 Kinase Inhibitors.

    Directory of Open Access Journals (Sweden)

    Katharina Rüben

    Full Text Available DYRK1A is a pleiotropic protein kinase with diverse functions in cellular regulation, including cell cycle control, neuronal differentiation, and synaptic transmission. Enhanced activity and overexpression of DYRK1A have been linked to altered brain development and function in Down syndrome and neurodegenerative diseases such as Alzheimer's disease. The β-carboline alkaloid harmine is a high affinity inhibitor of DYRK1A but suffers from the drawback of inhibiting monoamine oxidase A (MAO-A with even higher potency. Here we characterized a series of novel harmine analogs with minimal or absent MAO-A inhibitory activity. We identified several inhibitors with submicromolar potencies for DYRK1A and selectivity for DYRK1A and DYRK1B over the related kinases DYRK2 and HIPK2. An optimized inhibitor, AnnH75, inhibited CLK1, CLK4, and haspin/GSG2 as the only off-targets in a panel of 300 protein kinases. In cellular assays, AnnH75 dose-dependently reduced the phosphorylation of three known DYRK1A substrates (SF3B1, SEPT4, and tau without negative effects on cell viability. AnnH75 inhibited the cotranslational tyrosine autophosphorylation of DYRK1A and threonine phosphorylation of an exogenous substrate protein with similar potency. In conclusion, we have characterized an optimized β-carboline inhibitor as a highly selective chemical probe that complies with desirable properties of drug-like molecules and is suitable to interrogate the function of DYRK1A in biological studies.

  12. Quinolinone and pyridopyrimidinone inhibitors of DNA-dependent protein kinase.

    Science.gov (United States)

    Barbeau, Olivier R; Cano-Soumillac, Celine; Griffin, Roger J; Hardcastle, Ian R; Smith, Graeme C M; Richardson, Caroline; Clegg, William; Harrington, Ross W; Golding, Bernard T

    2007-08-21

    8-Substituted 2-morpholin-4-yl-quinolin-4-ones and 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones with selected aryl and heteroaryl groups as the substituent have been synthesised as potential inhibitors of DNA-dependent protein kinase. A multiple-parallel approach, employing Suzuki cross-coupling methodology, was utilised in the preparation of 8-substituted 2-morpholin-4-yl-quinolin-4-ones. For this purpose 8-bromo-2-morpholin-4-yl-quinolin-4-one was required as an intermediate. This compound was obtained by adapting a literature route in which thermal cyclocondensation of (2-bromoanilino)-morpholin-4-yl-5-methylene-2,2-dimethyl[1,3]dioxane-4,6-dione afforded 8-bromo-2-morpholin-4-yl-quinolin-4-one. A multiple-parallel approach, employing Suzuki cross-coupling methodology, was also utilised to prepare 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones using 9-hydroxy-2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-one O-trifluoromethanesulfonate as an intermediate. 8-Substituted 2-morpholin-4-yl-quinolin-4-ones and 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones were both inhibitors of DNA-dependent protein kinase. When the substituent was dibenzothiophen-4-yl, dibenzofuran-4-yl or biphen-3-yl, IC50 values in the low nanomolar range were observed. Interestingly, the pyridopyrimidinones and quinolinones were essentially equipotent with the corresponding 8-substituted 2-morpholin-4-yl-chromen-4-ones previously reported (I. R. Hardcastle, X. Cockcroft, N. J. Curtin, M. Desage El-Murr, J. J. J. Leahy, M. Stockley, B. T. Golding, L. Rigoreau, C. Richardson, G. C. M. Smith and R. J. Griffin, J. Med. Chem., 2005, 48, 7829-7846).

  13. Are Janus Kinase Inhibitors Superior over Classic Biologic Agents in RA Patients?

    Directory of Open Access Journals (Sweden)

    Przemyslaw J. Kotyla

    2018-01-01

    Full Text Available The Janus Kinases (JAKs are a family of intracellular tyrosine kinases that provide transmission signals from cytokine, interferons, and many hormones receptors to the nucleus resulting in synthesis of many biologically active compounds and changing cell metabolism and function. That was theoretical background to synthetize the JAK inhibitors (Jakinibs. In recent years a substantial battery of evidence has been collected indicating the potential role of Jakinibs to interact with the specific elements of the immune system, therefore changing the inflammatory response. JAK kinase blockade offers a unique opportunity to block most of the key cytokines enabling the deep interaction into immune system functioning. Following discovery first Jakinibs were intensively studied in various forms of autoimmune diseases, including rheumatoid arthritis, and finally two Jakinibs tofacitinib and Baricitinib have been approved for the treatment of rheumatoid arthritis. Some clinical data indicated that under special circumstances Jakinibs may be even superior to biologics in the treatment of RA; however this suggestion should be verified in large clinical and observational studies.

  14. The significance of major and stable molecular responses in chronic myeloid leukemia in the tyrosine kinase inhibitor era

    Directory of Open Access Journals (Sweden)

    Ilana Zalcberg Renault

    2011-12-01

    Full Text Available Tyrosine kinase inhibitors have changed the management and outcomes of chronic myeloid leukemia patients. Quantitative polymerase chain reaction is used to monitor molecular responses to tyrosine kinase inhibitors. Molecular monitoring represents the most sensitive tool to judge chronic myeloid leukemia disease course and allows early detection of relapse. Evidence of achieving molecular response is important for several reasons: 1. early molecular response is associated with major molecular response rates at 18-24 months; 2. patients achieving major molecular response are less likely to lose their complete cytogenetic response; 3. a durable, stable major molecular response is associated with increased progression-free survival. However, standardization of molecular techniques is still challenging.

  15. The significance of major and stable molecular responses in chronic myeloid leukemia in the tyrosine kinase inhibitor era

    Science.gov (United States)

    Renault, Ilana Zalcberg; Scholl, Vanesa; Hassan, Rocio; Capelleti, Paola; de Lima, Marcos; Cortes, Jorge

    2011-01-01

    Tyrosine kinase inhibitors have changed the management and outcomes of chronic myeloid leukemia patients. Quantitative polymerase chain reaction is used to monitor molecular responses to tyrosine kinase inhibitors. Molecular monitoring represents the most sensitive tool to judge chronic myeloid leukemia disease course and allows early detection of relapse. Evidence of achieving molecular response is important for several reasons: 1. early molecular response is associated with major molecular response rates at 18-24 months; 2. patients achieving major molecular response are less likely to lose their complete cytogenetic response; 3. a durable, stable major molecular response is associated with increased progression-free survival. However, standardization of molecular techniques is still challenging. PMID:23049363

  16. Staurosporine scaffold-based rational discovery of the wild-type sparing reversible inhibitors of EGFR T790M gatekeeper mutant in lung cancer with analog-sensitive kinase technology.

    Science.gov (United States)

    Song, Xiaoyun; Liu, Xingcai; Ding, Xi

    2017-04-01

    The human epidermal growth factor receptor (EGFR) has been established as an attractive target for lung cancer therapy. However, an acquired EGFR T790M gatekeeper mutation is frequently observed in patients treated with first-line anticancer agents such as gefitinib and erlotinib to cause drug resistance, largely limiting the application of small-molecule kinase inhibitors in EGFR-targeted chemotherapy. Previously, the reversible pan-kinase inhibitor staurosporine and its several analogs such as Gö6976 and K252a have been reported to selectively inhibit the EGFR T790M mutant (EGFR T790M ) over wild-type kinase (EGFR WT ), suggesting that the staurosporine scaffold is potentially to develop the wild-type sparing reversible inhibitors of EGFR T790M . Here, we systematically evaluated the inhibitor response of 28 staurosporine scaffold-based compounds to EGFR T790M mutation at structural, energetic, and molecular levels by using an integrated in silico-in vitro analog-sensitive (AS) kinase technology. With the strategy, we were able to identify 4 novel wild-type sparing inhibitors UCN-01, UCN-02, AFN941, and SB-218078 with high or moderate selectivity of 30-, 45-, 5-, and 8-fold for EGFR T790M over EGFR WT , respectively, which are comparable with or even better than that of the parent compound staurosporine (24-fold). Molecular modeling and structural analysis revealed that van der Waals contacts and hydrophobic forces can form between the side chain of mutated residue Met790 and the pyrrolidinone moiety of inhibitor ligand UCN-02, which may simultaneously improve the favorable interaction energy between the kinase and inhibitor, and reduce the unfavorable desolvation penalty upon the kinase-inhibitor binding. A hydroxyl group of UCN-02 additional to staurosporine locates at the pyrrolidinone moiety, which can largely alter the electronic distribution of pyrrolidinone moiety and thus promote the intermolecular interaction with Met790 residue. This can well explain

  17. Alteration of radiation response by two tyrosine kinase inhibitors: STI571 (Glivec) and BIBW 2992

    International Nuclear Information System (INIS)

    Huguet, F.

    2010-01-01

    Concurrent chemo-radiation is one of the main weapon in the treatment of cancer. The targeted therapies may act on the mechanisms of tumor resistance to radiation and are therefore very promising in combination with radiotherapy. The STI571 (imatinib or Gleevec) inhibits specifically the Bcr-Abl tyrosine kinase. It leads to radiosensitization in K562 chronic myeloid leukemia cell line by alterations of the cell cycle. The BIBW2992 is a selective inhibitor of EGFR and HER2. The BIBW 2992 shows cytotoxic and radiosensitizing effects on pancreatic adenocarcinoma cells BxPC3 and Capan-2, regardless of KRAS status. The mechanism underlying this radiosensitization is not unequivocal, involving both changes in the cell cycle and induction of mitotic death. Our results show that the combination of an inhibitor of tyrosine kinase with ionizing radiation may lead to a radiosensitization in vitro with mechanisms depending on the type of cell line. (author)

  18. Accurate calculation of mutational effects on the thermodynamics of inhibitor binding to p38α MAP kinase: a combined computational and experimental study.

    Science.gov (United States)

    Zhu, Shun; Travis, Sue M; Elcock, Adrian H

    2013-07-09

    A major current challenge for drug design efforts focused on protein kinases is the development of drug resistance caused by spontaneous mutations in the kinase catalytic domain. The ubiquity of this problem means that it would be advantageous to develop fast, effective computational methods that could be used to determine the effects of potential resistance-causing mutations before they arise in a clinical setting. With this long-term goal in mind, we have conducted a combined experimental and computational study of the thermodynamic effects of active-site mutations on a well-characterized and high-affinity interaction between a protein kinase and a small-molecule inhibitor. Specifically, we developed a fluorescence-based assay to measure the binding free energy of the small-molecule inhibitor, SB203580, to the p38α MAP kinase and used it measure the inhibitor's affinity for five different kinase mutants involving two residues (Val38 and Ala51) that contact the inhibitor in the crystal structure of the inhibitor-kinase complex. We then conducted long, explicit-solvent thermodynamic integration (TI) simulations in an attempt to reproduce the experimental relative binding affinities of the inhibitor for the five mutants; in total, a combined simulation time of 18.5 μs was obtained. Two widely used force fields - OPLS-AA/L and Amber ff99SB-ILDN - were tested in the TI simulations. Both force fields produced excellent agreement with experiment for three of the five mutants; simulations performed with the OPLS-AA/L force field, however, produced qualitatively incorrect results for the constructs that contained an A51V mutation. Interestingly, the discrepancies with the OPLS-AA/L force field could be rectified by the imposition of position restraints on the atoms of the protein backbone and the inhibitor without destroying the agreement for other mutations; the ability to reproduce experiment depended, however, upon the strength of the restraints' force constant

  19. Efficacy of cyclin dependent kinase 4 inhibitors as potent neuroprotective agents against insults relevant to Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Priyankar Sanphui

    Full Text Available Alzheimer's disease (AD is a progressive neurodegenerative disease with no cure till today. Aberrant activation of cell cycle regulatory proteins is implicated in neurodegenerative diseases including AD. We and others have shown that Cyclin dependent kinase 4 (Cdk4 is activated in AD brain and is required for neuron death. In this study, we tested the efficiency of commercially available Cdk4 specific inhibitors as well as a small library of synthetic molecule inhibitors targeting Cdk4 as neuroprotective agents in cellular models of neuron death. We found that several of these inhibitors significantly protected neuronal cells against death induced by nerve growth factor (NGF deprivation and oligomeric beta amyloid (Aβ that are implicated in AD. These neuroprotective agents inhibit specifically Cdk4 kinase activity, loss of mitochondrial integrity, induction of pro-apoptotic protein Bim and caspase3 activation in response to NGF deprivation. The efficacies of commercial and synthesized inhibitors are comparable. The synthesized molecules are either phenanthrene based or naphthalene based and they are synthesized by using Pschorr reaction and Buchwald coupling respectively as one of the key steps. A number of molecules of both kinds block neurodegeneration effectively. Therefore, we propose that Cdk4 inhibition would be a therapeutic choice for ameliorating neurodegeneration in AD and these synthetic Cdk4 inhibitors could lead to development of effective drugs for AD.

  20. Enantioselective synthesis of the novel chiral sulfoxide derivative as a glycogen synthase kinase 3beta inhibitor.

    Science.gov (United States)

    Saitoh, Morihisa; Kunitomo, Jun; Kimura, Eiji; Yamano, Toru; Itoh, Fumio; Kori, Masakuni

    2010-09-01

    Glycogen synthase kinase 3beta (GSK-3beta) inhibitors are expected to be attractive therapeutic agents for the treatment of Alzheimer's disease (AD). Recently we discovered sulfoxides (S)-1 as a novel GSK-3beta inhibitor having in vivo efficacy. We investigated practical asymmetric preparation methods for the scale-up synthesis of (S)-1. The highly enantioselective synthesis of (S)-1 (94% ee) was achieved by titanium-mediated oxidation with D-(-)-diethyl tartrate on gram scale.

  1. Fragment-based lead discovery of small molecule inhibitors for the EPHA4 receptor tyrosine kinase

    NARCIS (Netherlands)

    van Linden, O.P.J.; Farenc, C; Zoutman, W.H.; Hameetman, L; Wijtmans, M.; Leurs, R.; Tensen, C.P.; Siegal, G.; de Esch, I.J.P.

    2011-01-01

    The in silico identification, optimization and crystallographic characterization of a 6,7,8,9-tetrahydro-3H-pyrazolo[3,4-c]isoquinolin-1-amine scaffold as an inhibitor for the EPHA4 receptor tyrosine kinase is described. A database containing commercially available compounds was subjected to an in

  2. Protocols for the Design of Kinase-focused Compound Libraries.

    Science.gov (United States)

    Jacoby, Edgar; Wroblowski, Berthold; Buyck, Christophe; Neefs, Jean-Marc; Meyer, Christophe; Cummings, Maxwell D; van Vlijmen, Herman

    2018-05-01

    Protocols for the design of kinase-focused compound libraries are presented. Kinase-focused compound libraries can be differentiated based on the design goal. Depending on whether the library should be a discovery library specific for one particular kinase, a general discovery library for multiple distinct kinase projects, or even phenotypic screening, there exists today a variety of in silico methods to design candidate compound libraries. We address the following scenarios: 1) Datamining of SAR databases and kinase focused vendor catalogues; 2) Predictions and virtual screening; 3) Structure-based design of combinatorial kinase inhibitors; 4) Design of covalent kinase inhibitors; 5) Design of macrocyclic kinase inhibitors; and 6) Design of allosteric kinase inhibitors and activators. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Optimization of microtubule affinity regulating kinase (MARK) inhibitors with improved physical properties

    Energy Technology Data Exchange (ETDEWEB)

    Sloman, David L.; Noucti, Njamkou; Altman, Michael D.; Chen, Dapeng; Mislak, Andrea C.; Szewczak, Alexander; Hayashi, Mansuo; Warren, Lee; Dellovade, Tammy; Wu, Zhenhua; Marcus, Jacob; Walker, Deborah; Su, Hua-Poo; Edavettal, Suzanne C.; Munshi, Sanjeev; Hutton, Michael; Nuthall, Hugh; Stanton, Matthew G. (Merck)

    2016-09-01

    Inhibition of microtubule affinity regulating kinase (MARK) represents a potentially attractive means of arresting neurofibrillary tangle pathology in Alzheimer’s disease. This manuscript outlines efforts to optimize a pyrazolopyrimidine series of MARK inhibitors by focusing on improvements in potency, physical properties and attributes amenable to CNS penetration. A unique cylcyclohexyldiamine scaffold was identified that led to remarkable improvements in potency, opening up opportunities to reduce MW, Pgp efflux and improve pharmacokinetic properties while also conferring improved solubility.

  4. An in silico high-throughput screen identifies potential selective inhibitors for the non-receptor tyrosine kinase Pyk2

    Directory of Open Access Journals (Sweden)

    Meirson T

    2017-05-01

    Full Text Available Tomer Meirson, Abraham O Samson, Hava Gil-Henn Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Abstract: The non-receptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2 is a critical mediator of signaling from cell surface growth factor and adhesion receptors to cell migration, proliferation, and survival. Emerging evidence indicates that signaling by Pyk2 regulates hematopoietic cell response, bone density, neuronal degeneration, angiogenesis, and cancer. These physiological and pathological roles of Pyk2 warrant it as a valuable therapeutic target for invasive cancers, osteoporosis, Alzheimer’s disease, and inflammatory cellular response. Despite its potential as a therapeutic target, no potent and selective inhibitor of Pyk2 is available at present. As a first step toward discovering specific potential inhibitors of Pyk2, we used an in silico high-throughput screening approach. A virtual library of six million lead-like compounds was docked against four different high-resolution Pyk2 kinase domain crystal structures and further selected for predicted potency and ligand efficiency. Ligand selectivity for Pyk2 over focal adhesion kinase (FAK was evaluated by comparative docking of ligands and measurement of binding free energy so as to obtain 40 potential candidates. Finally, the structural flexibility of a subset of the docking complexes was evaluated by molecular dynamics simulation, followed by intermolecular interaction analysis. These compounds may be considered as promising leads for further development of highly selective Pyk2 inhibitors. Keywords: virtual screen, efficiency metrics, MM-GBSA, molecular dynamics

  5. Impact of Serine/Threonine Protein Kinases on the Regulation of Sporulation in Bacillus subtilis.

    Science.gov (United States)

    Pompeo, Frédérique; Foulquier, Elodie; Galinier, Anne

    2016-01-01

    Bacteria possess many kinases that catalyze phosphorylation of proteins on diverse amino acids including arginine, cysteine, histidine, aspartate, serine, threonine, and tyrosine. These protein kinases regulate different physiological processes in response to environmental modifications. For example, in response to nutritional stresses, the Gram-positive bacterium Bacillus subtilis can differentiate into an endospore; the initiation of sporulation is controlled by the master regulator Spo0A, which is activated by phosphorylation. Spo0A phosphorylation is carried out by a multi-component phosphorelay system. These phosphorylation events on histidine and aspartate residues are labile, highly dynamic and permit a temporal control of the sporulation initiation decision. More recently, another kind of phosphorylation, more stable yet still dynamic, on serine or threonine residues, was proposed to play a role in spore maintenance and spore revival. Kinases that perform these phosphorylation events mainly belong to the Hanks family and could regulate spore dormancy and spore germination. The aim of this mini review is to focus on the regulation of sporulation in B. subtilis by these serine and threonine phosphorylation events and the kinases catalyzing them.

  6. Stepwise high-throughput virtual screening of Rho kinase inhibitors from natural product library and potential therapeutics for pulmonary hypertension.

    Science.gov (United States)

    Su, Hao; Yan, Ji; Xu, Jian; Fan, Xi-Zhen; Sun, Xian-Lin; Chen, Kang-Yu

    2015-08-01

    Pulmonary hypertension (PH) is a devastating disease characterized by progressive elevation of pulmonary arterial pressure and vascular resistance due to pulmonary vasoconstriction and vessel remodeling. The activation of RhoA/Rho-kinase (ROCK) pathway plays a central role in the pathologic progression of PH and thus the Rho kinase, an essential effector of the ROCK pathway, is considered as a potential therapeutic target to attenuate PH. In the current study, a synthetic pipeline is used to discover new potent Rho inhibitors from various natural products. In the pipeline, the stepwise high-throughput virtual screening, quantitative structure-activity relationship (QSAR)-based rescoring, and kinase assay were integrated. The screening was performed against a structurally diverse, drug-like natural product library, from which six identified compounds were tested to determine their inhibitory potencies agonist Rho by using a standard kinase assay protocol. With this scheme, we successfully identified two potent Rho inhibitors, namely phloretin and baicalein, with activity values of IC50 = 0.22 and 0.95 μM, respectively. Structural examination suggested that complicated networks of non-bonded interactions such as hydrogen bonding, hydrophobic forces, and van der Waals contacts across the complex interfaces of Rho kinase are formed with the screened compounds.

  7. Tyrosine Kinase Inhibitor Treatment for Newly Diagnosed Chronic Myeloid Leukemia.

    Science.gov (United States)

    Radich, Jerald P; Mauro, Michael J

    2017-08-01

    Chronic myeloid leukemia (CML) is a myeloproliferative disorder that accounts for approximately 10% of new cases of leukemia. The introduction of tyrosine kinase inhibitors has led to a reduction in mortalities. Thus, the estimated prevalence of CML is increasing. The National Comprehensive Cancer Network and the European Leukemia Net guidelines incorporate frequent molecular monitoring of the fusion BCR-ABL transcript to ensure that patients reach and keep treatment milestones. Most patients with CML are diagnosed in the chronic phase, and approximately 10% to 30% of these patients will at some time in their course meet definition criteria of resistance to imatinib. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Phase 1 study of INNO-406, a dual Abl/Lyn kinase inhibitor, in Philadelphia chromosome-positive leukemias after imatinib resistance or intolerance.

    Science.gov (United States)

    Kantarjian, Hagop; le Coutre, Phillipp; Cortes, Jorge; Pinilla-Ibarz, Javier; Nagler, Arnon; Hochhaus, Andreas; Kimura, Shinya; Ottmann, Oliver

    2010-06-01

    : INNO-406, a dual v-abl Abelson murine leukemia viral oncogene homolog (Abl)/v-yes-1 Yamaguchi sarcoma viral-related oncogene homolog (Lyn) tyrosine kinase inhibitor (TKI), has demonstrated specific Lyn kinase inhibitory activity with no or limited activity against other sarcoma (Src) family member kinases. Several breakpoint cluster region (Bcr)-Abl kinase domain mutations are sensitive to INNO-406 in vitro, including mutations that involve a phenylalanine-to-leucine or phenylalanine-to-valine substitution at codon 317 (F317L and F317V, respectively). In the current study, the authors evaluated the use of INNO-406 in patients with Philadelphia (Ph) chromosome-positive chronic myelogenous leukemia (CML) or acute lymphocytic leukemia (ALL) after imatinib resistance or intolerance. : A dose-escalation study was conducted at a starting dose of oral INNO-406 30 mg once daily. Cohorts of at least 3 patients were treated at each dose level until the maximum tolerated dose (MTD) was reached. Twice-daily dosing also was evaluated. Therapy was allowed to continue for a maximum of 24 months. : INNO-406 was administered to 56 patients with imatinib resistance (n = 40) or intolerance (n = 16). Other previous treatments included nilotinib (n = 20 patients), dasatinib (n = 26 patients), and dasatinib/nilotinib (n = 9 patients). Common mutations at the time of study entry included a tyrosine-to-histidine substitution at codon 253 (Y253H) (n = 6 patients), a glycine-to-glutamic acid substitution at codon 250 (G250E) (n = 4 patients), a threonine-to-isoleucine substitution at codon 315 (T315I) (n = 4 patients), and F317L (n = 3 patients). Of 31 patients with CML in chronic phase who received INNO-406, the major cytogenetic response rate was 19%. No responses were observed in patients who had CML in accelerated phase, CML in blastic phase, or Ph-positive ALL. The dose-limiting toxicities (DLTs) at an INNO-406 dose of 480 mg twice daily were liver function abnormalities and

  9. Histone acetyltransferase inhibitors antagonize AMP-activated protein kinase in postmortem glycolysis

    Directory of Open Access Journals (Sweden)

    Qiong Li

    2017-06-01

    Full Text Available Objective The purpose of this study was to investigate the influence of AMP-activated protein kinase (AMPK activation on protein acetylation and glycolysis in postmortem muscle to better understand the mechanism by which AMPK regulates postmortem glycolysis and meat quality. Methods A total of 32 mice were randomly assigned to four groups and intraperitoneally injected with 5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR, a specific activator of AMPK, AICAR and histone acetyltransferase inhibitor II, or AICAR, Trichostatin A (TSA, an inhibitor of histone deacetylase I and II and Nicotinamide (NAM, an inhibitor of the Sirt family deacetylases. After mice were euthanized, the Longissimus dorsi muscle was collected at 0 h, 45 min, and 24 h postmortem. AMPK activity, protein acetylation and glycolysis in postmortem muscle were measured. Results Activation of AMPK by AICAR significantly increased glycolysis in postmortem muscle. At the same time, it increased the total acetylated proteins in muscle 45 min postmortem. Inhibition of protein acetylation by histone acetyltransferase inhibitors reduced AMPK activation induced increase in the total acetylated proteins and glycolytic rate in muscle early postmortem, while histone deacetylase inhibitors further promoted protein acetylation and glycolysis. Several bands of proteins were detected to be differentially acetylated in muscle with different glycolytic rates. Conclusion Protein acetylation plays an important regulatory role in postmortem glycolysis. As AMPK mediates the effects of pre-slaughter stress on postmortem glycolysis, protein acetylation is likely a mechanism by which antemortem stress influenced postmortem metabolism and meat quality though the exact mechanism is to be elucidated.

  10. The phosphoinositide 3-kinase α selective inhibitor BYL719 enhances the effect of the protein kinase C inhibitor AEB071 in GNAQ/GNA11-mutant uveal melanoma cells.

    Science.gov (United States)

    Musi, Elgilda; Ambrosini, Grazia; de Stanchina, Elisa; Schwartz, Gary K

    2014-05-01

    G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (sotrastaurin) and PI3K/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11-mutant cells with AEB071 versus no activity in wild-type cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of myristoylated alanine-rich C-kinase substrate, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal antiproliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11-mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ- and GNA11-mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ-mutant model. These findings suggest a new therapy treatment option for G-protein-mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy.

  11. Development of new mouse lung tumor models expressing EGFR T790M mutants associated with clinical resistance to kinase inhibitors.

    Science.gov (United States)

    Regales, Lucia; Balak, Marissa N; Gong, Yixuan; Politi, Katerina; Sawai, Ayana; Le, Carl; Koutcher, Jason A; Solit, David B; Rosen, Neal; Zakowski, Maureen F; Pao, William

    2007-08-29

    The EGFR T790M mutation confers acquired resistance to kinase inhibitors in human EGFR mutant lung adenocarcinoma, is occasionally detected before treatment, and may confer genetic susceptibility to lung cancer. To study further its role in lung tumorigenesis, we developed mice with inducible expression in type II pneumocytes of EGFR(T790M) alone or together with a drug-sensitive L858R mutation. Both transgenic lines develop lung adenocarcinomas that require mutant EGFR for tumor maintenance but are resistant to an EGFR kinase inhibitor. EGFR(L858R+T790M)-driven tumors are transiently targeted by hsp90 inhibition. Notably, EGFR(T790M)-expressing animals develop tumors with longer latency than EGFR(L858R+T790M)-bearing mice and in the absence of additional kinase domain mutations. These new mouse models of mutant EGFR-dependent lung adenocarcinomas provide insight into clinical observations. The models should also be useful for developing improved therapies for patients with lung cancers harboring EGFR(T790M) alone or in conjunction with drug-sensitive EGFR kinase domain mutations.

  12. Development of new mouse lung tumor models expressing EGFR T790M mutants associated with clinical resistance to kinase inhibitors.

    Directory of Open Access Journals (Sweden)

    Lucia Regales

    2007-08-01

    Full Text Available The EGFR T790M mutation confers acquired resistance to kinase inhibitors in human EGFR mutant lung adenocarcinoma, is occasionally detected before treatment, and may confer genetic susceptibility to lung cancer.To study further its role in lung tumorigenesis, we developed mice with inducible expression in type II pneumocytes of EGFR(T790M alone or together with a drug-sensitive L858R mutation. Both transgenic lines develop lung adenocarcinomas that require mutant EGFR for tumor maintenance but are resistant to an EGFR kinase inhibitor. EGFR(L858R+T790M-driven tumors are transiently targeted by hsp90 inhibition. Notably, EGFR(T790M-expressing animals develop tumors with longer latency than EGFR(L858R+T790M-bearing mice and in the absence of additional kinase domain mutations.These new mouse models of mutant EGFR-dependent lung adenocarcinomas provide insight into clinical observations. The models should also be useful for developing improved therapies for patients with lung cancers harboring EGFR(T790M alone or in conjunction with drug-sensitive EGFR kinase domain mutations.

  13. Phosphorylation sites of Arabidopsis MAP Kinase Substrate 1 (MKS1)

    DEFF Research Database (Denmark)

    Caspersen, M.B.; Qiu, J.-L.; Zhang, X.

    2007-01-01

    The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophore......The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified...... phosphopeptide detection. As MAP kinases generally phosphorylate serine or threonine followed by proline (Ser/Thr-Pro), theoretical masses of potentially phosphorylated peptides were calculated and mass spectrometric peaks matching these masses were fragmented and searched for a neutral-loss signal...... at approximately 98 Da indicative of phosphorylation. Additionally, mass spectrometric peaks present in the MPK4-treated MKS1, but not in the control peptide map of untreated MKS1, were fragmented. Fragmentation spectra were subjected to a MASCOT database search which identified three of the twelve Ser-Pro serine...

  14. Effects of kinase inhibitors and potassium phosphate (KPi) on site-specific phosphorylation of branched chain α-ketoacid dehydrogenase (BCKDH)

    International Nuclear Information System (INIS)

    Kuntz, M.J.; Shimomura, Y.; Ozawa, T.; Harris, R.A.

    1987-01-01

    BCKDH is phosphorylated by a copurifying kinase at two serine residues on the Elα subunit. Phosphorylation of both sites occurs at about the same rate initially, but inactivation is believed associated only with site 1 phosphorylation. The effects of KPi and known inhibitors of BCKDH kinase, α-chloroisocaproate (CIC) and branched chain α-ketoacids (BCKA), on the phosphorylation of purified rat liver BCKDH were studied. Site-specific phosphorylation was quantitated by thin-layer electrophoresis of tryptic peptides followed by densitometric scanning of autoradiograms. Addition of 5 mM KPi was found necessary to stabilize the BCKDH activity at 37 0 C. Increasing the KPi to 50 mM dramatically increased the CIC and BCKA inhibition of site 1 and site 2 phosphorylation. The finding of enhanced sensitivity of inhibitors with 50 mM KPi may facilitate identification of physiologically important kinase effectors. Regardless of the KPi concentration, CIC and the BCKA showed much more effective inhibition of site 2 than site 1 phosphorylation. Although site 1 is the primary inactivating site, predominant inhibition of site 2 phosphorylation may provide a means of modulating kinase/phosphatase control of BCKDH activity under steady state conditions

  15. Melanoma-associated antigen expression and the efficacy of tyrosine kinase inhibitors in head and neck cancer

    DEFF Research Database (Denmark)

    Hartmann, Stefan; Brands, Roman C; Küchler, Nora

    2015-01-01

    receptor (EGFR). The efficacy of tyrosine kinase inhibitors (TKI) in the context of melanoma-associated antigens is discussed in the present study. Five human squamous cell carcinoma cell lines were treated with the EGFR TKIs, erlotinib and gefitinib. The efficacy of these agents was measured using...

  16. The Link between Protein Kinase CK2 and Atypical Kinase Rio1

    Directory of Open Access Journals (Sweden)

    Konrad Kubiński

    2017-02-01

    Full Text Available The atypical kinase Rio1 is widespread in many organisms, ranging from Archaebacteria to humans, and is an essential factor in ribosome biogenesis. Little is known about the protein substrates of the enzyme and small-molecule inhibitors of the kinase. Protein kinase CK2 was the first interaction partner of Rio1, identified in yeast cells. The enzyme from various sources undergoes CK2-mediated phosphorylation at several sites and this modification regulates the activity of Rio1. The aim of this review is to present studies of the relationship between the two different kinases, with respect to CK2-mediated phosphorylation of Rio1, regulation of Rio1 activity, and similar susceptibility of the kinases to benzimidazole inhibitors.

  17. Amaryllidaceae Alkaloids as Potential Glycogen Synthase Kinase-3β Inhibitors

    Directory of Open Access Journals (Sweden)

    Daniela Hulcová

    2018-03-01

    Full Text Available Glycogen synthase kinase-3β (GSK-3β is a multifunctional serine/threonine protein kinase that was originally identified as an enzyme involved in the control of glycogen metabolism. It plays a key role in diverse physiological processes including metabolism, the cell cycle, and gene expression by regulating a wide variety of well-known substances like glycogen synthase, tau-protein, and β-catenin. Recent studies have identified GSK-3β as a potential therapeutic target in Alzheimer´s disease, bipolar disorder, stroke, more than 15 types of cancer, and diabetes. GSK-3β is one of the most attractive targets for medicinal chemists in the discovery, design, and synthesis of new selective potent inhibitors. In the current study, twenty-eight Amaryllidaceae alkaloids of various structural types were studied for their potency to inhibit GSK-3β. Promising results have been demonstrated by alkaloids of the homolycorine-{9-O-demethylhomolycorine (IC50 = 30.00 ± 0.71 µM, masonine (IC50 = 27.81 ± 0.01 μM}, and lycorine-types {caranine (IC50 = 30.75 ± 0.04 μM}.

  18. Novel Small Molecule Inhibitors of Choline Kinase Identified by Fragment-Based Drug Discovery.

    Science.gov (United States)

    Zech, Stephan G; Kohlmann, Anna; Zhou, Tianjun; Li, Feng; Squillace, Rachel M; Parillon, Lois E; Greenfield, Matthew T; Miller, David P; Qi, Jiwei; Thomas, R Mathew; Wang, Yihan; Xu, Yongjin; Miret, Juan J; Shakespeare, William C; Zhu, Xiaotian; Dalgarno, David C

    2016-01-28

    Choline kinase α (ChoKα) is an enzyme involved in the synthesis of phospholipids and thereby plays key roles in regulation of cell proliferation, oncogenic transformation, and human carcinogenesis. Since several inhibitors of ChoKα display antiproliferative activity in both cellular and animal models, this novel oncogene has recently gained interest as a promising small molecule target for cancer therapy. Here we summarize our efforts to further validate ChoKα as an oncogenic target and explore the activity of novel small molecule inhibitors of ChoKα. Starting from weakly binding fragments, we describe a structure based lead discovery approach, which resulted in novel highly potent inhibitors of ChoKα. In cancer cell lines, our lead compounds exhibit a dose-dependent decrease of phosphocholine, inhibition of cell growth, and induction of apoptosis at low micromolar concentrations. The druglike lead series presented here is optimizable for improvements in cellular potency, drug target residence time, and pharmacokinetic parameters. These inhibitors may be utilized not only to further validate ChoKα as antioncogenic target but also as novel chemical matter that may lead to antitumor agents that specifically interfere with cancer cell metabolism.

  19. Small tyrosine kinase inhibitors interrupt EGFR signaling by interacting with erbB3 and erbB4 in glioblastoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Carrasco-Garcia, Estefania; Saceda, Miguel [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Unidad de Investigacion, Hospital General Universitario de Elche, 03203 Elche (Alicante) (Spain); Grasso, Silvina; Rocamora-Reverte, Lourdes; Conde, Mariano; Gomez-Martinez, Angeles [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Garcia-Morales, Pilar [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Unidad de Investigacion, Hospital General Universitario de Elche, 03203 Elche (Alicante) (Spain); Ferragut, Jose A. [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Martinez-Lacaci, Isabel, E-mail: imlacaci@umh.es [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Unidad AECC de Investigacion Traslacional en Cancer, Hospital Universitario Virgen de la Arrixaca, 30120 Murcia (Spain)

    2011-06-10

    Signaling through the epidermal growth factor receptor (EGFR) is relevant in glioblastoma. We have determined the effects of the EGFR inhibitor AG1478 in glioblastoma cell lines and found that U87 and LN-229 cells were very sensitive to this drug, since their proliferation diminished and underwent a marked G{sub 1} arrest. T98 cells were a little more refractory to growth inhibition and A172 cells did not undergo a G{sub 1} arrest. This G{sub 1} arrest was associated with up-regulation of p27{sup kip1}, whose protein turnover was stabilized. EGFR autophosphorylation was blocked with AG1478 to the same extent in all the cell lines. Other small-molecule EGFR tyrosine kinase inhibitors employed in the clinic, such as gefitinib, erlotinib and lapatinib, were able to abrogate proliferation of glioblastoma cell lines, which underwent a G{sub 1} arrest. However, the EGFR monoclonal antibody, cetuximab had no effect on cell proliferation and consistently, had no effect on cell cycle either. Similarly, cetuximab did not inhibit proliferation of U87 {Delta}EGFR cells or primary glioblastoma cell cultures, whereas small-molecule EGFR inhibitors did. Activity of downstream signaling molecules of EGFR such as Akt and especially ERK1/2 was interrupted with EGFR tyrosine kinase inhibitors, whereas cetuximab treatment could not sustain this blockade over time. Small-molecule EGFR inhibitors were able to prevent phosphorylation of erbB3 and erbB4, whereas cetuximab only hindered EGFR phosphorylation, suggesting that EGFR tyrosine kinase inhibitors may mediate their anti-proliferative effects through other erbB family members. We can conclude that small-molecule EGFR inhibitors may be a therapeutic approach for the treatment of glioblastoma patients.

  20. Design and synthesis of imidazopyridine analogues as inhibitors of phosphoinositide 3-kinase signaling and angiogenesis.

    Science.gov (United States)

    Kim, Okseon; Jeong, Yujeong; Lee, Hyunseung; Hong, Sun-Sun; Hong, Sungwoo

    2011-04-14

    Phosphatidylinositol 3-kinase α (PI3Kα) is an important regulator of intracellular signaling pathways, controlling remarkably diverse arrays of physiological processes. Because the PI3K pathway is frequently up-regulated in human cancers, the inhibition of PI3Kα can be a promising approach to cancer therapy. In this study, we have designed and synthesized a new series of imidazo[1,2-a]pyridine derivatives as PI3Kα inhibitors through the fragment-growing strategy. By varying groups at the 3- and 6-positions of imidazo[1,2-a]pyridines, we studied the structure-activity relationships (SAR) profiles and identified a series of potent PI3Kα inhibitors. Representative derivatives showed good activity in cellular proliferation and apoptosis assays. Moreover, these inhibitors exhibited noteworthy antiangiogenic activity.

  1. Determination of Histidine pKa Values in the Propeptides of Furin and Proprotein Convertase 1/3 Using Histidine Hydrogen-Deuterium Exchange Mass Spectrometry.

    Science.gov (United States)

    Elferich, Johannes; Williamson, Danielle M; David, Larry L; Shinde, Ujwal

    2015-08-04

    Propeptides of proprotein convertases regulate activation of their protease domains by sensing the organellar pH within the secretory pathway. Earlier experimental work highlighted the importance of a conserved histidine residue within the propeptide of a widely studied member, furin. A subsequent evolutionary analysis found an increase in histidine content within propeptides of secreted eukaryotic proteases compared with their prokaryotic orthologs. However, furin activates in the trans-golgi network at a pH of 6.5 while a paralog, proprotein convertase 1/3, activates in secretory vesicles at a pH of 5.5. It is unclear how a conserved histidine can mediate activation at two different pH values. In this manuscript, we measured the pKa values of histidines within the propeptides of furin and proprotein convertase 1/3 using a histidine hydrogen-deuterium exchange mass spectrometry approach. The high density of histidine residues combined with an abundance of basic residues provided challenges for generation of peptide ions with unique histidine residues, which were overcome by employing ETD fragmentation. During this analysis, we found slow hydrogen-deuterium exchange in residues other than histidine at basic pH. Finally, we demonstrate that the pKa of the conserved histidine in proprotein convertase 1/3 is acid-shifted compared with furin and is consistent with its lower pH of activation.

  2. How tyrosine kinase inhibitors impair metabolism and endocrine system function: a systematic updated review.

    Science.gov (United States)

    Breccia, Massimo; Molica, Matteo; Alimena, Giuliana

    2014-12-01

    Tyrosine kinase inhibitors (TKIs) advent has deeply changed the outcome of chronic myeloid leukemia (CML) patients, with improved rates of response and overall survival. However, for this success some patients paid the price of a number of peculiar side effects, the so-called off-target side effects, specific for each one TKI. These effects are due to non-selective inhibition of other tyrosine kinase receptors, such as PDGFR, c-KIT, Src, VEGF. Consequences of this inhibition, some metabolic changes during the treatment with TKIs are reported. Aim of present review is to report metabolic changes and potential mechanisms involved in the pathogenesis related to imatinib, second (nilotinib and dasatinib) and third generation (bosutinib and ponatinib) TKIs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Antiproliferative activity of olomoucine II, a novel 2,6,9-trisubstituted purine cyclin-dependent kinase inhibitor

    Czech Academy of Sciences Publication Activity Database

    Kryštof, Vladimír; McNae, I. W.; Walkinshaw, M. D.; Fischer, P.M.; Müller, P.; Vojtešek, B.; Orság, Martin; Havlíček, Libor; Strnad, Miroslav

    2005-01-01

    Roč. 62, č. 15 (2005), s. 1763-1771 ISSN 1420-682X R&D Projects: GA ČR GP204/03/D231 Institutional research plan: CEZ:AV0Z50380511 Keywords : olomoucine II * roscovitine * cyclin-dependent kinase inhibitor Subject RIV: CE - Biochemistry Impact factor: 4.582, year: 2005

  4. The orally active and bioavailable ATR kinase inhibitor AZD6738 potentiates the anti-tumor effects of cisplatin to resolve ATM-deficient non-small cell lung cancer in vivo.

    Science.gov (United States)

    Vendetti, Frank P; Lau, Alan; Schamus, Sandra; Conrads, Thomas P; O'Connor, Mark J; Bakkenist, Christopher J

    2015-12-29

    ATR and ATM are DNA damage signaling kinases that phosphorylate several thousand substrates. ATR kinase activity is increased at damaged replication forks and resected DNA double-strand breaks (DSBs). ATM kinase activity is increased at DSBs. ATM has been widely studied since ataxia telangiectasia individuals who express no ATM protein are the most radiosensitive patients identified. Since ATM is not an essential protein, it is widely believed that ATM kinase inhibitors will be well-tolerated in the clinic. ATR has been widely studied, but advances have been complicated by the finding that ATR is an essential protein and it is widely believed that ATR kinase inhibitors will be toxic in the clinic. We describe AZD6738, an orally active and bioavailable ATR kinase inhibitor. AZD6738 induces cell death and senescence in non-small cell lung cancer (NSCLC) cell lines. AZD6738 potentiates the cytotoxicity of cisplatin and gemcitabine in NSCLC cell lines with intact ATM kinase signaling, and potently synergizes with cisplatin in ATM-deficient NSCLC cells. In contrast to expectations, daily administration of AZD6738 and ATR kinase inhibition for 14 consecutive days is tolerated in mice and enhances the therapeutic efficacy of cisplatin in xenograft models. Remarkably, the combination of cisplatin and AZD6738 resolves ATM-deficient lung cancer xenografts.

  5. Molecular characterization of c-Abl/c-Src kinase inhibitors targeted against murine tumour progenitor cells that express stem cell markers.

    Directory of Open Access Journals (Sweden)

    Thomas Kruewel

    Full Text Available BACKGROUND: The non-receptor tyrosine kinases c-Abl and c-Src are overexpressed in various solid human tumours. Inhibition of their hyperactivity represents a molecular rationale in the combat of cancerous diseases. Here we examined the effects of a new family of pyrazolo [3,4-d] pyrimidines on a panel of 11 different murine lung tumour progenitor cell lines, that express stem cell markers, as well as on the human lung adenocarcinoma cell line A549, the human hepatoma cell line HepG2 and the human colon cancer cell line CaCo2 to obtain insight into the mode of action of these experimental drugs. METHODOLOGY/PRINCIPAL FINDINGS: Treatment with the dual kinase inhibitors blocked c-Abl and c-Src kinase activity efficiently in the nanomolar range, induced apoptosis, reduced cell viability and caused cell cycle arrest predominantly at G0/G1 phase while western blot analysis confirmed repressed protein expression of c-Abl and c-Src as well as the interacting partners p38 mitogen activated protein kinase, heterogenous ribonucleoprotein K, cyclin dependent kinase 1 and further proteins that are crucial for tumour progression. Importantly, a significant repression of the epidermal growth factor receptor was observed while whole genome gene expression analysis evidenced regulation of many cell cycle regulated genes as well integrin and focal adhesion kinase (FAK signalling to impact cytoskeleton dynamics, migration, invasion and metastasis. CONCLUSIONS/SIGNIFICANCE: Our experiments and recently published in vivo engraftment studies with various tumour cell lines revealed the dual kinase inhibitors to be efficient in their antitumour activity.

  6. The Src family kinase inhibitor dasatinib delays pain-related behaviour and conserves bone in a rat model of cancer-induced bone pain

    DEFF Research Database (Denmark)

    Appel, Camilla Kristine; Gallego-Pedersen, Simone; Andersen, Line

    2017-01-01

    -induced bone pain, including cancer growth, osteoclastic bone degradation and nociceptive signalling. Here we investigate the role of dasatinib, an oral Src kinase family and Bcr-Abl tyrosine kinase inhibitor, in an animal model of cancer-induced bone pain. Daily administration of dasatinib (15 mg/kg, p...

  7. Navigating into the binding pockets of the HER family protein kinases: discovery of novel EGFR inhibitor as antitumor agent

    Directory of Open Access Journals (Sweden)

    Liu W

    2015-07-01

    Full Text Available Wei Liu,1,* Jin-Feng Ning,2,* Qing-Wei Meng,1 Jing Hu,1 Yan-Bin Zhao,1 Chao Liu,3 Li Cai11The Fourth Department of Medical Oncology, Harbin Medical University Cancer Hospital, 2The Thoracic Surgery Department, Harbin Medical University Cancer Hospital, Harbin, People’s Republic of China; 3General Surgery Department, Mudanjiang Guanliju Central Hospital, Mishan, Heilongjiang Province, People’s Republic of China*These authors contributed equally to this workAbstract: The epidermal growth factor receptor (EGFR family has been validated as a successful antitumor drug target for decades. Known EGFR inhibitors were exposed to distinct drug resistance against the various EGFR mutants within non-small-cell lung cancer (NSCLC, particularly the T790M mutation. Although so far a number of studies have been reported on the development of third-generation EGFR inhibitors for overcoming the resistance issue, the design procedure largely depends on the intuition of medicinal chemists. Here we retrospectively make a detailed analysis of the 42 EGFR family protein crystal complexes deposited in the Protein Data Bank (PDB. Based on the analysis of inhibitor binding modes in the kinase catalytic cleft, we identified a potent EGFR inhibitor (compound A-10 against drug-resistant EGFR through fragment-based drug design. This compound showed at least 30-fold more potency against EGFR T790M than the two control molecules erlotinib and gefitinib in vitro. Moreover, it could exhibit potent HER2 inhibitory activities as well as tumor growth inhibitory activity. Molecular docking studies revealed a structural basis for the increased potency and mutant selectivity of this compound. Compound A-10 may be selected as a promising candidate in further preclinical studies. In addition, our findings could provide a powerful strategy to identify novel selective kinase inhibitors on the basis of detailed kinase–ligand interaction space in the PDB.Keywords: EGFR, kinase

  8. Histidine protects against zinc and nickel toxicity in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    John T Murphy

    2011-03-01

    Full Text Available Zinc is an essential trace element involved in a wide range of biological processes and human diseases. Zinc excess is deleterious, and animals require mechanisms to protect against zinc toxicity. To identify genes that modulate zinc tolerance, we performed a forward genetic screen for Caenorhabditis elegans mutants that were resistant to zinc toxicity. Here we demonstrate that mutations of the C. elegans histidine ammonia lyase (haly-1 gene promote zinc tolerance. C. elegans haly-1 encodes a protein that is homologous to vertebrate HAL, an enzyme that converts histidine to urocanic acid. haly-1 mutant animals displayed elevated levels of histidine, indicating that C. elegans HALY-1 protein is an enzyme involved in histidine catabolism. These results suggest the model that elevated histidine chelates zinc and thereby reduces zinc toxicity. Supporting this hypothesis, we demonstrated that dietary histidine promotes zinc tolerance. Nickel is another metal that binds histidine with high affinity. We demonstrated that haly-1 mutant animals are resistant to nickel toxicity and dietary histidine promotes nickel tolerance in wild-type animals. These studies identify a novel role for haly-1 and histidine in zinc metabolism and may be relevant for other animals.

  9. Convergent Akt activation drives acquired EGFR inhibitor resistance in lung cancer

    DEFF Research Database (Denmark)

    Jacobsen, Kirstine; Bertran-Alamillo, Jordi; Molina, Miguel Angel

    2017-01-01

    Non-small-cell lung cancer patients with activating epidermal growth factor receptor (EGFR) mutations typically benefit from EGFR tyrosine kinase inhibitor treatment. However, virtually all patients succumb to acquired EGFR tyrosine kinase inhibitor resistance that occurs via diverse mechanisms....... The diversity and unpredictability of EGFR tyrosine kinase inhibitor resistance mechanisms presents a challenge for developing new treatments to overcome EGFR tyrosine kinase inhibitor resistance. Here, we show that Akt activation is a convergent feature of acquired EGFR tyrosine kinase inhibitor resistance......, across a spectrum of diverse, established upstream resistance mechanisms. Combined treatment with an EGFR tyrosine kinase inhibitor and Akt inhibitor causes apoptosis and synergistic growth inhibition in multiple EGFR tyrosine kinase inhibitor-resistant non-small-cell lung cancer models. Moreover...

  10. Evidence that Autophosphorylation of the Major Sporulation Kinase in Bacillus subtilis Is Able To Occur in trans.

    Science.gov (United States)

    Devi, Seram Nganbiton; Kiehler, Brittany; Haggett, Lindsey; Fujita, Masaya

    2015-08-01

    Entry into sporulation in Bacillus subtilis is governed by a multicomponent phosphorelay, a complex version of a two-component system which includes at least three histidine kinases (KinA to KinC), two phosphotransferases (Spo0F and Spo0B), and a response regulator (Spo0A). Among the three histidine kinases, KinA is known as the major sporulation kinase; it is autophosphorylated with ATP upon starvation and then transfers a phosphoryl group to the downstream components in a His-Asp-His-Asp signaling pathway. Our recent study demonstrated that KinA forms a homotetramer, not a dimer, mediated by the N-terminal domain, as a functional unit. Furthermore, when the N-terminal domain was overexpressed in the starving wild-type strain, sporulation was impaired. We hypothesized that this impairment of sporulation could be explained by the formation of a nonfunctional heterotetramer of KinA, resulting in the reduced level of phosphorylated Spo0A (Spo0A∼P), and thus, autophosphorylation of KinA could occur in trans. To test this hypothesis, we generated a series of B. subtilis strains expressing homo- or heterogeneous KinA protein complexes consisting of various combinations of the phosphoryl-accepting histidine point mutant protein and the catalytic ATP-binding domain point mutant protein. We found that the ATP-binding-deficient protein was phosphorylated when the phosphorylation-deficient protein was present in a 1:1 stoichiometry in the tetramer complex, while each of the mutant homocomplexes was not phosphorylated. These results suggest that ATP initially binds to one protomer within the tetramer complex and then the γ-phosphoryl group is transmitted to another in a trans fashion. We further found that the sporulation defect of each of the mutant proteins is complemented when the proteins are coexpressed in vivo. Taken together, these in vitro and in vivo results reinforce the evidence that KinA autophosphorylation is able to occur in a trans fashion

  11. Preclinical FLT-PET and FDG-PET imaging of tumor response to the multi-targeted Aurora B kinase inhibitor, TAK-901

    International Nuclear Information System (INIS)

    Cullinane, Carleen; Waldeck, Kelly L.; Binns, David; Bogatyreva, Ekaterina; Bradley, Daniel P.; Jong, Ron de; McArthur, Grant A.; Hicks, Rodney J.

    2014-01-01

    Introduction: The Aurora kinases play a key role in mitosis and have recently been identified as attractive targets for therapeutic intervention in cancer. The aim of this study was therefore to investigate the utility of 3′-[ 18 F]fluoro-3′-deoxythymidine (FLT) and 2-deoxy-2-[ 18 F]fluoro-D-glucose (FDG) for assessment of tumor response to the multi-targeted Aurora B kinase inhibitor, TAK-901. Methods: Balb/c nude mice bearing HCT116 colorectal xenografts were treated with up to 30 mg/kg TAK 901 or vehicle intravenously twice daily for two days on a weekly cycle. Tumor growth was monitored by calliper measurements and PET imaging was performed at baseline, day 4, 8, 11 and 15. Tumors were harvested at time points corresponding to days of PET imaging for analysis of ex vivo markers of cell proliferation and metabolism together with markers of Aurora B kinase inhibition including phospho-histone H3 (pHH3) and senescence associated β-galactosidase. Results: Tumor growth was inhibited by 60% on day 12 of 30 mg/kg TAK-901 therapy. FLT uptake was significantly reduced by day 4 of treatment and this corresponded with reduction in bromodeoxyuridine and pHH3 staining by immunohistochemistry. All biomarkers rebounded towards baseline levels by the commencement of the next treatment cycle, consistent with release of Aurora B kinase suppression. TAK-901 therapy had no impact on glucose metabolism as assessed by FDG uptake and GLUT1 staining by immunohistochemistry. Conclusions: FLT-PET, but not FDG-PET, is a robust non-invasive imaging biomarker of early HCT116 tumor response to the on-target effects of the multi-targeted Aurora B kinase inhibitor, TAK-901. Advances in knowledge and implications for patient care: This is the first report to demonstrate the impact of the multi-targeted Aurora B kinase inhibitor, TAK-901 on tumor FLT uptake. The findings provide a strong rationale for the evaluation of FLT-PET as an early biomarker of tumor response in the early phase

  12. The Src family kinase inhibitor dasatinib delays pain-related behaviour and conserves bone in a rat model of cancer-induced bone pain

    DEFF Research Database (Denmark)

    Appel, Camilla Kristine; Gallego-Pedersen, Simone; Andersen, Line

    2017-01-01

    Pain is a severe and debilitating complication of metastatic bone cancer. Current analgesics do not provide sufficient pain relief for all patients, creating a great need for new treatment options. The Src kinase, a non-receptor protein tyrosine kinase, is implicated in processes involved in cancer......-induced bone pain, including cancer growth, osteoclastic bone degradation and nociceptive signalling. Here we investigate the role of dasatinib, an oral Src kinase family and Bcr-Abl tyrosine kinase inhibitor, in an animal model of cancer-induced bone pain. Daily administration of dasatinib (15 mg/kg, p...

  13. Acute lymphoid and gastrointestinal toxicity induced by selective p38alpha map kinase and map kinase-activated protein kinase-2 (MK2) inhibitors in the dog.

    Science.gov (United States)

    Morris, Dale L; O'Neil, Shawn P; Devraj, Rajesh V; Portanova, Joseph P; Gilles, Richard W; Gross, Cindy J; Curtiss, Sandra W; Komocsar, Wendy J; Garner, Debra S; Happa, Fernando A; Kraus, Lori J; Nikula, Kristen J; Monahan, Joseph B; Selness, Shaun R; Galluppi, Gerald R; Shevlin, Kimberly M; Kramer, Jeffrey A; Walker, John K; Messing, Dean M; Anderson, David R; Mourey, Robert J; Whiteley, Laurence O; Daniels, John S; Yang, Jerry Z; Rowlands, Philip C; Alden, Carl L; Davis, John W; Sagartz, John E

    2010-06-01

    Exposure to moderately selective p38alpha mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38alpha MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38alpha MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38alpha MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.

  14. Design of inhibitors of thymidylate kinase from Variola virus as new selective drugs against smallpox.

    Science.gov (United States)

    Guimarães, Ana P; de Souza, Felipe R; Oliveira, Aline A; Gonçalves, Arlan S; de Alencastro, Ricardo B; Ramalho, Teodorico C; França, Tanos C C

    2015-02-16

    Recently we constructed a homology model of the enzyme thymidylate kinase from Variola virus (VarTMPK) and proposed it as a new target to the drug design against smallpox. In the present work, we used the antivirals cidofovir and acyclovir as reference compounds to choose eleven compounds as leads to the drug design of inhibitors for VarTMPK. Docking and molecular dynamics (MD) studies of the interactions of these compounds inside VarTMPK and human TMPK (HssTMPK) suggest that they compete for the binding region of the substrate and were used to propose the structures of ten new inhibitors for VarTMPK. Further docking and MD simulations of these compounds, inside VarTMPK and HssTMPK, suggest that nine among ten are potential selective inhibitors of VarTMPK. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  15. Repigmentation in vitiligo using the Janus kinase inhibitor tofacitinib may require concomitant light exposure.

    Science.gov (United States)

    Liu, Lucy Y; Strassner, James P; Refat, Maggi A; Harris, John E; King, Brett A

    2017-10-01

    Vitiligo is an autoimmune disease in which cutaneous depigmentation occurs. Existing therapies are often inadequate. Prior reports have shown benefit of the Janus kinase (JAK) inhibitors. To evaluate the efficacy of the JAK 1/3 inhibitor tofacitinib in the treatment of vitiligo. This is a retrospective case series of 10 consecutive patients with vitiligo treated with tofacitinib. Severity of disease was assessed by body surface area of depigmentation. Ten consecutive patients were treated with tofacitinib. Five patients achieved some repigmentation at sites of either sunlight exposure or low-dose narrowband ultraviolet B phototherapy. Suction blister sampling revealed that the autoimmune response was inhibited during treatment in both responding and nonresponding lesions, suggesting that light rather than immunosuppression was primarily required for melanocyte regeneration. Limitations include the small size of the study population, retrospective nature of the study, and lack of a control group. Treatment of vitiligo with JAK inhibitors appears to require light exposure. In contrast to treatment with phototherapy alone, repigmentation during treatment with JAK inhibitors may require only low-level light. Maintenance of repigmentation may be achieved with JAK inhibitor monotherapy. These results support a model wherein JAK inhibitors suppress T cell mediators of vitiligo and light exposure is necessary for stimulation of melanocyte regeneration. Copyright © 2017 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

  16. Systematic Kinase Inhibitor Profiling Identifies CDK9 as a Synthetic Lethal Target in NUT Midline Carcinoma

    Directory of Open Access Journals (Sweden)

    Johannes Brägelmann

    2017-09-01

    Full Text Available Kinase inhibitors represent the backbone of targeted cancer therapy, yet only a limited number of oncogenic drivers are directly druggable. By interrogating the activity of 1,505 kinase inhibitors, we found that BRD4-NUT-rearranged NUT midline carcinoma (NMC cells are specifically killed by CDK9 inhibition (CDK9i and depend on CDK9 and Cyclin-T1 expression. We show that CDK9i leads to robust induction of apoptosis and of markers of DNA damage response in NMC cells. While both CDK9i and bromodomain inhibition over time result in reduced Myc protein expression, only bromodomain inhibition induces cell differentiation and a p21-induced cell-cycle arrest in these cells. Finally, RNA-seq and ChIP-based analyses reveal a BRD4-NUT-specific CDK9i-induced perturbation of transcriptional elongation. Thus, our data provide a mechanistic basis for the genotype-dependent vulnerability of NMC cells to CDK9i that may be of relevance for the development of targeted therapies for NMC patients.

  17. Serum concentrations of nitrite and malondialdehyde as markers of oxidative stress in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors

    Directory of Open Access Journals (Sweden)

    Maria Juracy Petrola

    2012-01-01

    Full Text Available BACKGROUND: Chronic myeloid leukemia is a neoplasm characterized by clonal expansion of hematopoietic progenitor cells resulting from the (9:22(q34,11 translocation. The tyrosine kinase abl fusion protein,the initial leukemogenic event in chronic myeloid leukemia, is constitutively activated thus inducing the production of reactive oxygen species. Of particular relevance is the fact that an increase in reactive oxygen species can facilitate genomic instability and may contribute to disease progression. OBJETIVE: To evaluate oxidative stress by determining the levels of malondialdehyde and nitrite in chronic myeloid leukemia patients under treatment with 1st and 2nd generation tyrosine kinase inhibitors monitored at a referral hospital in Fortaleza, Ceará. METHODS: A cross-sectional study was performed of 64 male and female adults. Patients were stratified according to treatment. The levels of malondialdehyde and nitrite were determined by spectrophotometry. Statistical differences between groups were observed using the Student t-test and Fisher's exact test. The results are expressed as mean ± standard error of mean. The significance level was set for a p-value < 0.05 in all analyses. RESULTS: The results show significantly higher mean concentrations of nitrite and malondialdehyde in chronic myeloid leukemia patients using second-generation tyrosine kinase inhibitors compared to patients on imatinib. Conclusion: It follows that chronic myeloid leukemia patients present higher oxidative activity and that the increases in oxidative damage markers can indicate resistance to 1st generation tyrosine kinase inhibitors.

  18. Applying ligands profiling using multiple extended electron distribution based field templates and feature trees similarity searching in the discovery of new generation of urea-based antineoplastic kinase inhibitors.

    Directory of Open Access Journals (Sweden)

    Eman M Dokla

    Full Text Available This study provides a comprehensive computational procedure for the discovery of novel urea-based antineoplastic kinase inhibitors while focusing on diversification of both chemotype and selectivity pattern. It presents a systematic structural analysis of the different binding motifs of urea-based kinase inhibitors and the corresponding configurations of the kinase enzymes. The computational model depends on simultaneous application of two protocols. The first protocol applies multiple consecutive validated virtual screening filters including SMARTS, support vector-machine model (ROC = 0.98, Bayesian model (ROC = 0.86 and structure-based pharmacophore filters based on urea-based kinase inhibitors complexes retrieved from literature. This is followed by hits profiling against different extended electron distribution (XED based field templates representing different kinase targets. The second protocol enables cancericidal activity verification by using the algorithm of feature trees (Ftrees similarity searching against NCI database. Being a proof-of-concept study, this combined procedure was experimentally validated by its utilization in developing a novel series of urea-based derivatives of strong anticancer activity. This new series is based on 3-benzylbenzo[d]thiazol-2(3H-one scaffold which has interesting chemical feasibility and wide diversification capability. Antineoplastic activity of this series was assayed in vitro against NCI 60 tumor-cell lines showing very strong inhibition of GI(50 as low as 0.9 uM. Additionally, its mechanism was unleashed using KINEX™ protein kinase microarray-based small molecule inhibitor profiling platform and cell cycle analysis showing a peculiar selectivity pattern against Zap70, c-src, Mink1, csk and MeKK2 kinases. Interestingly, it showed activity on syk kinase confirming the recent studies finding of the high activity of diphenyl urea containing compounds against this kinase. Allover, the new series

  19. Comparative evaluation of bone marrow cells morpho-functional activity in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors of the first and second generation

    Directory of Open Access Journals (Sweden)

    I. O. Zhaleyko

    2014-07-01

    Full Text Available The efficiency of using the culture techniques of research for monitoring the patient’s response to the treatment by tyrosine kinase inhibitors of the first and second generation is shown. Thus, the functional activity of bone marrow cells in patients having the optimal treatment response to inhibitors of tyrosine kinases was significantly lower compared with patients with the acquired resistance to the drug, and patients who had CML diagnosed for first time. Furthermore, for patients with the optimal response to the nilotinib therapy, numbers of colonies in semi-solid agar in vitro was lower, than in patients with the optimal response to imatinib. When the leukaemic cell clone becomes resistant to tyrosine kinase inhibitors, the prevalence of early cells of granulocyte-macrophage hematopoietic stem cells is observed in CFU culture which can be an important prognostic factor for choosing the appropriate treatment strategy.

  20. Tyrosine kinase inhibitor BIBF1120 ameliorates inflammation, angiogenesis and fibrosis in CCl4-induced liver fibrogenesis mouse model

    NARCIS (Netherlands)

    Öztürk Akcora, Büsra; Storm, Gerrit; Prakash, Jai; Bansal, Ruchi

    2017-01-01

    Hepatic fibrosis, a progressive chronic disease mainly caused by hepatitis viral infections, alcohol abuse or metabolic syndrome leading to liver dysfunction and is the growing cause of mortality worldwide. Tyrosine kinase inhibitor BIBF1120 (Nintedanib) has been evaluated in clinical trials for

  1. Tyrosine kinase inhibitor SU6668 represses chondrosarcoma growth via antiangiogenesis in vivo

    International Nuclear Information System (INIS)

    Klenke, Frank M; Abdollahi, Amir; Bertl, Elisabeth; Gebhard, Martha-Maria; Ewerbeck, Volker; Huber, Peter E; Sckell, Axel

    2007-01-01

    As chondrosarcomas are resistant to chemotherapy and ionizing radiation, therapeutic options are limited. Radical surgery often cannot be performed. Therefore, additional therapies such as antiangiogenesis represent a promising strategy for overcoming limitations in chondrosarcoma therapy. There is strong experimental evidence that SU6668, an inhibitor of the angiogenic tyrosine kinases Flk-1/KDR, PDGFRbeta and FGFR1 can induce growth inhibition of various primary tumors. However, the effectiveness of SU6668 on malignant primary bone tumors such as chondrosarcomas has been rarely investigated. Therefore, the aim of this study was to investigate the effects of SU6668 on chondrosarcoma growth, angiogenesis and microcirculation in vivo. In 10 male severe combined immunodeficient (SCID) mice, pieces of SW1353 chondrosarcomas were implanted into a cranial window preparation where the calvaria serves as the site for the orthotopic implantation of bone tumors. From day 7 after tumor implantation, five animals were treated with SU6668 (250 mg/kg body weight, s.c.) at intervals of 48 hours (SU6668), and five animals with the equivalent amount of the CMC-based vehicle (Control). Angiogenesis, microcirculation, and growth of SW 1353 tumors were analyzed by means of intravital microscopy. SU6668 induced a growth arrest of chondrosarcomas within 7 days after the initiation of the treatment. Compared to Controls, SU6668 decreased functional vessel density and tumor size, respectively, by 37% and 53% on day 28 after tumor implantation. The time course of the experiments demonstrated that the impact on angiogenesis preceded the anti-tumor effect. Histological and immunohistochemical results confirmed the intravital microscopy findings. SU6668 is a potent inhibitor of chondrosarcoma tumor growth in vivo. This effect appears to be induced by the antiangiogenic effects of SU6668, which are mediated by the inhibition of the key angiogenic receptor tyrosine kinases Flk-1/KDR, PDGFRbeta

  2. A Novel, Broad-Spectrum Inhibitor of Enterovirus Replication That Targets Host Cell Factor Phosphatidylinositol 4-Kinase IIIβ

    Science.gov (United States)

    van der Schaar, Hilde M.; Leyssen, Pieter; Thibaut, Hendrik J.; de Palma, Armando; van der Linden, Lonneke; Lanke, Kjerstin H. W.; Lacroix, Céline; Verbeken, Erik; Conrath, Katja; MacLeod, Angus M.; Mitchell, Dale R.; Palmer, Nicholas J.; van de Poël, Hervé; Andrews, Martin

    2013-01-01

    Despite their high clinical and socioeconomic impacts, there is currently no approved antiviral therapy for the prophylaxis or treatment of enterovirus infections. Here we report on a novel inhibitor of enterovirus replication, compound 1, 2-fluoro-4-(2-methyl-8-(3-(methylsulfonyl)benzylamino)imidazo[1,2-a]pyrazin-3-yl)phenol. This compound exhibited a broad spectrum of antiviral activity, as it inhibited all tested species of enteroviruses and rhinoviruses, with 50% effective concentrations ranging between 4 and 71 nM. After a lengthy resistance selection process, coxsackievirus mutants resistant to compound 1 were isolated that carried substitutions in their 3A protein. Remarkably, the same substitutions were recently shown to provide resistance to inhibitors of phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ), a lipid kinase that is essential for enterovirus replication, suggesting that compound 1 may also target this host factor. Accordingly, compound 1 directly inhibited PI4KIIIβ in an in vitro kinase activity assay. Furthermore, the compound strongly reduced the PI 4-phosphate levels of the Golgi complex in cells. Rescue of coxsackievirus replication in the presence of compound 1 by a mutant PI4KIIIβ carrying a substitution in its ATP-binding pocket revealed that the compound directly binds the kinase at this site. Finally, we determined that an analogue of compound 1, 3-(3-fluoro-4-methoxyphenyl)-2-methyl-N-(pyridin-4-ylmethyl)imidazo[1,2-a]pyrazin-8-amine, is well tolerated in mice and has a dose-dependent protective activity in a coxsackievirus serotype B4-induced pancreatitis model. PMID:23896472

  3. Prevention of pulmonary vascular and myocardial remodeling by the combined tyrosine and serine-/threonine kinase inhibitor, sorafenib, in pulmonary hypertension and right heart failure

    Directory of Open Access Journals (Sweden)

    M. Klein

    2008-06-01

    Full Text Available Inhibition of tyrosine kinases can reverse pulmonary hypertension but little is known about the role of serine-/threonine kinases in vascular and myocardial remodeling. We investigated the effects of sorafenib, an inhibitor of the tyrosine kinases VEGFR, PDGFR and c-kit as well as the serine-/threonine kinase Raf-1, in pulmonary hypertension and right ventricular (RV pressure overload. In monocrotaline treated rats, sorafenib (10 mg·kg–1·d–1 p.o. reduced pulmonary arterial pressure, pulmonary artery muscularization and RV hypertrophy, and improved systemic hemodynamics (table 1. Sorafenib prevented phosphorylation of Raf-1 and suppressed activation of downstream signaling pathways (Erk 1/2. After pulmonary banding, sorafenib, but not the PDGFR/c-KIT/ABL-inhibitor imatinib reduced RV mass and RV filling pressure significantly. Congruent with these results, sorafenib only prevented ERK phosphorylation and vasopressin induced hypertrophy of the cardiomyocyte cell line H9c2 dose dependently (IC50 = 300 nM. Combined inhibition of tyrosine and serine-/threonine kinases by sorafenib prevents vascular and cardiac remodeling in pulmonary hypertension, which is partly mediated via inhibition of the Raf kinase pathway.

  4. Proteínas quinases: características estruturais e inibidores químicos Kinase protein: structural features and chemical inhibitors

    Directory of Open Access Journals (Sweden)

    Bárbara V. Silva

    2009-01-01

    Full Text Available Protein kinases are one of the largest protein families and they are responsible for regulation of a great number of signal transduction pathways in cells, through the phosphorylation of serine, threonine, or tyrosine residues. Deregulation of these enzymes is associated with several diseases including cancer, diabetes and inflammation. For this reason, specific inhibition of tyrosine or serine/threonine kinases may represent an interesting therapeutic approach. The most important types of protein kinases, their structural features and chemical inhibitors are discussed in this paper. Emphasis is given to the small-molecule drugs that target the ATP-binding sites of these enzymes.

  5. Effects of inhibitors of protein kinase C and NO-synthase on the radiation-induced cytogenetic adaptive response in Chinese hamster cells in culture

    International Nuclear Information System (INIS)

    Gil'yano, N.Ya.; Bondarev, G.N.; Bikineeva, E.G.; Krasotskaya, G.I.; Noskin, L.A.

    2001-01-01

    The effect of the serine-threonin kinase inhibitor - staurosporine and inhibitor of NO-synthase - L-NAME on the radiation-induced adaptive response were studied in fibroblasts of Chinese hamster in culture. It is shown that staurosporine and L-NAME inhibit cytogenetic adaptive response induced by β-particles in low doses. Inhibition is not connected with radiosensitizing effect of these agents. L-NAME decreases significantly the γ-rays-induced chromosome aberration yield also. Study confirms the role of protein kinase C in induction of the adaptive response and participation of NO-synthase in this process is noticed for the first time [ru

  6. Roles of Apicomplexan protein kinases at each life cycle stage.

    Science.gov (United States)

    Kato, Kentaro; Sugi, Tatsuki; Iwanaga, Tatsuya

    2012-06-01

    Inhibitors of cellular protein kinases have been reported to inhibit the development of Apicomplexan parasites, suggesting that the functions of protozoan protein kinases are critical for their life cycle. However, the specific roles of these protein kinases cannot be determined using only these inhibitors without molecular analysis, including gene disruption. In this report, we describe the functions of Apicomplexan protein kinases in each parasite life stage and the potential of pre-existing protein kinase inhibitors as Apicomplexan drugs against, mainly, Plasmodium and Toxoplasma. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  7. Activity of EGFR-tyrosine kinase and ALK inhibitors for EML4–ALK-rearranged non–small–cell lung cancer harbored coexisting EGFR mutation

    International Nuclear Information System (INIS)

    Miyanaga, Akihiko; Kawamoto, Masashi; Tsuchiya, Shinichi; Hagiwara, Koichi; Soda, Manabu; Takeuchi, Kengo; Yamamoto, Nobuyuki; Mano, Hiroyuki; Ishikawa, Yuichi; Gemma, Akihiko; Shimizu, Kumi; Noro, Rintaro; Seike, Masahiro; Kitamura, Kazuhiro; Kosaihira, Seiji; Minegishi, Yuji; Shukuya, Takehito; Yoshimura, Akinobu

    2013-01-01

    The EML4–ALK (echinoderm microtubule-associated protein-like 4 gene and the anaplastic lymphoma kinase gene) fusion oncogene represents a novel molecular target in a small subset of non–small–cell lung cancers (NSCLCs). The EML4–ALK fusion gene occurs generally in NSCLC without mutations in epidermal growth factor receptor (EGFR) and KRAS. We report that a case of EML4–ALK-positive NSCLC with EGFR mutation had a response of stable disease to both an EGFR tyrosine kinase inhibitor (EGFR-TKI) and ALK inhibitor. We described the first clinical report of a patient with EML4–ALK-positive NSCLC with EGFR mutation that had a response of stable disease to both single-agent EGFR-TKI and ALK inhibitor. EML4–ALK translocation may be associated with resistance to EGFR-TKI, and EGFR signaling may contribute to resistance to ALK inhibitor in EML4–ALK-positive NSCLC

  8. Tyrosine kinase inhibitors induced immune thrombocytopenia in chronic myeloid leukemia?

    Directory of Open Access Journals (Sweden)

    Avital F. Barak

    2011-12-01

    Full Text Available The outcome and quality of life of chronic myeloid leukemia (CML patients has remarkably changed with the treatment of tyrosine kinase inhibitors (TKIs. Currently, hematopoietic stem cell transplantation (HSCT is considered mainly as a third line salvage therapy in cases of TKIs resistance or intolerance. Here we describe a patient with chronic phase CML who developed both resistance and late occurrence of s severe thrombocytopenia on first and second generation TKIs and eventually underwent HSCT. Although the mechanism of the myelosuppression is not fully understood, we showed for the first time the development of dose dependent platelet antibodies in the presence of TKIs, suggesting the possibility of TKIs induced thrombocytopenia. Our case emphasizes that late development of severe myelosuppression during imatinib treatment is probably an important indication for consideration of early HSCT.

  9. Investigating the role of c-Jun N-terminal kinases in the proliferation of Werner syndrome fibroblasts using diaminopyridine inhibitors

    Directory of Open Access Journals (Sweden)

    Davis Terence

    2011-12-01

    Full Text Available Abstract Fibroblasts derived from the progeroid Werner syndrome show reduced replicative lifespan and a "stressed" morphology, both alleviated using the MAP kinase inhibitor SB203580. However, interpretation of these data is problematical because although SB203580 has the stress-activated kinases p38 and JNK1/2 as its preferred targets, it does show relatively low overall kinase selectivity. Several lines of data support a role for both p38 and JNK1/2 activation in the control of cellular proliferation and also the pathology of diseases of ageing, including type II diabetes, diseases to which Werner Syndrome individuals are prone, thus making the use of JNK inhibitors attractive as possible therapeutics. We have thus tested the effects of the widely used JNK inhibitor SP600125 on the proliferation and morphology of WS cells. In addition we synthesised and tested two recently described aminopyridine based inhibitors. SP600125 treatment resulted in the cessation of proliferation of WS cells and resulted in a senescent-like cellular phenotype that does not appear to be related to the inhibition of JNK1/2. In contrast, use of the more selective aminopyridine CMPD 6o at concentrations that fully inhibit JNK1/2 had a positive effect on cellular proliferation of immortalised WS cells, but no effect on the replicative lifespan of primary WS fibroblasts. In addition, CMPD 6o corrected the stressed WS cellular morphology. The aminopyridine CMPD 6r, however, had little effect on WS cells. CMDP 6o was also found to be a weak inhibitor of MK2, which may partially explain its effects on WS cells, since MK2 is known to be involved in regulating cellular morphology via HSP27 phosphorylation, and is thought to play a role in cell cycle arrest. These data suggest that total JNK1/2 activity does not play a substantial role in the proliferation control in WS cells.

  10. A Discovery Strategy for Selective Inhibitors of c-Src in Complex with the Focal Adhesion Kinase SH3/SH2-binding Region.

    Science.gov (United States)

    Moroco, Jamie A; Baumgartner, Matthew P; Rust, Heather L; Choi, Hwan Geun; Hur, Wooyoung; Gray, Nathanael S; Camacho, Carlos J; Smithgall, Thomas E

    2015-08-01

    The c-Src tyrosine kinase co-operates with the focal adhesion kinase to regulate cell adhesion and motility. Focal adhesion kinase engages the regulatory SH3 and SH2 domains of c-Src, resulting in localized kinase activation that contributes to tumor cell metastasis. Using assay conditions where c-Src kinase activity required binding to a tyrosine phosphopeptide based on the focal adhesion kinase SH3-SH2 docking sequence, we screened a kinase-biased library for selective inhibitors of the Src/focal adhesion kinase peptide complex versus c-Src alone. This approach identified an aminopyrimidinyl carbamate compound, WH-4-124-2, with nanomolar inhibitory potency and fivefold selectivity for c-Src when bound to the phospho-focal adhesion kinase peptide. Molecular docking studies indicate that WH-4-124-2 may preferentially inhibit the 'DFG-out' conformation of the kinase active site. These findings suggest that interaction of c-Src with focal adhesion kinase induces a unique kinase domain conformation amenable to selective inhibition. © 2014 John Wiley & Sons A/S.

  11. Crystal structure of human cyclin-dependent kinase-2 complex with MK2 inhibitor TEI-I01800: insight into the selectivity

    Energy Technology Data Exchange (ETDEWEB)

    Fujino, Aiko; Fukushima, Kei; Kubota, Takaharu; Kosugi, Tomomi; Takimoto-Kamimura, Midori, E-mail: m.kamimura@teijin.co.jp [Teijin Pharma Limited, 4-3-2 Asahigaoka, Hino-shi, Tokyo 191-8512 (Japan)

    2013-11-01

    The Gly-rich loop of cyclin-dependent kinase 2 (CDK2) bound to TEI-I01800 as an MK2 specific inhibitor forms a β-sheet which is a common structure in CDK2–ligand complexes. Here, the reason why TEI-I01800 does not become a strong inhibitor against CDK2 based on the conformation of TEI-I01800 is presented. Mitogen-activated protein kinase-activated protein kinase 2 (MK2 or MAPKAP-K2) is a Ser/Thr kinase from the p38 mitogen-activated protein kinase signalling pathway and plays an important role in inflammatory diseases. The crystal structure of the MK2–TEI-I01800 complex has been reported; its Gly-rich loop was found to form an α-helix, not a β-sheet as has been observed for other Ser/Thr kinases. TEI-I01800 is 177-fold selective against MK2 compared with CDK2; in order to understand the inhibitory mechanism of TEI-I01800, the cyclin-dependent kinase 2 (CDK2) complex structure with TEI-I01800 was determined at 2.0 Å resolution. Interestingly, the Gly-rich loop of CDK2 formed a β-sheet that was different from that of MK2. In MK2, TEI-I01800 changed the secondary structure of the Gly-rich loop from a β-sheet to an α-helix by collision between Leu70 and a p-ethoxyphenyl group at the 7-position and bound to MK2. However, for CDK2, TEI-I01800 bound to CDK2 without this structural change and lost the interaction with the substituent at the 7-position. In summary, the results of this study suggest that the reason for the selectivity of TEI-I01800 is the favourable conformation of TEI-I01800 itself, making it suitable for binding to the α-form MK2.

  12. The Phosphoinositide 3-Kinaseα Selective Inhibitor, BYL719, Enhances the Effect of the Protein Kinase C Inhibitor, AEB071, in GNAQ/GNA11 Mutant Uveal Melanoma Cells

    Science.gov (United States)

    Musi, Elgilda; Ambrosini, Grazia; de Stanchina, Elisa; Schwartz, Gary K.

    2014-01-01

    G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-Kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (Sotrastaurin) and PI3k/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11 mutant cells with AEB071 versus no activity in WT cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of MARCKS, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal anti-proliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11 mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ and GNA11 mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ mutant model. These findings suggest a new therapy treatment option for G-protein mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy. PMID:24563540

  13. Discovery of Brigatinib (AP26113), a Phosphine Oxide-Containing, Potent, Orally Active Inhibitor of Anaplastic Lymphoma Kinase.

    Science.gov (United States)

    Huang, Wei-Sheng; Liu, Shuangying; Zou, Dong; Thomas, Mathew; Wang, Yihan; Zhou, Tianjun; Romero, Jan; Kohlmann, Anna; Li, Feng; Qi, Jiwei; Cai, Lisi; Dwight, Timothy A; Xu, Yongjin; Xu, Rongsong; Dodd, Rory; Toms, Angela; Parillon, Lois; Lu, Xiaohui; Anjum, Rana; Zhang, Sen; Wang, Frank; Keats, Jeffrey; Wardwell, Scott D; Ning, Yaoyu; Xu, Qihong; Moran, Lauren E; Mohemmad, Qurish K; Jang, Hyun Gyung; Clackson, Tim; Narasimhan, Narayana I; Rivera, Victor M; Zhu, Xiaotian; Dalgarno, David; Shakespeare, William C

    2016-05-26

    In the treatment of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase positive (ALK+) non-small-cell lung cancer (NSCLC), secondary mutations within the ALK kinase domain have emerged as a major resistance mechanism to both first- and second-generation ALK inhibitors. This report describes the design and synthesis of a series of 2,4-diarylaminopyrimidine-based potent and selective ALK inhibitors culminating in identification of the investigational clinical candidate brigatinib. A unique structural feature of brigatinib is a phosphine oxide, an overlooked but novel hydrogen-bond acceptor that drives potency and selectivity in addition to favorable ADME properties. Brigatinib displayed low nanomolar IC50s against native ALK and all tested clinically relevant ALK mutants in both enzyme-based biochemical and cell-based viability assays and demonstrated efficacy in multiple ALK+ xenografts in mice, including Karpas-299 (anaplastic large-cell lymphomas [ALCL]) and H3122 (NSCLC). Brigatinib represents the most clinically advanced phosphine oxide-containing drug candidate to date and is currently being evaluated in a global phase 2 registration trial.

  14. Protein Kinase C Inhibitors as Modulators of Vascular Function and Their Application in Vascular Disease

    Directory of Open Access Journals (Sweden)

    Raouf A. Khalil

    2013-03-01

    Full Text Available Blood pressure (BP is regulated by multiple neuronal, hormonal, renal and vascular control mechanisms. Changes in signaling mechanisms in the endothelium, vascular smooth muscle (VSM and extracellular matrix cause alterations in vascular tone and blood vessel remodeling and may lead to persistent increases in vascular resistance and hypertension (HTN. In VSM, activation of surface receptors by vasoconstrictor stimuli causes an increase in intracellular free Ca2+ concentration ([Ca2+]i, which forms a complex with calmodulin, activates myosin light chain (MLC kinase and leads to MLC phosphorylation, actin-myosin interaction and VSM contraction. Vasoconstrictor agonists could also increase the production of diacylglycerol which activates protein kinase C (PKC. PKC is a family of Ca2+-dependent and Ca2+-independent isozymes that have different distributions in various blood vessels, and undergo translocation from the cytosol to the plasma membrane, cytoskeleton or the nucleus during cell activation. In VSM, PKC translocation to the cell surface may trigger a cascade of biochemical events leading to activation of mitogen-activated protein kinase (MAPK and MAPK kinase (MEK, a pathway that ultimately increases the myofilament force sensitivity to [Ca2+]i, and enhances actin-myosin interaction and VSM contraction. PKC translocation to the nucleus may induce transactivation of various genes and promote VSM growth and proliferation. PKC could also affect endothelium-derived relaxing and contracting factors as well as matrix metalloproteinases (MMPs in the extracellular matrix further affecting vascular reactivity and remodeling. In addition to vasoactive factors, reactive oxygen species, inflammatory cytokines and other metabolic factors could affect PKC activity. Increased PKC expression and activity have been observed in vascular disease and in certain forms of experimental and human HTN. Targeting of vascular PKC using PKC inhibitors may function in

  15. A targeted enzyme approach to sensitization of tyrosine kinase inhibitor-resistant breast cancer cells.

    Science.gov (United States)

    Giordano, Courtney R; Mueller, Kelly L; Terlecky, Laura J; Krentz, Kendra A; Bollig-Fischer, Aliccia; Terlecky, Stanley R; Boerner, Julie L

    2012-10-01

    Gefitinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) of potential use in patients with breast cancer. Unfortunately, in clinical studies, gefitinib is often ineffective indicating that resistance to EGFR inhibitors may be a common occurrence in cancer of the breast. EGFR has been shown to be overexpressed in breast cancer, and in particular remains hyperphosphorylated in cell lines such as MDA-MB-468 that are resistant to EGFR inhibitors. Here, we investigate the cause of this sustained phosphorylation and the molecular basis for the ineffectiveness of gefitinib. We show that reactive oxygen species (ROS), known to damage cellular macromolecules and to modulate signaling cascades in a variety of human diseases including cancers, appear to play a critical role in mediating EGFR TKI-resistance. Furthermore, elimination of these ROS through use of a cell-penetrating catalase derivative sensitizes the cells to gefitinib. These results suggest a new approach for the treatment of TKI-resistant breast cancer patients specifically, the targeting of ROS and attendant downstream oxidative stress and their effects on signaling cascades. Copyright © 2012. Published by Elsevier Inc.

  16. Adventures in Scaffold Morphing: Discovery of Fused Ring Heterocyclic Checkpoint Kinase 1 (CHK1) Inhibitors.

    Science.gov (United States)

    Yang, Bin; Vasbinder, Melissa M; Hird, Alexander W; Su, Qibin; Wang, Haixia; Yu, Yan; Toader, Dorin; Lyne, Paul D; Read, Jon A; Breed, Jason; Ioannidis, Stephanos; Deng, Chun; Grondine, Michael; DeGrace, Nancy; Whitston, David; Brassil, Patrick; Janetka, James W

    2018-02-08

    Checkpoint kinase 1 (CHK1) inhibitors are potential cancer therapeutics that can be utilized for enhancing the efficacy of DNA damaging agents. Multiple small molecule CHK1 inhibitors from different chemical scaffolds have been developed and evaluated in clinical trials in combination with chemotherapeutics and radiation treatment. Scaffold morphing of thiophene carboxamide ureas (TCUs), such as AZD7762 (1) and a related series of triazoloquinolines (TZQs), led to the identification of fused-ring bicyclic CHK1 inhibitors, 7-carboxamide thienopyridines (7-CTPs), and 7-carboxamide indoles. X-ray crystal structures reveal a key intramolecular noncovalent sulfur-oxygen interaction in aligning the hinge-binding carboxamide group to the thienopyridine core in a coplanar fashion. An intramolecular hydrogen bond to an indole NH was also effective in locking the carboxamide in the preferred bound conformation to CHK1. Optimization on the 7-CTP series resulted in the identification of lead compound 44, which displayed respectable drug-like properties and good in vitro and in vivo potency.

  17. The Pim kinases: new targets for drug development.

    Science.gov (United States)

    Swords, Ronan; Kelly, Kevin; Carew, Jennifer; Nawrocki, Stefan; Mahalingam, Devalingam; Sarantopoulos, John; Bearss, David; Giles, Francis

    2011-12-01

    The three Pim kinases are a small family of serine/threonine kinases regulating several signaling pathways that are fundamental to cancer development and progression. They were first recognized as pro-viral integration sites for the Moloney Murine Leukemia virus. Unlike other kinases, they possess a hinge region which creates a unique binding pocket for ATP. Absence of a regulatory domain means that these proteins are constitutively active once transcribed. Pim kinases are critical downstream effectors of the ABL (ableson), JAK2 (janus kinase 2), and Flt-3 (FMS related tyrosine kinase 1) oncogenes and are required by them to drive tumorigenesis. Recent investigations have established that the Pim kinases function as effective inhibitors of apoptosis and when overexpressed, produce resistance to the mTOR (mammalian target of rapamycin) inhibitor, rapamycin . Overexpression of the PIM kinases has been reported in several hematological and solid tumors (PIM 1), myeloma, lymphoma, leukemia (PIM 2) and adenocarcinomas (PIM 3). As such, the Pim kinases are a very attractive target for pharmacological inhibition in cancer therapy. Novel small molecule inhibitors of the human Pim kinases have been designed and are currently undergoing preclinical evaluation.

  18. Epigallocatechin gallate (EGCG), a major component of green tea, is a dual phosphoinositide-3-kinase/mTOR inhibitor

    International Nuclear Information System (INIS)

    Van Aller, Glenn S.; Carson, Jeff D.; Tang, Wei; Peng, Hao; Zhao, Lin; Copeland, Robert A.; Tummino, Peter J.; Luo, Lusong

    2011-01-01

    Research highlights: → Epigallocatechin-3-gallate (EGCG) is an ATP-competitive inhibitor of PI3K and mTOR with Ki values around 300 nM. → EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231and A549 cells. → Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site. → These results suggest another important molecular mechanism for the anticancer activities of EGCG. -- Abstract: The PI3K signaling pathway is activated in a broad spectrum of human cancers, either directly by genetic mutation or indirectly via activation of receptor tyrosine kinases or inactivation of the PTEN tumor suppressor. The key nodes of this pathway have emerged as important therapeutic targets for the treatment of cancer. In this study, we show that (-)-epigallocatechin-3-gallate (EGCG), a major component of green tea, is an ATP-competitive inhibitor of both phosphoinositide-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) with K i values of 380 and 320 nM respectively. The potency of EGCG against PI3K and mTOR is within physiologically relevant concentrations. In addition, EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231 and A549 cells. Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site, agreeing with the finding that EGCG competes for ATP binding. Our results suggest another important molecular mechanism for the anticancer activities of EGCG.

  19. β-Catenin is required for intrinsic but not extrinsic BCR-ABL1 kinase-independent resistance to tyrosine kinase inhibitors in chronic myeloid leukemia.

    Science.gov (United States)

    Eiring, A M; Khorashad, J S; Anderson, D J; Yu, F; Redwine, H M; Mason, C C; Reynolds, K R; Clair, P M; Gantz, K C; Zhang, T Y; Pomicter, A D; Kraft, I L; Bowler, A D; Johnson, K; Partlin, M Mac; O'Hare, T; Deininger, M W

    2015-12-01

    Activation of nuclear β-catenin and expression of its transcriptional targets promotes chronic myeloid leukemia (CML) progression, tyrosine kinase inhibitor (TKI) resistance, and leukemic stem cell self-renewal. We report that nuclear β-catenin has a role in leukemia cell-intrinsic but not -extrinsic BCR-ABL1 kinase-independent TKI resistance. Upon imatinib inhibition of BCR-ABL1 kinase activity, β-catenin expression was maintained in intrinsically resistant cells grown in suspension culture and sensitive cells cultured in direct contact (DC) with bone marrow (BM) stromal cells. Thus, TKI resistance uncouples β-catenin expression from BCR-ABL1 kinase activity. In β-catenin reporter assays, intrinsically resistant cells showed increased transcriptional activity versus parental TKI-sensitive controls, and this was associated with restored expression of β-catenin target genes. In contrast, DC with BM stromal cells promoted TKI resistance, but had little effects on Lef/Tcf reporter activity and no consistent effects on cytoplasmic β-catenin levels, arguing against a role for β-catenin in extrinsic TKI resistance. N-cadherin or H-cadherin blocking antibodies abrogated DC-based resistance despite increasing Lef/Tcf reporter activity, suggesting that factors other than β-catenin contribute to extrinsic, BM-derived TKI resistance. Our data indicate that, while nuclear β-catenin enhances survival of intrinsically TKI-resistant CML progenitors, it is not required for extrinsic resistance mediated by the BM microenvironment.

  20. Allosteric inhibition enhances the efficacy of ABL kinase inhibitors to target unmutated BCR-ABL and BCR-ABL-T315I

    Directory of Open Access Journals (Sweden)

    Mian Afsar

    2012-09-01

    Full Text Available Abstract Background Chronic myelogenous leukemia (CML and Philadelphia chromosome-positive (Ph+ acute lymphatic leukemia (Ph + ALL are caused by the t(9;22, which fuses BCR to ABL resulting in deregulated ABL-tyrosine kinase activity. The constitutively activated BCR/ABL-kinase “escapes” the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. The ABL-kinase inhibitors (AKIs Imatinib, Nilotinib or Dasatinib, which target the ATP-binding site, are effective in Ph + leukemia. Another molecular therapy approach targeting BCR/ABL restores allosteric inhibition. Given the fact that all AKIs fail to inhibit BCR/ABL harboring the ‘gatekeeper’ mutation T315I, we investigated the effects of AKIs in combination with the allosteric inhibitor GNF2 in Ph + leukemia. Methods The efficacy of this approach on the leukemogenic potential of BCR/ABL was studied in Ba/F3 cells, primary murine bone marrow cells, and untransformed Rat-1 fibroblasts expressing BCR/ABL or BCR/ABL-T315I as well as in patient-derived long-term cultures (PDLTC from Ph + ALL-patients. Results Here, we show that GNF-2 increased the effects of AKIs on unmutated BCR/ABL. Interestingly, the combination of Dasatinib and GNF-2 overcame resistance of BCR/ABL-T315I in all models used in a synergistic manner. Conclusions Our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants using a combination of AKIs and allosteric inhibitors.

  1. Differential effects of Rho-kinase inhibitor and angiotensin II type-1 receptor antagonist on the vascular function in hypertensive rats induced by chronic l-NAME treatment

    Directory of Open Access Journals (Sweden)

    Bainian Chen

    2012-10-01

    Full Text Available Little attention has been paid to the effect of Rho-kinase inhibitor on the vascular dysfunction of nitric oxide-deficient hypertension. We aimed to investigate whether the Rho-kinase inhibitor fasudil showed beneficial effect on the vascular dysfunction of the NG-nitro-l-arginine methyl ester (l-NAME treated rat, as well as to compare the differential effects of fasudil and angiotensin II receptor antagonist valsartan on vascular function. In the present study, both valsartan and fasudil exerted antihypertensive action on the l-NAME-treated rats, while only valsartan attenuated the cardiac hypertrophy. Treatment with valsartan showed improvement on vascular reactivity to norepinephrine, KCl and CaCl2, whereas fasudil therapy showed little effect on vasoconstriction. Endothelium-dependent vasodilation to acetylcholine was reduced in the NO-deficient group but was normalized by the fasudil therapy. The increased expression of RhoA and Rho-kinase (ROCK in the vasculature was corrected well to normal level by either valsartan or fasudil administration, which seemed to be at least partially responsible for the beneficial effect of the drug infusion. These findings suggest that the angiotensin II receptor antagonist interferes more with the contractile response than Rho-kinase inhibitor, whereas inhibition of Rho-kinase activity exhibits a better improvement on vasorelaxation than blockade of angiotensin II receptor.

  2. Mussel-inspired histidine-based transient network metal coordination hydrogels

    Science.gov (United States)

    Fullenkamp, Dominic E.; He, Lihong; Barrett, Devin G.; Burghardt, Wesley R.; Messersmith, Phillip B.

    2013-01-01

    Transient network hydrogels cross-linked through histidine-divalent cation coordination bonds were studied by conventional rheologic methods using histidine-modified star poly(ethylene glycol) (PEG) polymers. These materials were inspired by the mussel, which is thought to use histidine-metal coordination bonds to impart self-healing properties in the mussel byssal thread. Hydrogel viscoelastic mechanical properties were studied as a function of metal, pH, concentration, and ionic strength. The equilibrium metal-binding constants were determined by dilute solution potentiometric titration of monofunctional histidine-modified methoxy-PEG and were found to be consistent with binding constants of small molecule analogs previously studied. pH-dependent speciation curves were then calculated using the equilibrium constants determined by potentiometric titration, providing insight into the pH dependence of histidine-metal ion coordination and guiding the design of metal coordination hydrogels. Gel relaxation dynamics were found to be uncorrelated with the equilibrium constants measured, but were correlated to the expected coordination bond dissociation rate constants. PMID:23441102

  3. Group X hybrid histidine kinase Chk1 is dispensable for stress adaptation, host-pathogen interactions and virulence in the opportunistic yeast Candida guilliermondii.

    Science.gov (United States)

    Navarro-Arias, María J; Dementhon, Karine; Defosse, Tatiana A; Foureau, Emilien; Courdavault, Vincent; Clastre, Marc; Le Gal, Solène; Nevez, Gilles; Le Govic, Yohann; Bouchara, Jean-Philippe; Giglioli-Guivarc'h, Nathalie; Noël, Thierry; Mora-Montes, Hector M; Papon, Nicolas

    2017-09-01

    Hybrid histidine kinases (HHKs) progressively emerge as prominent sensing proteins in the fungal kingdom and as ideal targets for future therapeutics. The group X HHK is of major interest, since it was demonstrated to play an important role in stress adaptation, host-pathogen interactions and virulence in some yeast and mold models, and particularly Chk1, that corresponds to the sole group X HHK in Candida albicans. In the present work, we investigated the role of Chk1 in the low-virulence species Candida guilliermondii, in order to gain insight into putative conservation of the role of group X HHK in opportunistic yeasts. We demonstrated that disruption of the corresponding gene CHK1 does not influence growth, stress tolerance, drug susceptibility, protein glycosylation or cell wall composition in C. guilliermondii. In addition, we showed that loss of CHK1 does not affect C. guilliermondii ability to interact with macrophages and to stimulate cytokine production by human peripheral blood mononuclear cells. Finally, the C. guilliermondii chk1 null mutant was found to be as virulent as the wild-type strain in the experimental model Galleria mellonella. Taken together, our results demonstrate that group X HHK function is not conserved in Candida species. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  4. An allostatic mechanism for M2 pyruvate kinase as an amino-acid sensor.

    Science.gov (United States)

    Yuan, Meng; McNae, Iain W; Chen, Yiyuan; Blackburn, Elizabeth A; Wear, Martin A; Michels, Paul A M; Fothergill-Gilmore, Linda A; Hupp, Ted; Walkinshaw, Malcolm D

    2018-05-10

    We have tested the effect of all 20 proteinogenic amino acids on the activity of the M2 isoenzyme of pyruvate kinase (M2PYK) and show that within physiologically relevant concentrations, phenylalanine, alanine, tryptophan, methionine, valine, and proline act as inhibitors while histidine and serine act as activators. Size exclusion chromatography has been used to show that all amino acids, whether activators or inhibitors, stabilise the tetrameric form of M2PYK. In the absence of amino-acid ligands an apparent tetramer-monomer dissociation K d is estimated to be ~0.9 µM with a slow dissociation rate (t 1/2 ~ 15 min). X-ray structures of M2PYK complexes with alanine, phenylalanine, and tryptophan show the M2PYK locked in an inactive T-state conformation, while activators lock the M2PYK tetramer in the active R-state conformation. Amino-acid binding in the allosteric pocket triggers rigid body rotations (11°) stabilising either T or R-states. The opposing inhibitory and activating effects of the non-essential amino acids serine and alanine suggest that M2PYK could act as a rapid-response nutrient sensor to rebalance cellular metabolism. This competition at a single allosteric site between activators and inhibitors provides a novel regulatory mechanism by which M2PYK activity is finely tuned by the relative (but not absolute) concentrations of activator and inhibitor amino acids. Such 'allostatic' regulation may be important in metabolic reprogramming and influencing cell fate. ©2018 The Author(s).

  5. Receptor tyrosine kinase (c-Kit inhibitors: a potential therapeutic target in cancer cells

    Directory of Open Access Journals (Sweden)

    Abbaspour Babaei M

    2016-08-01

    Full Text Available Maryam Abbaspour Babaei,1 Behnam Kamalidehghan,2,3 Mohammad Saleem,4–6 Hasniza Zaman Huri,1,7 Fatemeh Ahmadipour1 1Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; 2Department of Medical Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB, Shahrak-e Pajoohesh, 3Medical Genetics Department, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran; 4Department of Urology, 5Department of Laboratory Medicine and Pathology, Masonic Cancer Center, University of Minnesota, 6Section of Molecular Therapeutics & Cancer Health Disparity, The Hormel Institute, Austin, MN, USA; 7Clinical Investigation Centre, University Malaya Medical Centre, Lembah Pantai, Kuala Lumpur, Malaysia Abstract: c-Kit, a receptor tyrosine kinase, is involved in intracellular signaling, and the mutated form of c-Kit plays a crucial role in occurrence of some cancers. The function of c-Kit has led to the concept that inhibiting c-Kit kinase activity can be a target for cancer therapy. The promising results of inhibition of c-Kit for treatment of cancers have been observed in some cancers such as gastrointestinal stromal tumor, acute myeloid leukemia, melanoma, and other tumors, and these results have encouraged attempts toward improvement of using c-Kit as a capable target for cancer therapy. This paper presents the findings of previous studies regarding c-Kit as a receptor tyrosine kinase and an oncogene, as well as its gene targets and signaling pathways in normal and cancer cells. The c-Kit gene location, protein structure, and the role of c-Kit in normal cell have been discussed. Comprehending the molecular mechanism underlying c-Kit-mediated tumorogenesis is consequently essential and may lead to the identification of future novel drug targets. The potential mechanisms by which c-Kit induces cellular transformation have been described. This study aims to elucidate the function of c

  6. Cyclooxygenase inhibitors potentiate receptor tyrosine kinase therapies in bladder cancer cells in vitro

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    Bourn J

    2018-06-01

    Full Text Available Jennifer Bourn,1,2 Maria Cekanova1,2 1Department of Small Animal Clinical Sciences, College of Veterinary Medicine, The University of Tennessee, Knoxville, TN, USA; 2UT-ORNL Graduate School of Genome Science and Technology, The University of Tennessee, Knoxville, TN, USA Purpose: Receptor tyrosine kinase inhibitors (RTKIs are used as targeted therapies for patients diagnosed with cancer with highly expressed receptor tyrosine kinases (RTKs, including the platelet-derived growth factor receptor (PDGFR and c-Kit receptor. Resistance to targeted therapies is partially due to the activation of alternative pro-survival signaling pathways, including cyclooxygenase (COX-2. In this study, we validated the effects of two RTKIs, axitinib and AB1010, in combination with COX inhibitors on the V-akt murine thymoma oncogene homolog 1 (Akt and COX-2 signaling pathways in bladder cancer cells.Methods: The expression of several RTKs and their downstream signaling targets was analyzed by Western blot (WB analysis in human and canine bladder transitional cell carcinoma (TCC cell lines. The effects of RTKIs and COX inhibitors in bladder TCC cells were assessed by MTS for cell viability, by Caspase-3/7 and Annexin V assay for apoptosis, by WB analysis for detection of COX-2 and Akt signaling pathways, and by enzyme-linked immunosorbent assay for detection of prostaglandin E2 (PGE2 levels.Results: All tested TCC cells expressed the c-Kit and PDGFRα receptors, except human 5637 cells that had low RTKs expression. In addition, all tested cells expressed COX-1, COX-2, Akt, extracellular signal regulated kinases 1/2, and nuclear factor kappa-light-chain-enhance of activated B cells proteins, except human UM-UC-3 cells, where no COX-2 expression was detected by WB analysis. Both RTKIs inhibited cell viability and increased apoptosis in a dose-dependent manner in tested bladder TCC cells, which positively correlated with their expression levels of the PDGFRα and c

  7. New therapeutic modality for corneal endothelial disease using Rho-associated kinase inhibitor eye drops.

    Science.gov (United States)

    Koizumi, Noriko; Okumura, Naoki; Ueno, Morio; Kinoshita, Shigeru

    2014-11-01

    Corneal endothelial dysfunction accompanied by visual disturbance is a primary indication for corneal endothelial transplantation. However, despite the value and potential of endothelial graft surgery, a strictly pharmacological approach for treating corneal endothelial dysfunction remains an attractive proposition. Previously, we reported that the selective Rho-associated kinase (ROCK) inhibitor Y-27632 promotes cell adhesion and proliferation, and inhibits the apoptosis of primate corneal endothelial cells in culture. These findings have led us to develop a novel medical treatment for the early phase of corneal endothelial disease using ROCK inhibitor eye drops. In rabbit and monkey models of partial endothelial dysfunction, we showed that corneal endothelial wound healing was accelerated via the topical application of ROCK inhibitor to the ocular surface, resulting in the regeneration of a corneal endothelial monolayer with a high endothelial cell density. Based on these animal studies, we are now attempting to advance the clinical application of ROCK inhibitor eye drops for patients with corneal endothelial dysfunction. A pilot clinical study was performed at the Kyoto Prefectural University of Medicine, and the effects of Y-27632 eye drops after transcorneal freezing were evaluated in 8 patients with corneal endothelial dysfunction. We observed a positive effect of ROCK inhibitor eye drops in treating patients with central edema caused by Fuchs corneal endothelial dystrophy. We believe that our new findings will contribute to the establishment of a new approach for the treatment of corneal endothelial dysfunction.

  8. ESCMID Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biological therapies: an infectious diseases perspective (Intracellular signaling pathways: tyrosine kinase and mTOR inhibitors).

    Science.gov (United States)

    Reinwald, M; Silva, J T; Mueller, N J; Fortún, J; Garzoni, C; de Fijter, J W; Fernández-Ruiz, M; Grossi, P; Aguado, J M

    2018-06-01

    The present review is part of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biologic therapies. To review, from an infectious diseases perspective, the safety profile of therapies targeting different intracellular signaling pathways and to suggest preventive recommendations. Computer-based Medline searches with MeSH terms pertaining to each agent or therapeutic family. Although BCR-ABL tyrosine kinase inhibitors modestly increase the overall risk of infection, dasatinib has been associated with cytomegalovirus and hepatitis B virus reactivation. BRAF/MEK kinase inhibitors do not significantly affect infection susceptibility. The effect of Bruton tyrosine kinase inhibitors (ibrutinib) among patients with B-cell malignancies is difficult to distinguish from that of previous immunosuppression. However, cases of Pneumocystis jirovecii pneumonia (PCP), invasive fungal infection and progressive multifocal leukoencephalopathy have been occasionally reported. Because phosphatidylinositol-3-kinase inhibitors (idelalisib) may predispose to opportunistic infections, anti-Pneumocystis prophylaxis and prevention strategies for cytomegalovirus are recommended. No increased rates of infection have been observed with venetoclax (antiapoptotic protein Bcl-2 inhibitor). Therapy with Janus kinase inhibitors markedly increases the incidence of infection. Pretreatment screening for chronic hepatitis B virus and latent tuberculosis infection must be performed, and anti-Pneumocystis prophylaxis should be considered for patients with additional risk factors. Cancer patients receiving mTOR inhibitors face an increased incidence of overall infection, especially those with additional risk factors (prior therapies or delayed wound healing). Specific preventive approaches are warranted in view of the increased risk of infection associated with some of the

  9. Protective effect of the poly(ADP-ribose polymerase inhibitor PJ34 on mitochondrial depolarization-mediated cell death in hepatocellular carcinoma cells involves attenuation of c-Jun N-terminal kinase-2 and protein kinase B/Akt activation

    Directory of Open Access Journals (Sweden)

    Radnai Balazs

    2012-05-01

    Full Text Available Abstract Background 2,4-Dimethoxyphenyl-E-4-arylidene-3-isochromanone (IK11 was previously described to induce apoptotic death of A431 tumor cells. In this report, we investigated the molecular action of IK11 in the HepG2 human hepatocellular carcinoma cell line to increase our knowledge of the role of poly (ADP-ribose-polymerase (PARP, protein kinase B/Akt and mitogen activated protein kinase (MAPK activation in the survival and death of tumor cells and to highlight the possible role of PARP-inhibitors in co-treatments with different cytotoxic agents in cancer therapy. Results We found that sublethal concentrations of IK11 prevented proliferation, migration and entry of the cells into their G2 phase. At higher concentrations, IK11 induced reactive oxygen species (ROS production, mitochondrial membrane depolarization, activation of c-Jun N-terminal kinase 2 (JNK2, and substantial loss of HepG2 cells. ROS production appeared marginal in mediating the cytotoxicity of IK11 since N-acetyl cysteine was unable to prevent it. However, the PARP inhibitor PJ34, although not a ROS scavenger, strongly inhibited both IK11-induced ROS production and cell death. JNK2 activation seemed to be a major mediator of the effect of IK11 since inhibition of JNK resulted in a substantial cytoprotection while inhibitors of the other kinases failed to do so. Inhibition of Akt slightly diminished the effect of IK11, while the JNK and Akt inhibitor and ROS scavenger trans-resveratrol completely protected against it. Conclusions These results indicate significant involvement of PARP, a marginal role of ROS and a pro-apoptotic role of Akt in this system, and raise attention to a novel mechanism that should be considered when cancer therapy is augmented with PARP-inhibition, namely the cytoprotection by inhibition of JNK2.

  10. Differential Effects of Phosphatidylinositol 4-Kinase (PI4K and 3-Kinase (PI3K Inhibitors on Stomatal Responses to Environmental Signals

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    Koh Iba

    2017-05-01

    Full Text Available Specific cellular components including products of phosphatidylinositol (PI metabolism play an important role as signaling molecules in stomatal responses to environmental signals. In this study, pharmacological inhibitors of a set of cellular components, including PI4-kinase (PI4K and PI3K, were used to investigate stomatal closure in response to CO2, darkness, and abscisic acid (ABA. Treatment with PAO, a specific inhibitor of PI4K, specifically inhibited the stomatal response to CO2 compared with that to darkness and ABA. In contrast, treatment with LY294002, a PI3K-specific inhibitor, specifically inhibited the stomatal response to darkness compared with that to CO2 and ABA. The specific inhibitory effects of PAO and LY294002 were also observed as changes in the spatial density of dot-like structures labeled by green fluorescent protein-tagged PATROL1, a protein that controls stomatal aperture possibly via regulation of H+-ATPase amount in guard cell plasma membranes. Our results suggest an important role for PI4K and PI3K in the CO2 and darkness signal transduction pathways, respectively, that mediate PATROL1 dynamics.

  11. EXPERIMENT ON EFFECTS OF LOE-PROTEIN DIET SUPPLEMENTED WITH α-KETOACIDS ON HYPERTROPHY OF DIABETIC GLOMERULUS AND ITS RELATIONSHIP WITH THE LEVEL OF CYCLIN KINASE INHIBITOR P27

    OpenAIRE

    Zhou, Yi; Yuan, Weijie; Dong, Ting; Wang, Ling

    2012-01-01

    Low-protein diet supplemented with α-keto acids was reported to have renoprotective roles in diabetic nephropathy via inhibiting glomerular hypertrophy, however, the mechanism has not yet been fully clarified. the cyclin kinase inhibitor p27 play an important role in hypertrophy of diabetic glomerulus, The objective of the present study was to investigate the relationship between the cyclin kinase inhibitor p27 and the effect of low-protein diet supplemented with α-keto acids on hypertrophy o...

  12. Oral protein kinase c β inhibition using ruboxistaurin

    DEFF Research Database (Denmark)

    Aiello, Lloyd Paul; Vignati, Louis; Sheetz, Matthew J

    2011-01-01

    To evaluate efficacy, safety, and causes of vision loss among 813 patients (1,392 eyes) with moderately severe to very severe nonproliferative diabetic retinopathy from the Protein Kinase C β Inhibitor-Diabetic Retinopathy Study and Protein Kinase C β Inhibitor-Diabetic Retinopathy Study 2 ruboxi...

  13. Rho-kinase inhibitor and nicotinamide adenine dinucleotide phosphate oxidase inhibitor prevent impairment of endothelium-dependent cerebral vasodilation by acute cigarette smoking in rats.

    Science.gov (United States)

    Iida, Hiroki; Iida, Mami; Takenaka, Motoyasu; Fukuoka, Naokazu; Dohi, Shuji

    2008-06-01

    We previously reported that acute cigarette smoking can cause a dysfunction of endothelium-dependent vasodilation in cerebral vessels, and that blocking the angiotensin II (Ang II) type 1 (AT1) receptor with valsartan prevented this impairment. Our aim was to investigate the effects of a Rho-kinase inhibitor (fasudil) and a Nicotinamide Adenine Dinucleotide PHosphate (NADPH) oxidase inhibitor (apocynin) on smoking-induced endothelial dysfunction in cerebral arterioles. In Sprague-Dawley rats, we used a closed cranial window preparation to measure changes in pial vessel diameters following topical acetylcholine (ACh) before smoking. After one-minute smoking, we again examined the arteriolar responses to ACh. Finally, after intravenous fasudil or apocynin pre-treatment we re-examined the vasodilator responses to topical ACh (before and after cigarette smoking). Under control conditions, cerebral arterioles were dose-dependently dilated by topical ACh (10(-6) M and 10(-5) M). One hour after a one-minute smoking (1 mg-nicotine cigarette), 10(-5) M ACh constricted cerebral arterioles. However, one hour after a one-minute smoking, 10(-5) M ACh dilated cerebral pial arteries both in the fasudil pre-treatment and the apocynin pre-treatment groups, responses that were significantly different from those obtained without fasudil or apocynin pre-treatment. Thus, inhibition of Rho-kinase and NADPH oxidase activities may prevent the above smoking-induced impairment of endothelium-dependent vasodilation.

  14. Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase

    DEFF Research Database (Denmark)

    Biondi, R M; Walz, K; Issinger, O G

    1996-01-01

    of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe...

  15. Safety and efficacy of the bumped kinase inhibitor BKI-1553 in pregnant sheep experimentally infected with Neospora caninum tachyzoites

    Directory of Open Access Journals (Sweden)

    Roberto Sánchez-Sánchez

    2018-04-01

    Full Text Available Neospora caninum is one of the main causes of abortion in cattle, and recent studies have highlighted its relevance as an abortifacient in small ruminants. Vaccines or drugs for the control of neosporosis are lacking. Bumped kinase inhibitors (BKIs, which are ATP-competitive inhibitors of calcium dependent protein kinase 1 (CDPK1, were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. We here present the pharmacokinetics, safety and efficacy of BKI-1553 in pregnant ewes and foetuses using a pregnant sheep model of N. caninum infection. BKI-1553 showed exposure in pregnant ewes with trough concentrations of approximately 4 μM, and of 1  μM in foetuses. Subcutaneous BKI-1553 administration increased rectal temperatures shortly after treatment, and resulted in dermal nodules triggering a slight monocytosis after repeated doses at short intervals. BKI-1553 treatment decreased fever in infected pregnant ewes already after two applications, resulted in a 37–50% reduction in foetal mortality, and modulated immune responses; IFNγ levels were increased early after infection and IgG levels were reduced subsequently. N. caninum was abundantly found in placental tissues; however, parasite detection in foetal brain tissue decreased from 94% in the infected/untreated group to 69–71% in the treated groups. In summary, BKI-1553 confers partial protection against abortion in a ruminant experimental model of N. caninum infection during pregnancy. In addition, reduced parasite detection, parasite load and lesions in foetal brains were observed. Keywords: Neospora caninum, Sheep, Pregnancy, Treatment, Protein kinase inhibitor, BKI-1553

  16. Immunological changes with kinase inhibitor therapy for chronic lymphocytic leukemia.

    Science.gov (United States)

    Pleyer, Christopher; Wiestner, Adrian; Sun, Clare

    2018-05-15

    Ibrutinib and idelalisib are kinase inhibitors that have revolutionized the treatment of chronic lymphocytic leukemia (CLL). Capable of inducing durable remissions, these agents also modulate the immune system. Both ibrutinib and idelalisib abrogate the tumor-supporting microenvironment by disrupting cell-cell interactions, modulating the T-cell compartment, and altering the cytokine milieu. Ibrutinib also partially restores T-cell and myeloid defects associated with CLL. In contrast, immune-related adverse effects, including pneumonitis, colitis, hepatotoxicity, and infections are of particular concern with idelalisib. While opportunistic infections and viral reactivations occur with both ibrutinib and idelalisib, these complications are less common and less severe with ibrutinib, especially when used as monotherapy without additional immunosuppressive agents. This review discusses the impact of ibrutinib and idelalisib on the immune system, including infectious and auto-immune complications as well as their specific effects on the B-cell, T-cell, and myeloid compartment.

  17. Tumor-targeted Nanobullets: Anti-EGFR nanobody-liposomes loaded with anti-IGF-1R kinase inhibitor for cancer treatment.

    Science.gov (United States)

    van der Meel, Roy; Oliveira, Sabrina; Altintas, Isil; Haselberg, Rob; van der Veeken, Joris; Roovers, Rob C; van Bergen en Henegouwen, Paul M P; Storm, Gert; Hennink, Wim E; Schiffelers, Raymond M; Kok, Robbert J

    2012-04-30

    The epidermal growth factor receptor (EGFR) is a validated target for anti-cancer therapy and several EGFR inhibitors are used in the clinic. Over the years, an increasing number of studies have reported on the crosstalk between EGFR and other receptors that can contribute to accelerated cancer development or even acquisition of resistance to anti-EGFR therapies. Combined targeting of EGFR and insulin-like growth factor 1 receptor (IGF-1R) is a rational strategy to potentiate anti-cancer treatment and possibly retard resistance development. In the present study, we have pursued this by encapsulating the kinase inhibitor AG538 in anti-EGFR nanobody-liposomes. The thus developed dual-active nanobody-liposomes associated with EGFR-(over)expressing cells in an EGFR-specific manner and blocked both EGFR and IGF-1R activation, due to the presence of the EGFR-blocking nanobody EGa1 and the anti-IGF-1R kinase inhibitor AG538 respectively. AG538-loaded nanobody-liposomes induced a strong inhibition of tumor cell proliferation even upon short-term exposure followed by a drug-free wash-out period. Therefore, AG538-loaded nanobody-liposomes are a promising anti-cancer formulation due to efficient intracellular delivery of AG538 in combination with antagonistic and downregulating properties of the EGa1 nanobody-liposomes. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Salt effects on ionization equilibria of histidines in myoglobin.

    Science.gov (United States)

    Kao, Y H; Fitch, C A; Bhattacharya, S; Sarkisian, C J; Lecomte, J T; García-Moreno E, B

    2000-09-01

    The salt dependence of histidine pK(a) values in sperm whale and horse myoglobin and in histidine-containing peptides was measured by (1)H-NMR spectroscopy. Structure-based pK(a) calculations were performed with continuum methods to test their ability to capture the effects of solution conditions on pK(a) values. The measured pK(a) of most histidines, whether in the protein or in model compounds, increased by 0.3 pH units or more between 0.02 M and 1.5 M NaCl. In myoglobin two histidines (His(48) and His(36)) exhibited a shallower dependence than the average, and one (His(113)) showed a steeper dependence. The (1)H-NMR data suggested that the salt dependence of histidine pK(a) values in the protein was determined primarily by the preferential stabilization of the charged form of histidine with increasing salt concentrations rather than by screening of electrostatic interactions. The magnitude and salt dependence of interactions between ionizable groups were exaggerated in pK(a) calculations with the finite-difference Poisson-Boltzmann method applied to a static structure, even when the protein interior was treated with arbitrarily high dielectric constants. Improvements in continuum methods for calculating salt effects on pK(a) values will require explicit consideration of the salt dependence of model compound pK(a) values used for reference in the calculations.

  19. Alignment independent 3D-QSAR, quantum calculations and molecular docking of Mer specific tyrosine kinase inhibitors as anticancer drugs

    Directory of Open Access Journals (Sweden)

    Fereshteh Shiri

    2016-03-01

    Full Text Available Mer receptor tyrosine kinase is a promising novel cancer therapeutic target in many human cancers, because abnormal activation of Mer has been implicated in survival signaling and chemoresistance. 3D-QSAR analyses based on alignment independent descriptors were performed on a series of 81 Mer specific tyrosine kinase inhibitors. The fractional factorial design (FFD and the enhanced replacement method (ERM were applied and tested as variable selection algorithms for the selection of optimal subsets of molecular descriptors from a much greater pool of such regression variables. The data set was split into 65 molecules as the training set and 16 compounds as the test set. All descriptors were generated by using the GRid INdependent descriptors (GRIND approach. After variable selection, GRIND were correlated with activity values (pIC50 by PLS regression. Of the two applied variable selection methods, ERM had a noticeable improvement on the statistical parameters of PLS model, and yielded a q2 value of 0.77, an rpred2 of 0.94, and a low RMSEP value of 0.25. The GRIND information contents influencing the affinity on Mer specific tyrosine kinase were also confirmed by docking studies. In a quantum calculation study, the energy difference between HOMO and LUMO (gap implied the high interaction of the most active molecule in the active site of the protein. In addition, the molecular electrostatic potential energy at DFT level confirmed results obtained from the molecular docking. The identified key features obtained from the molecular modeling, enabled us to design novel kinase inhibitors.

  20. Alignment independent 3D-QSAR, quantum calculations and molecular docking of Mer specific tyrosine kinase inhibitors as anticancer drugs.

    Science.gov (United States)

    Shiri, Fereshteh; Pirhadi, Somayeh; Ghasemi, Jahan B

    2016-03-01

    Mer receptor tyrosine kinase is a promising novel cancer therapeutic target in many human cancers, because abnormal activation of Mer has been implicated in survival signaling and chemoresistance. 3D-QSAR analyses based on alignment independent descriptors were performed on a series of 81 Mer specific tyrosine kinase inhibitors. The fractional factorial design (FFD) and the enhanced replacement method (ERM) were applied and tested as variable selection algorithms for the selection of optimal subsets of molecular descriptors from a much greater pool of such regression variables. The data set was split into 65 molecules as the training set and 16 compounds as the test set. All descriptors were generated by using the GRid INdependent descriptors (GRIND) approach. After variable selection, GRIND were correlated with activity values (pIC50) by PLS regression. Of the two applied variable selection methods, ERM had a noticeable improvement on the statistical parameters of PLS model, and yielded a q (2) value of 0.77, an [Formula: see text] of 0.94, and a low RMSEP value of 0.25. The GRIND information contents influencing the affinity on Mer specific tyrosine kinase were also confirmed by docking studies. In a quantum calculation study, the energy difference between HOMO and LUMO (gap) implied the high interaction of the most active molecule in the active site of the protein. In addition, the molecular electrostatic potential energy at DFT level confirmed results obtained from the molecular docking. The identified key features obtained from the molecular modeling, enabled us to design novel kinase inhibitors.

  1. Identification of a novel multiple kinase inhibitor with potent antiviral activity against influenza virus by reducing viral polymerase activity

    Energy Technology Data Exchange (ETDEWEB)

    Sasaki, Yutaka; Kakisaka, Michinori; Chutiwitoonchai, Nopporn [Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); Tajima, Shigeru [Department of Virology I, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640 (Japan); Hikono, Hirokazu; Saito, Takehiko [Influenza and Prion Disease Research Center, National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856 (Japan); Aida, Yoko, E-mail: aida@riken.jp [Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan)

    2014-07-18

    Highlights: • Screening of 50,000 compounds and subsequent lead optimization identified WV970. • WV970 has antiviral effects against influenza A, B and highly pathogenic viral strains. • WV970 inhibits viral genome replication and transcription. • A target database search suggests that WV970 may bind to a number of kinases. • KINOMEscan screening revealed that WV970 has inhibitory effects on 15 kinases. - Abstract: Neuraminidase inhibitors are the only currently available influenza treatment, although resistant viruses to these drugs have already been reported. Thus, new antiviral drugs with novel mechanisms of action are urgently required. In this study, we identified a novel antiviral compound, WV970, through cell-based screening of a 50,000 compound library and subsequent lead optimization. This compound exhibited potent antiviral activity with nanomolar IC{sub 50} values against both influenza A and B viruses but not non-influenza RNA viruses. Time-of-addition and indirect immunofluorescence assays indicated that WV970 acted at an early stage of the influenza life cycle, but likely after nuclear entry of viral ribonucleoprotein (vRNP). Further analyses of viral RNA expression and viral polymerase activity indicated that WV970 inhibited vRNP-mediated viral genome replication and transcription. Finally, structure-based virtual screening and comprehensive human kinome screening were used to demonstrate that WV970 acts as a multiple kinase inhibitor, many of which are associated with influenza virus replication. Collectively, these results strongly suggest that WV970 is a promising anti-influenza drug candidate and that several kinases associated with viral replication are promising drug targets.

  2. Identification of a novel multiple kinase inhibitor with potent antiviral activity against influenza virus by reducing viral polymerase activity

    International Nuclear Information System (INIS)

    Sasaki, Yutaka; Kakisaka, Michinori; Chutiwitoonchai, Nopporn; Tajima, Shigeru; Hikono, Hirokazu; Saito, Takehiko; Aida, Yoko

    2014-01-01

    Highlights: • Screening of 50,000 compounds and subsequent lead optimization identified WV970. • WV970 has antiviral effects against influenza A, B and highly pathogenic viral strains. • WV970 inhibits viral genome replication and transcription. • A target database search suggests that WV970 may bind to a number of kinases. • KINOMEscan screening revealed that WV970 has inhibitory effects on 15 kinases. - Abstract: Neuraminidase inhibitors are the only currently available influenza treatment, although resistant viruses to these drugs have already been reported. Thus, new antiviral drugs with novel mechanisms of action are urgently required. In this study, we identified a novel antiviral compound, WV970, through cell-based screening of a 50,000 compound library and subsequent lead optimization. This compound exhibited potent antiviral activity with nanomolar IC 50 values against both influenza A and B viruses but not non-influenza RNA viruses. Time-of-addition and indirect immunofluorescence assays indicated that WV970 acted at an early stage of the influenza life cycle, but likely after nuclear entry of viral ribonucleoprotein (vRNP). Further analyses of viral RNA expression and viral polymerase activity indicated that WV970 inhibited vRNP-mediated viral genome replication and transcription. Finally, structure-based virtual screening and comprehensive human kinome screening were used to demonstrate that WV970 acts as a multiple kinase inhibitor, many of which are associated with influenza virus replication. Collectively, these results strongly suggest that WV970 is a promising anti-influenza drug candidate and that several kinases associated with viral replication are promising drug targets

  3. Exploiting polypharmacology for improving therapeutic outcome of kinase inhibitors (KIs): An update of recent medicinal chemistry efforts.

    Science.gov (United States)

    Ma, Xiaodong; Lv, Xiaoqing; Zhang, Jiankang

    2018-01-01

    Polypharmacology has been increasingly advocated for the therapeutic intervention in complex pathological conditions, exemplified by cancer. Although kinase inhibitors (KIs) have revolutionized the treatment for certain types of malignancies, some major medical needs remain unmet due to the relentless advance of drug resistance and insufficient efficacy of mono-target KIs. Hence, "multiple targets, multi-dimensional activities" represents an emerging paradigm for innovative anti-cancer drug discovery. Over recent years, considerable leaps have been made in pursuit of kinase-centric polypharmacological anti-cancer therapeutics, providing avenues to tackling the limitation of mono-target KIs. In the review, we summarize the clinically important mechanisms inducing KI resistance and depict a landscape of recent medicinal chemistry efforts on exploring kinase-centric polypharmacological anti-cancer agents that targeting multiple cancer-related processes. In parallel, some inevitable challenges are emphasized for the sake of more accurate and efficient drug discovery in the field. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  4. Molecular modeling-driven approach for identification of Janus kinase 1 inhibitors through 3D-QSAR, docking and molecular dynamics simulations.

    Science.gov (United States)

    Itteboina, Ramesh; Ballu, Srilata; Sivan, Sree Kanth; Manga, Vijjulatha

    2017-10-01

    Janus kinase 1 (JAK 1) belongs to the JAK family of intracellular nonreceptor tyrosine kinase. JAK-signal transducer and activator of transcription (JAK-STAT) pathway mediate signaling by cytokines, which control survival, proliferation and differentiation of a variety of cells. Three-dimensional quantitative structure activity relationship (3 D-QSAR), molecular docking and molecular dynamics (MD) methods was carried out on a dataset of Janus kinase 1(JAK 1) inhibitors. Ligands were constructed and docked into the active site of protein using GLIDE 5.6. Best docked poses were selected after analysis for further 3 D-QSAR analysis using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methodology. Employing 60 molecules in the training set, 3 D-QSAR models were generate that showed good statistical reliability, which is clearly observed in terms of r 2 ncv and q 2 loo values. The predictive ability of these models was determined using a test set of 25 molecules that gave acceptable predictive correlation (r 2 Pred ) values. The key amino acid residues were identified by means of molecular docking, and the stability and rationality of the derived molecular conformations were also validated by MD simulation. The good consonance between the docking results and CoMFA/CoMSIA contour maps provides helpful clues about the reasonable modification of molecules in order to design more efficient JAK 1 inhibitors. The developed models are expected to provide some directives for further synthesis of highly effective JAK 1 inhibitors.

  5. Autoregulation of kinase dephosphorylation by ATP binding in AGC protein kinases.

    Science.gov (United States)

    Chan, Tung O; Pascal, John M; Armen, Roger S; Rodeck, Ulrich

    2012-02-01

    AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non- ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.

  6. Structural basis of Zn(II induced metal detoxification and antibiotic resistance by histidine kinase CzcS in Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Dan Wang

    2017-07-01

    Full Text Available Pseudomonas aeruginosa (P. aeruginosa is a major opportunistic human pathogen, causing serious nosocomial infections among immunocompromised patients by multi-determinant virulence and high antibiotic resistance. The CzcR-CzcS signal transduction system in P. aeruginosa is primarily involved in metal detoxification and antibiotic resistance through co-regulating cross-resistance between Zn(II and carbapenem antibiotics. Although the intracellular regulatory pathway is well-established, the mechanism by which extracellular sensor domain of histidine kinase (HK CzcS responds to Zn(II stimulus to trigger downstream signal transduction remains unclear. Here we determined the crystal structure of the CzcS sensor domain (CzcS SD in complex with Zn(II at 1.7 Å resolution. This is the first three-dimensional structural view of Zn(II-sensor domain of the two-component system (TCS. The CzcS SD is of α/β-fold in nature, and it senses the Zn(II stimulus at micromole level in a tetrahedral geometry through its symmetry-related residues (His55 and Asp60 on the dimer interface. Though the CzcS SD resembles the PhoQ-DcuS-CitA (PDC superfamily member, it interacts with the effector in a novel domain with the N-terminal α-helices rather than the conserved β-sheets pocket. The dimerization of the N-terminal H1 and H1' α-helices is of primary importance for the activity of HK CzcS. This study provides preliminary insight into the molecular mechanism of Zn(II sensing and signaling transduction by the HK CzcS, which will be beneficial to understand how the pathogen P. aeruginosa resists to high levels of heavy metals and antimicrobial agents.

  7. pH-dependent structural change of the extracellular sensor domain of the DraK histidine kinase from Streptomyces coelicolor

    International Nuclear Information System (INIS)

    Yeo, Kwon Joo; Kim, Eun Hye; Hwang, Eunha; Han, Young-Hyun; Eo, Yumi; Kim, Hyun Jung; Kwon, Ohsuk; Hong, Young-Soo; Cheong, Chaejoon; Cheong, Hae-Kap

    2013-01-01

    Highlights: ► We described the biochemical and biophysical properties of the extracellular sensory domain (ESD) of DraK histidine kinase. ► The ESD of DraK showed a reversible pH-dependent conformational change in a wide pH range. ► The E83 is an important residue for the pH-dependent conformational change. -- Abstract: Recently, the DraR/DraK (Sco3063/Sco3062) two-component system (TCS) of Streptomycescoelicolor has been reported to be involved in the differential regulation of antibiotic biosynthesis. However, it has not been shown that under which conditions and how the DraR/DraK TCS is activated to initiate the signal transduction process. Therefore, to understand the sensing mechanism, structural study of the sensory domain of DraK is highly required. Here, we report the biochemical and biophysical properties of the extracellular sensory domain (ESD) of DraK. We observed a reversible pH-dependent conformational change of the ESD in a pH range of 2.5–10. Size-exclusion chromatography and AUC (analytical ultracentrifugation) data indicated that the ESD is predominantly monomeric in solution and exists in equilibrium between monomer and dimer states in acidic condition. Using NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy, our findings suggest that the structure of the ESD at low pH is more structured than that at high pH. In particular, the glutamate at position 83 is an important residue for the pH-dependent conformational change. These results suggest that this pH-dependent conformational change of ESD may be involved in signal transduction process of DraR/DraK TCS

  8. pH-dependent structural change of the extracellular sensor domain of the DraK histidine kinase from Streptomyces coelicolor

    Energy Technology Data Exchange (ETDEWEB)

    Yeo, Kwon Joo [Division of Magnetic Resonance, Korea Basic Science Institute (KBSI), 16 Yeongudanji-Ro, Ochang, Chungbuk 363-883 (Korea, Republic of); Kim, Eun Hye [Systems and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-Ro, Yuseong-Gu, Daejeon 305-333 (Korea, Republic of); Hwang, Eunha; Han, Young-Hyun; Eo, Yumi; Kim, Hyun Jung [Division of Magnetic Resonance, Korea Basic Science Institute (KBSI), 16 Yeongudanji-Ro, Ochang, Chungbuk 363-883 (Korea, Republic of); Kwon, Ohsuk [Systems and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-Ro, Yuseong-Gu, Daejeon 305-333 (Korea, Republic of); Hong, Young-Soo [Chemical Biology Research Center, KRIBB, 30 Yeongudanji-Ro, Ochang, Chungbuk 363-883 (Korea, Republic of); Cheong, Chaejoon, E-mail: cheong@kbsi.re.kr [Division of Magnetic Resonance, Korea Basic Science Institute (KBSI), 16 Yeongudanji-Ro, Ochang, Chungbuk 363-883 (Korea, Republic of); Cheong, Hae-Kap, E-mail: haekap@kbsi.re.kr [Division of Magnetic Resonance, Korea Basic Science Institute (KBSI), 16 Yeongudanji-Ro, Ochang, Chungbuk 363-883 (Korea, Republic of)

    2013-02-15

    Highlights: ► We described the biochemical and biophysical properties of the extracellular sensory domain (ESD) of DraK histidine kinase. ► The ESD of DraK showed a reversible pH-dependent conformational change in a wide pH range. ► The E83 is an important residue for the pH-dependent conformational change. -- Abstract: Recently, the DraR/DraK (Sco3063/Sco3062) two-component system (TCS) of Streptomycescoelicolor has been reported to be involved in the differential regulation of antibiotic biosynthesis. However, it has not been shown that under which conditions and how the DraR/DraK TCS is activated to initiate the signal transduction process. Therefore, to understand the sensing mechanism, structural study of the sensory domain of DraK is highly required. Here, we report the biochemical and biophysical properties of the extracellular sensory domain (ESD) of DraK. We observed a reversible pH-dependent conformational change of the ESD in a pH range of 2.5–10. Size-exclusion chromatography and AUC (analytical ultracentrifugation) data indicated that the ESD is predominantly monomeric in solution and exists in equilibrium between monomer and dimer states in acidic condition. Using NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy, our findings suggest that the structure of the ESD at low pH is more structured than that at high pH. In particular, the glutamate at position 83 is an important residue for the pH-dependent conformational change. These results suggest that this pH-dependent conformational change of ESD may be involved in signal transduction process of DraR/DraK TCS.

  9. Patients harboring EGFR mutation after primary resistance to crizotinib and response to EGFR-tyrosine kinase inhibitor

    Directory of Open Access Journals (Sweden)

    Wang WX

    2016-01-01

    Full Text Available Wenxian Wang,1 Xiaowen Jiang,1 Zhengbo Song,1,2 Yiping Zhang1,2 1Department of Chemotherapy, Zhejiang Cancer Hospital, 2Key Laboratory Diagnosis and Treatment Technology on Thoracic Oncology, Hangzhou, Zhejiang, People’s Republic of China Abstract: Anaplastic lymphoma kinase (ALK rearrangement lung cancer responds to ALK tyrosine kinase inhibitors. It is known that many cases ultimately acquired resistance to crizotinib. However, a case of primary resistance is rare. We present a case of harboring exon 19 deletion in epidermal growth factor receptor in ALK rearranged lung adenocarcinoma, who experienced a partial tumor response to icotinib after failure with crizotinib therapy and chemotherapy. Considering the partial response, we conclude that it is important to find the cause of resistance to crizotinib. We detected gene mutations with plasma by the next-generation sequencing; the next-generation sequencing demonstrates an attractive system to identify mutations improving the outcome of patients with a deadly disease. Keywords: non-small cell lung cancer, anaplastic lymphoma kinase, crizotinib, epidermal growth factor receptor

  10. Furan-2-ylmethylene thiazolidinediones as novel, potent, and selective inhibitors of phosphoinositide 3-kinase gamma.

    Science.gov (United States)

    Pomel, Vincent; Klicic, Jasna; Covini, David; Church, Dennis D; Shaw, Jeffrey P; Roulin, Karen; Burgat-Charvillon, Fabienne; Valognes, Delphine; Camps, Montserrat; Chabert, Christian; Gillieron, Corinne; Françon, Bernard; Perrin, Dominique; Leroy, Didier; Gretener, Denise; Nichols, Anthony; Vitte, Pierre Alain; Carboni, Susanna; Rommel, Christian; Schwarz, Matthias K; Rückle, Thomas

    2006-06-29

    Class I phosphoinositide 3-kinases (PI3Ks), in particular PI3Kgamma, have become attractive drug targets for inflammatory and autoimmune diseases. Here, we disclose a novel series of furan-2-ylmethylene thiazolidinediones as selective, ATP-competitive PI3Kgamma inhibitors. Structure-based design and X-ray crystallography of complexes formed by inhibitors bound to PI3Kgamma identified key pharmacophore features for potency and selectivity. An acidic NH group on the thiazolidinedione moiety and a hydroxy group on the furan-2-yl-phenyl part of the molecule play crucial roles in binding to PI3K and contribute to class IB PI3K selectivity. Compound 26 (AS-252424), a potent and selective small-molecule PI3Kgamma inhibitor emerging from these efforts, was further profiled in three different cellular PI3K assays and shown to be selective for class IB PI3K-mediated cellular effects. Oral administration of 26 in a mouse model of acute peritonitis led to a significant reduction of leukocyte recruitment.

  11. PKI-179: an orally efficacious dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor.

    Science.gov (United States)

    Venkatesan, Aranapakam M; Chen, Zecheng; dos Santos, Osvaldo; Dehnhardt, Christoph; Santos, Efren Delos; Ayral-Kaloustian, Semiramis; Mallon, Robert; Hollander, Irwin; Feldberg, Larry; Lucas, Judy; Yu, Ker; Chaudhary, Inder; Mansour, Tarek S

    2010-10-01

    A series of mono-morpholino 1,3,5-triazine derivatives (8a-8q) bearing a 3-oxa-8-azabicyclo[3.2.1]octane were prepared and evaluated for PI3-kinase/mTOR activity. Replacement of one of the bis-morpholines in lead compound 1 (PKI-587) with 3-oxa-8-azabicyclo[3.2.1]octane and reduction of the molecular weight yielded 8m (PKI-179), an orally efficacious dual PI3-kinase/mTOR inhibitor. The in vitro activity, in vivo efficacy, and PK properties of 8m are discussed. Copyright © 2010. Published by Elsevier Ltd.

  12. Semiconductor technology in protein kinase research and drug discovery: sensing a revolution.

    Science.gov (United States)

    Bhalla, Nikhil; Di Lorenzo, Mirella; Estrela, Pedro; Pula, Giordano

    2017-02-01

    Since the discovery of protein kinase activity in 1954, close to 600 kinases have been discovered that have crucial roles in cell physiology. In several pathological conditions, aberrant protein kinase activity leads to abnormal cell and tissue physiology. Therefore, protein kinase inhibitors are investigated as potential treatments for several diseases, including dementia, diabetes, cancer and autoimmune and cardiovascular disease. Modern semiconductor technology has recently been applied to accelerate the discovery of novel protein kinase inhibitors that could become the standard-of-care drugs of tomorrow. Here, we describe current techniques and novel applications of semiconductor technologies in protein kinase inhibitor drug discovery. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Expression of a TGF-{beta} regulated cyclin-dependent kinase inhibitor in normal and immortalized airway epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Tierney, L.A.; Bloomfield, C.; Johnson, N.F. [and others

    1995-12-01

    Tumors arising from epithelial cells, including lung cancers are frequently resistant to factors that regulate growth and differentiation in normal in normal cells. Once such factor is transforming growth factor-{Beta} (TGF-{Beta}). Escape from the growth-inhibitory effects of TGF-{Beta} is thought to be a key step in the transformation of airway epithelial cells. most lung cancer cell lines require serum for growth. In contrast, normal human bronchial epithelial (NHBE) cells are exquisitely sensitive to growth-inhibitory and differentiating effects of TGF-{Beta}. The recent identification of a novel cyclin-dependent kinase inhibitor, p15{sup INK4B}, which is regulated by TGF-{Beta}, suggests a mechanism by which TGF-{Beta} mediates growth arrest in NHBE cells. The purpose of this study was two-fold: (1) to determine if p15{sup INK4B} is induced by TGF-{Beta} in NHBE cells or immortalized bronchial epithelial (R.1) cells and if that induction corresponds to a G1/S cell-cycle arrest; (2) to determine the temporal relationship between p15{sup INK4B} induction, cell-cycle arrest, and the phosphorylation state of the pRB because it is thought that p15{sup INK4B} acts indirectly by preventing phosphorylation of the RB gene product. In this study, expression of p15{sup INK4B} was examined in NHBE cells and R.1 cells at different time intervals following TGF-{Beta} treatment. The expression of this kinase inhibitor and its relationship to the cell and the pRb phosphorylation state were examined in cells that were both sensitive (NHBE) and resistant (R.1) to the effects of TGF-{Beta}. These results suggest that the cyclin-dependent kinase inhibitor, p15{sup INK4B}, is involved in airway epithelial cell differentiation and that loss or reduction of expression plays a role in the resistance of transformed or neoplastic cells to the growth-inhibitory effects of TGF-{Beta}.

  14. Neratinib, A Novel HER2-Targeted Tyrosine Kinase Inhibitor.

    Science.gov (United States)

    Tiwari, Shruti Rakesh; Mishra, Prasun; Abraham, Jame

    2016-10-01

    HER2 gene amplification and receptor overexpression is identified in 20% to 25% of human breast cancers. Use of targeted therapy for HER2-amplified breast cancer has led to improvements in disease-free and overall survival in this subset of patients. Neratinib is an oral pan HER inhibitor, that irreversibly inhibits the tyrosine kinase activity of epidermal growth factor receptor (EGFR or HER1), HER2, and HER4, which leads to reduced phosphorylation and activation of downstream signaling pathways. Neratinib is currently being tested in a number of clinical trials for its safety and efficacy in lung cancer, and colorectal, bladder, and breast cancers. In this review we discuss the available phase I, II, and III data for use of neratinib in the metastatic, adjuvant, neoadjuvant, and extended adjuvant settings along with the ongoing clinical trials of neratinib in breast cancer. We also elaborate on the side effect profile of this relatively new drug and provide guidelines for its use in clinical practice. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Carboxamide Spleen Tyrosine Kinase (Syk) Inhibitors: Leveraging Ground State Interactions To Accelerate Optimization

    Energy Technology Data Exchange (ETDEWEB)

    Ellis, J. Michael; Altman, Michael D.; Cash, Brandon; Haidle, Andrew M.; Kubiak, Rachel L.; Maddess, Matthew L.; Yan, Youwei; Northrup, Alan B. (Merck)

    2016-12-08

    Optimization of a series of highly potent and kinome selective carbon-linked carboxamide spleen tyrosine kinase (Syk) inhibitors with favorable drug-like properties is described. A pervasive Ames liability in an analogous nitrogen-linked carboxamide series was obviated by replacement with a carbon-linked moiety. Initial efforts lacked on-target potency, likely due to strain induced between the hinge binding amide and solvent front heterocycle. Consideration of ground state and bound state energetics allowed rapid realization of improved solvent front substituents affording subnanomolar Syk potency and high kinome selectivity. These molecules were also devoid of mutagenicity risk as assessed via the Ames test using the TA97a Salmonella strain.

  16. Liquid chromatography-tandem mass spectrometric assay for the tyrosine kinase inhibitor afatinib in mouse plasma using salting-out liquid-liquid extraction

    NARCIS (Netherlands)

    Sparidans, Rolf W; van Hoppe, Stephanie; Rood, Johannes J M; Schinkel, Alfred H; Schellens, Jan H M; Beijnen, Jos H

    2016-01-01

    A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for afatinib, an irreversible inhibitor of the ErbB (erythroblastic leukemia viral oncogene homolog) tyrosine kinase family, was developed and validated. Plasma samples were pre-treated using salting-out

  17. Exogenous addition of histidine reduces copper availability in the yeast Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Daisuke Watanabe

    2014-07-01

    Full Text Available The basic amino acid histidine inhibited yeast cell growth more severely than lysine and arginine. Overexpression of CTR1, which encodes a high-affinity copper transporter on the plasma membrane, or addition of copper to the medium alleviated this cytotoxicity. However, the intracellular level of copper ions was not decreased in the presence of excess histidine. These results indicate that histidine cytotoxicity is associated with low copper availability inside cells, not with impaired copper uptake. Furthermore, histidine did not affect cell growth under limited respiration conditions, suggesting that histidine cytotoxicity is involved in deficiency of mitochondrial copper.

  18. Exogenous addition of histidine reduces copper availability in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Watanabe, Daisuke; Kikushima, Rie; Aitoku, Miho; Nishimura, Akira; Ohtsu, Iwao; Nasuno, Ryo; Takagi, Hiroshi

    2014-07-07

    The basic amino acid histidine inhibited yeast cell growth more severely than lysine and arginine. Overexpression of CTR1 , which encodes a high-affinity copper transporter on the plasma membrane, or addition of copper to the medium alleviated this cytotoxicity. However, the intracellular level of copper ions was not decreased in the presence of excess histidine. These results indicate that histidine cytotoxicity is associated with low copper availability inside cells, not with impaired copper uptake. Furthermore, histidine did not affect cell growth under limited respiration conditions, suggesting that histidine cytotoxicity is involved in deficiency of mitochondrial copper.

  19. Autoregulation of kinase dephosphorylation by ATP binding to AGC protein kinases

    Science.gov (United States)

    Pascal, John M; Armen, Roger S

    2012-01-01

    AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non-ATP-competitive kinase inhibitors that discriminate within and between protein kinase families. PMID:22262182

  20. Icotinib, a selective EGF receptor tyrosine kinase inhibitor, for the treatment of non-small-cell lung cancer.

    Science.gov (United States)

    Tan, Fenlai; Shi, Yuankai; Wang, Yinxiang; Ding, Lieming; Yuan, Xiaobin; Sun, Yan

    2015-01-01

    Advanced non-small-cell lung cancer (NSCLC) is the main cause for cancer-related mortality. Treatments for advanced NSCLC are largely palliative and a benefit plateau appears to have reached with the platinum-based chemotherapy regimens. EGF receptor (EGFR) tyrosine kinase inhibitors gefitinib, erlotinib and afatinib came up with prolonged progression-free survival and improved quality of life, especially in EGFR-mutated patients. Icotinib is an oral selective EGFR tyrosine kinase, which was approved by China Food and Drug administration in June 2011 for treating advanced NSCLC. Its approval was based on the registered Phase III trial (ICOGEN), which showed icotinib is noninferior to gefitinib. This review will discuss the role of icotinib in NSCLC, and its potential application and ongoing investigations.

  1. Contractions activate hormone-sensitive lipase in rat muscle by protein kinase C and mitogen-activated protein kinase

    DEFF Research Database (Denmark)

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia

    2003-01-01

    and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction......Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline......-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50% by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None...

  2. Disseminated Infections with Talaromyces marneffei in Non-AIDS Patients Given Monoclonal Antibodies against CD20 and Kinase Inhibitors

    OpenAIRE

    Chan, Jasper F.W.; Chan, Thomas S.Y.; Gill, Harinder; Lam, Frank Y.F.; Trendell-Smith, Nigel J.; Sridhar, Siddharth; Tse, Herman; Lau, Susanna K.P.; Hung, Ivan F.N.; Yuen, Kwok-Yung; Woo, Patrick C.Y.

    2015-01-01

    Infections with the fungus Talaromyces (formerly Penicillium) marneffei are rare in patients who do not have AIDS. We report disseminated T. marneffei infection in 4 hematology patients without AIDS who received targeted therapy with monoclonal antibodies against CD20 or kinase inhibitors during the past 2 years. Clinicians should be aware of this emerging complication, especially in patients from disease-endemic regions.

  3. The new InsP3Kinase inhibitor BIP-4 is competitive to InsP3 and blocks proliferation and adhesion of lung cancer cells.

    Science.gov (United States)

    Schröder, Dominik; Tödter, Klaus; Gonzalez, Beatriz; Franco-Echevarría, Elsa; Rohaly, Gabor; Blecher, Christine; Lin, Hong-Ying; Mayr, Georg W; Windhorst, Sabine

    2015-07-15

    As ectopic expression of the neuronal inositol-1,4,5-trisphosphate-3-kinase A (InsP3Kinase) in tumor cells increases the metastatic potential, InsP3Kinase is an interesting target for tumor therapy. Recently, we have identified a membrane-permeable InsP3Kinase inhibitor (BAMB-4) exhibiting an IC50-value of 20 μM. Here we characterized a new InsP3Kinase inhibitor which shows a 130-fold lower IC50 value (157 ± 57 nM) as compared to BAMB-4. We demonstrate that this nitrophenolic compound, BIP-4, is non-competitive to ATP but competitive to InsP3, thus exhibits a high selectivity for inhibition of InsP3Kinase activity. Docking analysis suggested a putative binding mode of this molecule into the InsP3Kinase active site. Determination of cellular uptake in lung cancer cells (H1299) revealed that 6% of extracellular BIP-4 is internalized by non-endosomal uptake, showing that BIP-4 is not trapped inside endo/lysosomes but is available to inhibit cellular InsP3Kinase activity. Interestingly, we found that BIP-4 mediated inhibition of InsP3Kinase activity in the two lung cancer cell lines H1299 and LN4323 inhibited proliferation and adhesion at IC50 values of 3 μM or 2 μM, respectively. InsP3Kinase inhibition did not alter ATP-induced calcium signals but significantly reduced the level of Ins(1,3,4,5,6)P5. From these data we conclude that the inhibitory effect of BIP-4 on proliferation and adhesion of lung cancer cells does not result from alterations of calcium but from alterations of inositol phosphate signals. In summary, we reveal that inhibition of cellular InsP3Kinase by BIP-4 impairs proliferation and adhesion and therefore BIP-4 might be a promising compound to reduce the metastatic potential of lung carcinoma cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Tyrosine and aurora kinase inhibitors diminish transport function of multidrug resistance-associated protein (MRP 4 and breast cancer resistance protein (BCRP

    Directory of Open Access Journals (Sweden)

    Rhiannon N. Hardwick

    2016-12-01

    Full Text Available Tyrosine and aurora kinases are important effectors in signal transduction pathways that are often involved in aberrant cancer cell growth. Tyrosine (TKI and aurora (AKI kinase inhibitors are anti-cancer agents specifically designed to target such signaling pathways through TKI/AKI binding to the ATP-binding pocket of kinases thereby leading to diminished kinase activity. Some TKIs have been identified as inhibitors of ATP-binding cassette (ABC transporters such as P-glycoprotein and breast cancer resistance protein (BCRP, which are commonly upregulated in malignant cells. TKI/AKIs have been investigated as ABC transporter inhibitors in order to facilitate the accumulation of concomitantly administered chemo-therapeutics within cancer cells. However, ABC transporters are prominently expressed in the liver and other eliminating organs, and their inhibition has been linked to intracellular accumulation of drugs, altered disposition, and toxicity. The potential for TKIs/AKIs to inhibit other important hepatic efflux transporters, particularly multidrug resistance-associated proteins (MRPs, remains unknown. The aim of the current study was to compare the inhibitory potency of 20 selected TKI/AKIs against MRP4 and BCRP through the use of inverted membrane vesicle assays. Relative IC50 values were estimated by determining TKI/AKI inhibition of MRP4-mediated [3H]-dehydroepiandrosterone sulfate uptake and BCRP-mediated [3H]-estrone sulfate uptake. To provide insight to the clinical relevance of TKI/AKI inhibition of ABC efflux transporters, the ratio of the steady-state maximum total plasma concentration (Css to the IC50 for each compound was calculated with Css/IC50 ratio >0.1 deemed potentially clinically relevant. Such analysis identified several potentially clinically relevant inhibitors of MRP4: alisertib, danusertib, erlotinib, lapatinib, neratinib, nilotinib, pazopanib, sorafenib, and tozasertib. The potentially clinically relevant inhibition of

  5. Synthesis and SAR of 1-acetanilide-4-aminopyrazole-substituted quinazolines: selective inhibitors of Aurora B kinase with potent anti-tumor activity.

    Science.gov (United States)

    Foote, Kevin M; Mortlock, Andrew A; Heron, Nicola M; Jung, Frédéric H; Hill, George B; Pasquet, Georges; Brady, Madeleine C; Green, Stephen; Heaton, Simon P; Kearney, Sarah; Keen, Nicholas J; Odedra, Rajesh; Wedge, Stephen R; Wilkinson, Robert W

    2008-03-15

    A new class of 1-acetanilide-4-aminopyrazole-substituted quinazoline Aurora kinase inhibitors has been discovered possessing highly potent cellular activity. Continuous infusion into athymic mice bearing SW620 tumors of the soluble phosphate derivative 2 led to dose-proportional exposure of the des-phosphate compound 8 with a high-unbound fraction. The combination of potent cell activity and high free-drug exposure led to pharmacodynamic changes in the tumor at low doses, indicative of Aurora B-kinase inhibition and a reduction in tumor volume.

  6. The selectivity and promiscuity of brain-neuroregenerative inhibitors between ROCK1 and ROCK2 isoforms: An integration of SB-QSSR modelling, QM/MM analysis and in vitro kinase assay.

    Science.gov (United States)

    Zhu, L; Yang, Y; Lu, X

    2016-01-01

    The Rho-associated kinases (ROCKs) have long been recognized as an attractive therapeutic target for various neurological diseases; selective inhibition of ROCK1 and ROCK2 isoforms would result in distinct biological effects on neurogenesis, neuroplasticity and neuroregeneration after brain surgery and traumatic brain injury. However, the discovery and design of isoform-selective inhibitors remain a great challenge due to the high conservation and similarity between the kinase domains of ROCK1 and ROCK2. Here, a structure-based quantitative structure-selectivity relationship (SB-QSSR) approach was used to correlate experimentally measured selectivity with the difference in inhibitor binding to the two kinase isoforms. The resulting regression models were examined rigorously through both internal cross-validation and external blind validation; a nonlinear predictor was found to have high fitting stability and strong generalization ability, which was then employed to perform virtual screening against a structurally diverse, drug-like compound library. Consequently, five and seven hits were identified as promising candidates of 1-o-2 and 2-o-1 selective inhibitors, respectively, from which seven purchasable compounds were tested in vitro using a standard kinase assay protocol to determine their inhibitory activity against and selectivity between ROCK1 and ROCK2. The structural basis, energetic property and biological implication underlying inhibitor selectivity and promiscuity were also investigated systematically using a hybrid quantum mechanics/molecular mechanics (QM/MM) scheme.

  7. Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway

    DEFF Research Database (Denmark)

    Kolkova, K; Novitskaya, V; Pedersen, N

    2000-01-01

    , inhibitors of the nonreceptor tyrosine kinase p59(fyn), PLC, PKC and MEK and an activator of PKC, phorbol-12-myristate-13-acetate (PMA). MEK2 transfection rescued cells treated with all inhibitors. The same was found for PMA treatment, except when cells concomitantly were treated with the MEK inhibitor....... Arachidonic acid rescued cells treated with antibodies to the FGF receptor or the PLC inhibitor, but not cells in which the activity of PKC, p59(fyn), FAK, Ras, or MEK was inhibited. Interaction of NCAM with a synthetic NCAM peptide ligand, known to induce neurite outgrowth, was shown to stimulate...... phosphorylation of the MAP kinases extracellular signal-regulated kinases ERK1 and ERK2. The MAP kinase activation was sustained, because ERK1 and ERK2 were phosphorylated in PC12-E2 cells and primary hippocampal neurons even after 24 hr of cultivation on NCAM-expressing fibroblasts. Based on these results, we...

  8. Dabrafenib, an inhibitor of RIP3 kinase-dependent necroptosis, reduces ischemic brain injury

    Directory of Open Access Journals (Sweden)

    Shelly A Cruz

    2018-01-01

    Full Text Available Ischemic brain injury triggers neuronal cell death by apoptosis via caspase activation and by necroptosis through activation of the receptor-interacting protein kinases (RIPK associated with the tumor necrosis factor-alpha (TNF-α/death receptor. Recent evidence shows RIPK inhibitors are neuroprotective and alleviate ischemic brain injury in a number of animal models, however, most have not yet undergone clinical trials and safety in humans remains in question. Dabrafenib, originally identified as a B-raf inhibitor that is currently used to treat melanoma, was later revealed to be a potent RIPK3 inhibitor at micromolar concentrations. Here, we investigated whether Dabrafenib would show a similar neuroprotective effect in mice subjected to ischemic brain injury by photothrombosis. Dabrafenib administered intraperitoneally at 10 mg/kg one hour after photothrombosis-induced focal ischemic injury significantly reduced infarct lesion size in C57BL6 mice the following day, accompanied by a markedly attenuated upregulation of TNF-α. However, subsequent lower doses (5 mg/kg/day failed to sustain this neuroprotective effect after 4 days. Dabrafenib blocked lipopolysaccharides-induced activation of TNF-α in bone marrow-derived macrophages, suggesting that Dabrafenib may attenuate TNF-α-induced necroptotic pathway after ischemic brain injury. Since Dabrafenib is already in clinical use for the treatment of melanoma, it might be repurposed for stroke therapy.

  9. The discovery and the structural basis of an imidazo[4,5-b]pyridine-based p21-activated kinase 4 inhibitor.

    Science.gov (United States)

    Park, Jeung Kuk; Kim, Sunmin; Han, Yu Jin; Kim, Seong Hwan; Kang, Nam Sook; Lee, Hyuk; Park, SangYoun

    2016-06-01

    p21-Activated kinases (PAKs) which belong to the family of ste20 serine/threonine protein kinases regulate cytoskeletal reorganization, cell motility, cell proliferation, and oncogenic transformation which are all related to the cellular functions during cancer induction and metastasis. The fact that PAK mutations are detected in multiple tumor tissues makes PAKs a novel therapeutic drug target. In this study, an imidazo[4,5-b]pyridine-based PAK4 inhibitor, KY-04045 (6-Bromo-2-(3-isopropyl-1-methyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-b]pyridine), was discovered using a virtual site-directed fragment-based drug design and was validated using an inhibition assay. Although PAK4 affinity to KY-04045 seems much weaker than that of the reported PAK4 inhibitors, the location of KY-04045 is clearly defined in the structure of PAK4 co-crystallized with KY-04045. The crystal structure illustrates that the pyrazole and imidazopyridine rings of KY-04045 are sufficient for mediating PAK4 hinge loop interaction. Hence, we believe that KY-04045 can be exploited as a basic building block in designing novel imidazo[4,5-b]pyridine-based PAK4 inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Os inibidores de tirosino quinase de segunda geração The inhibitors of tyrosine kinase

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    Márcia T. Delamain

    2008-04-01

    Full Text Available O imatinibe tem sido confirmado como terapia de primeira linha para a Leucemia Mielóide Crônica (LMC por apresentar respostas duradouras na maior parte dos pacientes, principalmente nos que se encontram em fase precoce da doença. Entretanto, resistência ou intolerância ao imatinibe podem ocorrer. A resistência ao imatinibe ocorre com muito mais freqüência em fases mais avançadas da doença, sendo a causa mais comum o desenvolvimento de mutações no sítio BCR-ABL. Em face deste problema, novos inibidores de tirosino quinase têm sido desenvolvidos, com maior potência, diminuindo assim a chance de desenvolvimento de resistência ao tratamento. O nilotinibe e o dasatinibe são dois exemplos de inibidores de segunda geração de tirosino quinase recentemente aprovados. Ambos têm demonstrado excelentes resultados em pacientes que desenvolvem resistência ou são intolerantes ao imatinibe.Despite the success with imatinib as the first choice treatment of chronic myeloid leukemia (CML, there is still a subset of patients that do not respond optimally to or are intolerant of this drug or lose response. Imatinib resistance can occur at any phase, but it is more frequent in advanced phases, with mutations in the BCR-ABL kinase domain being the most common mechanism of resistance. More potent tyrosine kinase inhibitors have been developed that can overcome resistance to imatinib. Nilotinib and dasatinib are good examples of new tyrosine kinase inhibitors that are available. With these new agents, patients who develop imatinib resistance or those unable to tolerate imatinib treatment can achieve significant clinical responses.

  11. Sequential treatment of tyrosine kinase inhibitors and chemotherapy for EGFR-mutated non-small cell lung cancer: a meta-analysis of Phase III trials

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    Zhang Y

    2013-11-01

    Full Text Available Yiliang Zhang,1,* Yihua Sun,1,* Lei Wang,1 Ting Ye,1 Yunjian Pan,1 Haichuan Hu,1 Yongfu Yu,2 Naiqing Zhao,2 Yanyan Song,3 David Garfield,4 Haiquan Chen1 1Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, 2Department of Biostatistics, School of Public Health, Fudan University, 3Department of Pharmacology and Biostatistics, Institute of Medical Science, Shanghai Jiaotong University School of Medicine, 4ProMed Cancer Centers, Shanghai, People’s Republic of China *These authors contributed equally to this work Background: This aim of this study was to compare the efficacy of first-line tyrosine kinase inhibitor therapy followed, upon progression, by chemotherapy with the reverse sequence in patients with EGFR-mutated non-small cell lung cancer (NSCLC in terms of overall survival. Methods: We performed a meta-analysis of studies that met the following criteria: Phase III clinical trial comparing the sequencing of epidermal growth factor receptor (EGFR tyrosine kinase inhibitors with chemotherapy in the treatment of advanced EGFR-mutated NSCLC; activating mutations reported; and availability of hazard ratio estimates with 95% confidence intervals (CIs for overall survival. Results: Six clinical trials were included in this study. The pooled hazard ratio for overall survival of the EGFR-mutated population that completed sequential treatment was 1.03 (95% CI 0.86–1.22, P=0.776. There was no statistically significant heterogeneity between the studies (tau2 =0; I2=0, 95% CI 0–0.37, P=0.548. Evidence of marked publication bias for the two treatment sequences was insufficient (P=0.145. Conclusion: In patients with advanced NSCLC and activating EGFR mutations, first-line chemotherapy followed upon progression by a tyrosine kinase inhibitor was not inferior in terms of overall survival compared with the inverse sequence. This may serve as an indication that

  12. Feeding filaggrin: effects of L-histidine supplementation in atopic dermatitis

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    Tan SP

    2017-10-01

    Full Text Available Siao Pei Tan,1,2 Simon B Brown,1,2 Christopher EM Griffiths,3 Richard B Weller,1,2 Neil K Gibbs3,4 1MRC Centre for Inflammation Research, 2Department of Dermatology, The University of Edinburgh, Edinburgh, 3Dermatology Centre, Division of Musculoskeletal and Dermatological Sciences, Salford Royal NHS Foundation Trust, University of Manchester, Manchester, 4Curapel, Life Sciences Hub Wales, Cardiff, UK Abstract: Atopic dermatitis (AD, also known as eczema, is one of the most common chronic skin conditions worldwide, affecting up to 16% of children and 10% of adults. It is incurable and has significant psychosocial and economic impacts on the affected individuals. AD etiology has been linked to deficiencies in the skin barrier protein, filaggrin. In mammalian skin, l-histidine is rapidly incorporated into filaggrin. Subsequent filaggrin proteolysis releases l-histidine as an important natural moisturizing factor (NMF. In vitro studies were conducted to investigate the influence of l-histidine on filaggrin processing and barrier function in human skin-equivalent models. Our further aim was to examine the effects of daily oral l-histidine supplementation on disease severity in adult AD patients. We conducted a randomized, double-blind, placebo-controlled, crossover, nutritional supplementation pilot study to explore the effects of oral l-histidine in adult AD patients (n=24. In vitro studies demonstrated that l-histidine significantly increased both filaggrin formation and skin barrier function (P<0.01, respectively. Data from the clinical study indicated that once daily oral l-histidine significantly reduced (P<0.003 AD disease severity by 34% (physician assessment using the SCORingAD tool and 39% (patient self-assessment using the Patient Oriented Eczema Measure tool after 4 weeks of treatment. No improvement was noted with the placebo (P>0.32. The clinical effect of oral l-histidine in AD was similar to that of mid-potency topical corticosteroids

  13. L-histidine enhances learning in stressed zebrafish

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    L.P.V. Cofiel

    2009-01-01

    Full Text Available The aim of the present study was to determine the effect of the histaminergic precursor L-histidine and the H3 receptor antagonist thioperamide on the learning process of zebrafish submitted or not to confinement stress. On each of the 5 consecutive days of experiment (D1, D2, D3, D4, D5, animals had to associate an interruption of the aquarium air supply with food offering. Non-stressed zebrafish received an intraperitoneal injection of 100 mg/kg L-histidine, 10 mg/kg thioperamide or saline after training. Stressed animals received drug treatment and then were submitted to confinement stress for 1 h before the learning procedure. Time to approach the feeder was measured (in seconds and was considered to be indicative of learning. A decrease in time to approach the feeder was observed in the saline-treated group (D1 = 141.92 ± 13.57; D3 = 55 ± 13.54, indicating learning. A delay in learning of stressed animals treated with saline was observed (D1 = 217.5 ± 25.66. L-histidine facilitated learning in stressed (D1 = 118.68 ± 13.9; D2 = 45.88 ± 8.2 and non-stressed (D1 = 151.11 ± 19.20; D5 = 62 ± 14.68 animals. Thioperamide inhibited learning in non-stressed (D1 = 110.38 ± 9.49; D4 = 58.79 ± 16.83 and stressed animals (D1 = 167.3 ± 26.39; D5 = 172.15 ± 27.35. L-histidine prevented the increase in blood glucose after one session of confinement (L-histidine = 65.88 ± 4.50; control = 53 ± 3.50 mg/dL. These results suggest that the histaminergic system enhances learning and modulates stress responses in zebrafish.

  14. Combined treatment of tyrosine kinase inhibitor labeled gold nanorod encapsulated albumin with laser thermal ablation in a renal cell carcinoma model

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    This manuscript served to characterize and evaluate Human Serum Albumin-encapsulated Nanoparticles (NPs) for drug delivery of a tyrosine kinase inhibitor combined with induction of photothermal ablation (PTA) combination therapy of Renal Cell Carcinoma (RCC). RCC is the most common type of kidney c...

  15. Raman Microspectroscopic Evidence for the Metabolism of a Tyrosine Kinase Inhibitor, Neratinib, in Cancer Cells.

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    Aljakouch, Karim; Lechtonen, Tatjana; Yosef, Hesham K; Hammoud, Mohamad K; Alsaidi, Wissam; Kötting, Carsten; Mügge, Carolin; Kourist, Robert; El-Mashtoly, Samir F; Gerwert, Klaus

    2018-06-11

    Tyrosine kinase receptors are one of the main targets in cancer therapy. They play an essential role in the modulation of growth factor signaling and thereby inducing cell proliferation and growth. Tyrosine kinase inhibitors such as neratinib bind to EGFR and HER2 receptors and exhibit antitumor activity. However, little is known about their detailed cellular uptake and metabolism. Here, we report for the first time the intracellular spatial distribution and metabolism of neratinib in different cancer cells using label-free Raman imaging. Two new neratinib metabolites were detected and fluorescence imaging of the same cells indicate that neratinib accumulates in lysosomes. The results also suggest that both EGFR and HER2 follow the classical endosome lysosomal pathway for degradation. A combination of Raman microscopy, DFT calculations, and LC-MS was used to identify the chemical structure of neratinib metabolites. These results show the potential of Raman microscopy to study drug pharmacokinetics. © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  16. Role of the VirA histidine autokinase of Agrobacterium tumefaciens in the initial steps of pathogenesis

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    Yi-Han eLin

    2014-05-01

    Full Text Available Histidine kinases serve as critical environmental sensing modules, and despite their designation as simple two-component modules, their functional roles are remarkably diverse. In Agrobacterium tumefaciens pathogenesis, VirA serves with VirG as the initiating sensor/transcriptional activator for inter-kingdom gene transfer and transformation of higher plants. Through responses to three separate signal inputs, low pH, sugars, and phenols, A. tumefaciens commits to pathogenesis in virtually all flowering plants. However, how these three signals are integrated to regulate the response and why these signals might be diagnostic for susceptible cells across such a broad host-range remains poorly understood. Using a homology model of the VirA linker region, we provide evidence for coordinated long-range transmission of inputs perceived both outside and inside the cell through the creation of targeted VirA truncations. Further, our evidence is consistent with signal inputs weakening associations between VirA domains to position the active site histidine for phosphate transfer. This mechanism requires long-range regulation of inter-domain stability and the transmission of input signals through a common integrating domain for VirA signal transduction.

  17. The phosphatidylinositol-3-phosphate 5-kinase inhibitor apilimod blocks filoviral entry and infection.

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    Elizabeth A Nelson

    2017-04-01

    Full Text Available Phosphatidylinositol-3-phosphate 5-kinase (PIKfyve is a lipid kinase involved in endosome maturation that emerged from a haploid genetic screen as being required for Ebola virus (EBOV infection. Here we analyzed the effects of apilimod, a PIKfyve inhibitor that was reported to be well tolerated in humans in phase 2 clinical trials, for its effects on entry and infection of EBOV and Marburg virus (MARV. We first found that apilimod blocks infections by EBOV and MARV in Huh 7, Vero E6 and primary human macrophage cells, with notable potency in the macrophages (IC50, 10 nM. We next observed that similar doses of apilimod block EBOV-glycoprotein-virus like particle (VLP entry and transcription-replication competent VLP infection, suggesting that the primary mode of action of apilimod is as an entry inhibitor, preventing release of the viral genome into the cytoplasm to initiate replication. After providing evidence that the anti-EBOV action of apilimod is via PIKfyve, we showed that it blocks trafficking of EBOV VLPs to endolysosomes containing Niemann-Pick C1 (NPC1, the intracellular receptor for EBOV. Concurrently apilimod caused VLPs to accumulate in early endosome antigen 1-positive endosomes. We did not detect any effects of apilimod on bulk endosome acidification, on the activity of cathepsins B and L, or on cholesterol export from endolysosomes. Hence by antagonizing PIKfyve, apilimod appears to block EBOV trafficking to its site of fusion and entry into the cytoplasm. Given the drug's observed anti-filoviral activity, relatively unexplored mechanism of entry inhibition, and reported tolerability in humans, we propose that apilimod be further explored as part of a therapeutic regimen to treat filoviral infections.

  18. Unbinding Kinetics of a p38 MAP Kinase Type II Inhibitor from Metadynamics Simulations.

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    Casasnovas, Rodrigo; Limongelli, Vittorio; Tiwary, Pratyush; Carloni, Paolo; Parrinello, Michele

    2017-04-05

    Understanding the structural and energetic requisites of ligand binding toward its molecular target is of paramount relevance in drug design. In recent years, atomistic free energy calculations have proven to be a valid tool to complement experiments in characterizing the thermodynamic and kinetic properties of protein/ligand interaction. Here, we investigate, through a recently developed metadynamics-based protocol, the unbinding mechanism of an inhibitor of the pharmacologically relevant target p38 MAP kinase. We provide a thorough description of the ligand unbinding pathway identifying the most stable binding mode and other thermodynamically relevant poses. From our simulations, we estimated the unbinding rate as k off = 0.020 ± 0.011 s -1 . This is in good agreement with the experimental value (k off = 0.14 s -1 ). Next, we developed a Markov state model that allowed identifying the rate-limiting step of the ligand unbinding process. Our calculations further show that the solvation of the ligand and that of the active site play crucial roles in the unbinding process. This study paves the way to investigations on the unbinding dynamics of more complex p38 inhibitors and other pharmacologically relevant inhibitors in general, demonstrating that metadynamics can be a powerful tool in designing new drugs with engineered binding/unbinding kinetics.

  19. Crosstalk between Substrates and Rho-Associated Kinase Inhibitors in Cryopreservation of Tissue-Engineered Constructs

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    Arindam Bit

    2017-01-01

    Full Text Available It is documented that human mesenchymal stem cells (hMSCs can be differentiated into various types of cells to present a tool for tissue engineering and regenerative medicine. Thus, the preservation of stem cells is a crucial factor for their effective long-term storage that further facilitates their continuous supply and transportation for application in regenerative medicine. Cryopreservation is the most important, practicable, and the only established mechanism for long-term preservation of cells, tissues, and organs, and engineered tissues; thus, it is the key step for the improvement of tissue engineering. A significant portion of MSCs loses cellular viability while freeze-thawing, which represents an important technical limitation to achieving sufficient viable cell numbers for maximum efficacy. Several natural and synthetic materials are extensively used as substrates for tissue engineering constructs and cryopreservation because they promote cell attachment and proliferation. Rho-associated kinase (ROCK inhibitors can improve the physiological function and postthaw viability of cryopreserved MSCs. This review proposes a crosstalk between substrate topology and interaction of cells with ROCK inhibitors. It is shown that incorporation of ionic nanoparticles in the presence of an external electrical field improves the generation of ROCK inhibitors to safeguard cellular viability for the enhanced cryopreservation of engineered tissues.

  20. [INHIBITORS OF MAP-KINASE PATHWAY U0126 AND PD98059 DIFFERENTLY AFFECT ORGANIZATION OF TUBULIN CYTOSKELETON AFTER STIMULATION OF EGF RECEPTOR ENDOCYTOSIS].

    Science.gov (United States)

    Zlobina, M V; Steblyanko, Yu Yu; Shklyaeva, M A; Kharchenko, V V; Salova, A V; Kornilova, E S

    2015-01-01

    To confirm the hypothesis about the involvement of EGF-stimulated MAP-kinase ERK1/2 in the regulation of microtubule (MT) system, the influence of two widely used ERK1/2 inhibitors, U0126 and PD98059, on the organization of tubulin cytoskeleton in interphase HeLa cells during EGF receptor endocytosis has been investigated. We have found that addition of U0126 or PD98059 to not-stimulated with EGF ells for 30 min has no effect on radially organized MT system. However, in the case of U0126 addition before EGF endocytosis stimulation, the number of MT per cell decreased within 15 min after such stimulation and was followed by complete MT depolymerization by 60-90 min. Stimulation of EGF endocytosis in the presence of PD98059 resulted only in insignificant depolymerization of MT and it could be detected mainly from their minus-ends. At the same time, MT regions close to plasma membrane became stabilized, which was proved by increase in tubulin acetylation level. This situation was characteristic for all period of the experiment. It has been also found that the inhibitors affect endocytosis dynamics of EGF-receptor complexes. Quantitative analysis demonstrated that the stimulation of endocytosis in the presence of U0126 generated a greater number of endosomes compared to control cells, and their number did not change significantly during the experiment. All these endosomes were localized peripherally. Effect of PD98059 resulted in the formation of lower number of endosomes that in control, but they demonstrated very slow clusterization despite the presence of some intact MT. Both inhibitors decreased EGFR colocolization with early endosomal marker EEA1, which indicated a delay in endosome fusions and maturation. The inhibitors were also shown to affect differently phospho-ERK 1 and 2 forms: U0126 completely inhibited phospho-ERK1 and 2, white, in the presence of PD98059, the two ERK forms demonstrated sharp transient activation in 15 min after stimulation, but only

  1. Fasting potentiates the anticancer activity of tyrosine kinase inhibitors by strengthening MAPK signaling inhibition

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    Caffa, Irene; D'Agostino, Vito; Damonte, Patrizia; Soncini, Debora; Cea, Michele; Monacelli, Fiammetta; Odetti, Patrizio; Ballestrero, Alberto; Provenzani, Alessandro; Longo, Valter D.; Nencioni, Alessio

    2015-01-01

    Tyrosine kinase inhibitors (TKIs) are now the mainstay of treatment in many types of cancer. However, their benefit is frequently short-lived, mandating the search for safe potentiation strategies. Cycles of fasting enhance the activity of chemo-radiotherapy in preclinical cancer models and dietary approaches based on fasting are currently explored in clinical trials. Whether combining fasting with TKIs is going to be potentially beneficial remains unknown. Here we report that starvation conditions increase the ability of commonly administered TKIs, including erlotinib, gefitinib, lapatinib, crizotinib and regorafenib, to block cancer cell growth, to inhibit the mitogen-activated protein kinase (MAPK) signaling pathway and to strengthen E2F-dependent transcription inhibition. In cancer xenografts models, both TKIs and cycles of fasting slowed tumor growth, but, when combined, these interventions were significantly more effective than either type of treatment alone. In conclusion, cycles of fasting or of specifically designed fasting-mimicking diets should be evaluated in clinical studies as a means to potentiate the activity of TKIs in clinical use. PMID:25909220

  2. Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

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    Sanne H. Olesen

    2015-01-01

    Full Text Available The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein–protein interaction (PPI inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2 did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM, while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.

  3. Histidine augments the suppression of hepatic glucose production by central insulin action.

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    Kimura, Kumi; Nakamura, Yusuke; Inaba, Yuka; Matsumoto, Michihiro; Kido, Yoshiaki; Asahara, Shun-Ichiro; Matsuda, Tomokazu; Watanabe, Hiroshi; Maeda, Akifumi; Inagaki, Fuyuhiko; Mukai, Chisato; Takeda, Kiyoshi; Akira, Shizuo; Ota, Tsuguhito; Nakabayashi, Hajime; Kaneko, Shuichi; Kasuga, Masato; Inoue, Hiroshi

    2013-07-01

    Glucose intolerance in type 2 diabetes is related to enhanced hepatic glucose production (HGP) due to the increased expression of hepatic gluconeogenic enzymes. Previously, we revealed that hepatic STAT3 decreases the expression of hepatic gluconeogenic enzymes and suppresses HGP. Here, we show that increased plasma histidine results in hepatic STAT3 activation. Intravenous and intracerebroventricular (ICV) administration of histidine-activated hepatic STAT3 reduced G6Pase protein and mRNA levels and augmented HGP suppression by insulin. This suppression of hepatic gluconeogenesis by histidine was abolished by hepatic STAT3 deficiency or hepatic Kupffer cell depletion. Inhibition of HGP by histidine was also blocked by ICV administration of a histamine H1 receptor antagonist. Therefore, histidine activates hepatic STAT3 and suppresses HGP via central histamine action. Hepatic STAT3 phosphorylation after histidine ICV administration was attenuated in histamine H1 receptor knockout (Hrh1KO) mice but not in neuron-specific insulin receptor knockout (NIRKO) mice. Conversely, hepatic STAT3 phosphorylation after insulin ICV administration was attenuated in NIRKO but not in Hrh1KO mice. These findings suggest that central histidine action is independent of central insulin action, while both have additive effects on HGP suppression. Our results indicate that central histidine/histamine-mediated suppression of HGP is a potential target for the treatment of type 2 diabetes.

  4. [Literature review and presentation of our own research results regarding the effects on bone of tyrosine kinase inhibitors imatinib and nilotinib used in the treatment of oncohematological diseases].

    Science.gov (United States)

    Kirschner, Gyöngyi; Balla, Bernadett; Kósa, János; Horváth, Péter; Kövesdi, Andrea; Lakatos, Gergely; Takács, István; Nagy, Zsolt; Tóbiás, Bálint; Árvai, Kristóf; Lakatos, Péter

    2016-09-01

    Tyrosine kinase inhibitors are widely used for treatment of certain oncohematological diseases. Several clinical studies have confirmed that specific BCR-ABL tyrosine kinase inhibitors alter the physiological process of bone tissue in a complex and unclearly identified manner. Since these treatments are being given to more and more patients, and the therapy takes decades or lasts even lifelong, it is justifiable to obtain more detailed knowledge of the molecular background of these mechanisms. In this article the authors summarize preliminary research results and human clinical observations on imatinib and nilotinib which are related to bone metabolism, and present the results of their own experiments in in vitro osteoblast cultures. Based on the presented results, the effects of imatinib and nilotinib on bone cells depend on the concentration of imatinib and nilotinib, the maturation stage of the cells and the distribution ratio of receptor tyrosine kinase signaling pathways. In this study the authors firstly prepared a stop-gap, comprehensive review in the Hungarian literature, regarding the effects of tyrosine kinase inhibitors on bone metabolism. In addition they firstly performed whole transcriptome analysis on osteoblasts in order to obtain a better understanding of the cellular molecular mechanisms. Orv. Hetil., 2016, 157(36), 1429-1437.

  5. Targeting Polo-Like Kinases: A Promising Therapeutic Approach for Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Xiaoqi Liu

    2015-06-01

    Full Text Available Polo-like kinases (Plks are a family of serine-threonine kinases that regulate multiple intracellular processes including DNA replication, mitosis, and stress response. Plk1, the most well understood family member, regulates numerous stages of mitosis and is overexpressed in many cancers. Plk inhibitors are currently under clinical investigation, including phase III trials of volasertib, a Plk inhibitor, in acute myeloid leukemia and rigosertib, a dual inhibitor of Plk1/phosphoinositide 3-kinase signaling pathways, in myelodysplastic syndrome. Other Plk inhibitors, including the Plk1 inhibitors GSK461364A, TKM-080301, GW843682, purpurogallin, and poloxin and the Plk4 inhibitor CFI-400945 fumarate, are in earlier clinical development. This review discusses the biologic roles of Plks in cell cycle progression and cancer, and the mechanisms of action of Plk inhibitors currently in development as cancer therapies.

  6. Structural basis for decreased induction of class IB PI3-kinases expression by MIF inhibitors

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    Singh, Abhay Kumar [Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis MO USA; Pantouris, Georgios [Department of Pharmacology, Yale University School of Medicine, New Haven CT USA; Borosch, Sebastian [Institute of Biochemistry and Molecular Cell Biology, RWTH Aachen University, Aachen Germany; Rojanasthien, Siripong [Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis MO USA; Cho, Thomas Yoonsang [Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis MO USA

    2016-09-13

    Macrophage migration inhibitory factor (MIF) is a master regulator of proinflammatory cytokines and plays pathological roles when not properly regulated in rheumatoid arthritis, lupus, atherosclerosis, asthma and cancer. Unlike canonical cytokines, MIF has vestigial keto-enol tautomerase activity. Most of the current MIF inhibitors were screened for the inhibition of this enzymatic activity. However, only some of the enzymatic inhibitors inhibit receptor-mediated biological functions of MIF, such as cell recruitment, through an unknown molecular mechanism. The goal of this study was to understand the molecular basis underlying the pharmacological inhibition of biological functions of MIF. Here, we demonstrate how the structural changes caused upon inhibitor binding translate into the alteration of MIF-induced downstream signalling. Macrophage migration inhibitory factor activates phosphoinositide 3-kinases (PI3Ks) that play a pivotal role in immune cell recruitment in health and disease. There are several different PI3K isoforms, but little is known about how they respond to MIF. We demonstrate that MIF up-regulates the expression of Class IB PI3Ks in leucocytes. We also demonstrate that MIF tautomerase active site inhibitors down-regulate the expression of Class IB PI3Ks as well as leucocyte recruitment in vitro and in vivo. Finally, based on our MIF:inhibitor complex crystal structures, we hypothesize that the reduction in Class IB PI3K expression occurs because of the displacement of Pro1 towards the second loop of MIF upon inhibitor binding, which results in increased flexibility of the loop 2 and sub-optimal MIF binding to its receptors. These results will provide molecular insights for fine-tuning the biological functions of MIF.

  7. Clinical review: kinase inhibitors: adverse effects related to the endocrine system.

    Science.gov (United States)

    Lodish, Maya B

    2013-04-01

    The use of kinase inhibitors (KIs) in the treatment of cancer has become increasingly common, and practitioners must be familiar with endocrine-related side effects associated with these agents. This review provides an update to the clinician regarding the management of potential endocrinological effects of KIs. PubMed was employed to identify relevant manuscripts. A review of the literature was conducted, and data were summarized and incorporated. KIs, including small molecule KIs and monoclonal antibodies directed against kinases, have emerged over the past decade as an important class of anticancer agents. KIs specifically interfere with signaling pathways that are dysregulated in certain types of cancers and also target common mechanisms of growth, invasion, metastasis, and angiogenesis. Currently, at least 20 KIs are approved as cancer therapeutics. However, KIs may affect a broad spectrum of targets and may have additional, unidentified mechanisms of action at the cellular level due to overlap between signaling pathways in the tumor cell and endocrine system. Recent reports in the literature have identified side effects associated with KIs, including alterations in thyroid function, bone metabolism, linear growth, gonadal function, fetal development, adrenal function, and glucose metabolism. Clinicians need to monitor the thyroid functions of patients on KIs. In addition, bone density and vitamin D status should be assessed. Special care should be taken to follow linear growth and development in children taking these agents. Clinicians should counsel patients appropriately on the potential adverse effects of KIs on fetal development.

  8. ARPP-16 Is a Striatal-Enriched Inhibitor of Protein Phosphatase 2A Regulated by Microtubule-Associated Serine/Threonine Kinase 3 (Mast 3 Kinase).

    Science.gov (United States)

    Andrade, Erika C; Musante, Veronica; Horiuchi, Atsuko; Matsuzaki, Hideo; Brody, A Harrison; Wu, Terence; Greengard, Paul; Taylor, Jane R; Nairn, Angus C

    2017-03-08

    ARPP-16 (cAMP-regulated phospho-protein of molecular weight 16 kDa) is one of several small acid-soluble proteins highly expressed in medium spiny neurons of striatum that are phosphorylated in response to dopamine acting via D1 receptor/protein kinase A (PKA) signaling. We show here that ARPP-16 is also phosphorylated in vitro and in vivo by microtubule-associated serine/threonine kinase 3 (MAST3 kinase), an enzyme of previously unknown function that is enriched in striatum. We find that ARPP-16 interacts directly with the scaffolding A subunit of the serine/threonine protein phosphatase, PP2A, and that phosphorylation of ARPP-16 at Ser46 by MAST3 kinase converts the protein into a selective inhibitor of B55α- and B56δ-containing heterotrimeric forms of PP2A. Ser46 of ARPP-16 is phosphorylated to a high basal stoichiometry in striatum, suggestive of basal inhibition of PP2A in striatal neurons. In support of this hypothesis, conditional knock-out of ARPP-16 in CaMKIIα::cre/floxed ARPP-16/19 mice results in dephosphorylation of a subset of PP2A substrates including phospho-Thr75-DARPP-32, phospho-T308-Akt, and phospho-T202/Y204-ERK. Conditional knock-out of ARPP-16/19 is associated with increased motivation measured on a progressive ratio schedule of food reinforcement, yet an attenuated locomotor response to acute cocaine. Our previous studies have shown that ARPP-16 is phosphorylated at Ser88 by PKA. Activation of PKA in striatal slices leads to phosphorylation of Ser88, and this is accompanied by marked dephosphorylation of Ser46. Together, these studies suggest that phospho-Ser46-ARPP-16 acts to basally control PP2A in striatal medium spiny neurons but that dopamine acting via PKA inactivates ARPP-16 leading to selective potentiation of PP2A signaling. SIGNIFICANCE STATEMENT We describe a novel mechanism of signal transduction enriched in medium spiny neurons of striatum that likely mediates effects of the neurotransmitter dopamine acting on these cells. We

  9. Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Qiao, Lan [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Pharmaceutical Sciences, Jilin University, Changchun 130021 (China); Paul, Pritha; Lee, Sora [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Qiao, Jingbo [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Wang, Yongsheng [Department of Pharmaceutical Sciences, Jilin University, Changchun 130021 (China); Chung, Dai H., E-mail: dai.chung@vanderbilt.edu [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States)

    2013-05-31

    Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated that GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma.

  10. Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

    International Nuclear Information System (INIS)

    Qiao, Lan; Paul, Pritha; Lee, Sora; Qiao, Jingbo; Wang, Yongsheng; Chung, Dai H.

    2013-01-01

    Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated that GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma

  11. Novel Indications for Bruton’s Tyrosine Kinase Inhibitors, beyond Hematological Malignancies

    Directory of Open Access Journals (Sweden)

    Robert Campbell

    2018-03-01

    Full Text Available Bruton’s tyrosine kinase (BTK is a critical terminal enzyme in the B-cell antigen receptor (BCR pathway. BTK activation has been implicated in the pathogenesis of certain B-cell malignancies. Targeting this pathway has emerged as a novel target in B-cell malignancies, of which ibrutinib is the first-in-class agent. A few other BTK inhibitors (BTKi are also under development (e.g., acalabrutinib. While the predominant action of BTKi is the blockade of B-cell receptor pathway within malignant B-cells, increasing the knowledge of off-target effects as well as a potential role for B-cells in proliferation of solid malignancies is expanding the indication of BTKi into non-hematological malignancies. In addition to the expansion of the role of BTKi monotherapy, combination therapy strategies utilizing ibrutinib with established regimens and combination with modern immunotherapy compounds are being explored.

  12. Tofacitinib, an Oral Janus Kinase Inhibitor: Perspectives in Dermatology.

    Science.gov (United States)

    Kostovic, Kresimir; Gulin, Sandra J; Mokos, Zrinka B; Ceovic, Romana

    2017-05-31

    Tofacitinib (formerly known as CP-690,550, CP690550, tasocitinib), a novel selective immunosuppressant, is a small molecule classified as Janus kinase inhibitor. The aim of this review article is to present updated data summary on the tofacitinib in the field of dermatology. We undertook a structured search of bibliographic databases for peer-reviewed scientific articles, including review articles, original research articles as well as case report articles based on inclusion/exclusion criteria. Technical reports on tofacitinib from U.S. Food and Drug Administration and European Medical Agency were also included. Forty-three papers were included in this review. We report current data on tofacitinib chemical properties, pharmacology, non-clinical toxicity, as well as efficacy and safety in potential new indications in dermatology: psoriasis, alopecia areata, vitiligo, atopic dermatitis and nail dystrophy associated with alopecia areata. JAK/STAT pathway has an important role in the pathogenesis of psoriasis, alopecia areata, atopic dermatitis, and vitiligo. Despite encouraging efficacy, due to concerns about the overall safety profile of tofacitinib, additional studies will have to determine the adequate risk-to-benefit ratio. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  13. Discovery of imidazopyridine derivatives as novel c-Met kinase inhibitors: Synthesis, SAR study, and biological activity.

    Science.gov (United States)

    Yang, Yifei; Zhang, Yuan; Yang, LingYun; Zhao, Leilei; Si, Lianghui; Zhang, Huibin; Liu, Qingsong; Zhou, Jinpei

    2017-02-01

    Receptor tyrosine kinase c-Met acts as an alternative angiogenic pathway in the process and contents of cancers. A series of imidazopyridine derivatives were designed and synthesized according to the established docking studies as possible c-Met inhibitors. Most of these imidazopyridine derivatives displayed nanomolar potency against c-Met in both biochemical enzymatic screens and cellular pharmacology studies. Especially, compound 7g exhibited the most inhibitory activity against c-Met with IC 50 of 53.4nM and 253nM in enzymatic and cellular level, respectively. Following that, the compound 7g was docked into the protein of c-Met and the structure-activity relationship was analyzed in detail. These findings indicated that the novel imidazopyridine derivative compound 7g was a potential c-Met inhibitor deserving further investigation for cancer treatment. Copyright © 2016. Published by Elsevier Inc.

  14. AT13148, a first-in-class multi-AGC kinase inhibitor, potently inhibits gastric cancer cells both in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Xi, Yu [Tongji Hospital, Tongji Medical School, Huazhong University of Science and Technology, Wuhan, Hubei 430030 (China); Department of General Surgery, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi, Xinjiang 832008 (China); Niu, Jianhua [Department of General Surgery, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi, Xinjiang 832008 (China); Shen, Yun [Department of Gastroenterology, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi, Xinjiang 832008 (China); Li, Dongmei [Department of Biochemistry and Molecular Biology, School of Medicine, Shihezi University, Shihezi, Xinjiang 832008 (China); Peng, Xinyu, E-mail: pppengxinyu@sina.com [Department of General Surgery, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi, Xinjiang 832008 (China); Wu, Xiangwei, E-mail: wuxiangweiys@126.com [Department of General Surgery, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi, Xinjiang 832008 (China)

    2016-09-09

    The AGC kinase family is important cell proliferation and survival. Dysregulation of this family contributes to gastric cancer progression. Here, we evaluated the potential activity of AT13148, a first-in-class multi-AGC kinase inhibitor, against gastric cancer cells. Our results showed that AT13148 exerted potent cytotoxic and anti-proliferative activities against a panel human gastric cancer cell lines (HGC-27, AGS, SNU-601, N87 and MKN-28), possibly via inducing cancer cell apoptotic death. Apoptosis inhibition by the Caspase blockers dramatically attenuated AT13148-caused cytotoxicity against gastric cancer cells. Intriguingly, same AT13148 treatment was not cytotoxic/pro-apoptotic to the non-cancerous human gastric epithelial GEC-1 cells. At the signaling level, AT13148 treatment in gastric cancer cells dramatically suppressed activation of multiple AGC kinases, including Akt (at p-Thr-308), p70S6 kinase (p70S6K), glycogen synthase kinase 3β (GSK-3β) and p90 ribosomal S6 kinase (RSK). Our in vivo studies demonstrated that daily oral gavage of AT13148 at well-tolerated doses significantly inhibited HGC27 xenograft tumor growth in nude mice. AGC activity was also dramatically decreased in AT13148-administrated HGC27 tumors. Therefore, targeting AGC kinases by AT13148 demonstrates superior anti-gastric cancer activity both in vitro and in vivo. The preclinical results of this study support the progression of this molecule into future evaluation as a valuable anti-gastric cancer candidate. - Highlights: • AT13148 is cytotoxic and anti-proliferative to human gastric cancer cells. • AT13148 induces gastric cancer cell apoptotic death, inhibited by Caspase inhibitors. • AT13148 inactivates multiple AGC kinases in human gastric cancer cells. • AT13148 oral administration suppresses HGC27 xenograft growth in nude mice. • AT13148 oral administration inhibits multiple AGC kinases in HGC27 xenograft tumors.

  15. Enzastaurin (LY317615), a Protein Kinase C Beta Selective Inhibitor, Enhances Antiangiogenic Effect of Radiation

    International Nuclear Information System (INIS)

    Willey, Christopher D.; Xiao Dakai; Tu Tianxiang; Kim, Kwang Woon; Moretti, Luigi; Niermann, Kenneth J.; Tawtawy, Mohammed N.; Quarles, Chad C. Ph.D.; Lu Bo

    2010-01-01

    Purpose: Angiogenesis has generated interest in oncology because of its important role in cancer growth and progression, particularly when combined with cytotoxic therapies, such as radiotherapy. Among the numerous pathways influencing vascular growth and stability, inhibition of protein kinase B(Akt) or protein kinase C(PKC) can influence tumor blood vessels within tumor microvasculature. Therefore, we wanted to determine whether PKC inhibition could sensitize lung tumors to radiation. Methods and Materials: The combination of the selective PKCβ inhibitor Enzastaurin (ENZ, LY317615) and ionizing radiation were used in cell culture and a mouse model of lung cancer. Lung cancer cell lines and human umbilical vascular endothelial cells (HUVEC) were examined using immunoblotting, cytotoxic assays including cell proliferation and clonogenic assays, and Matrigel endothelial tubule formation. In vivo, H460 lung cancer xenografts were examined for tumor vasculature and proliferation using immunohistochemistry. Results: ENZ effectively radiosensitizes HUVEC within in vitro models. Furthermore, concurrent ENZ treatment of lung cancer xenografts enhanced radiation-induced destruction of tumor vasculature and proliferation by IHC. However, tumor growth delay was not enhanced with combination treatment compared with either treatment alone. Analysis of downstream effectors revealed that HUVEC and the lung cancer cell lines differed in their response to ENZ and radiation such that only HUVEC demonstrate phosphorylated S6 suppression, which is downstream of mTOR. When ENZ was combined with the mTOR inhibitor, rapamycin, in H460 lung cancer cells, radiosensitization was observed. Conclusion: PKC appears to be crucial for angiogenesis, and its inhibition by ENZ has potential to enhance radiotherapy in vivo.

  16. Thromboembolism with Janus Kinase (JAK) Inhibitors for Rheumatoid Arthritis: How Real is the Risk?

    Science.gov (United States)

    Scott, Ian C; Hider, Samantha L; Scott, David L

    2018-03-02

    Two different Janus kinase (JAK) inhibitors-baricitinib and tofacitinib-are effective and licensed in active rheumatoid arthritis (RA). There have been recent concerns about potential thromboembolic risks with these drugs. Concerns about baricitinib focus on clinical trial findings. Using all publically available data, we estimate thromboembolic risks are approximately five events per 1000 patient years with 4 mg baricitinib daily. Concerns about tofacitinib have been raised by analyses of the Federal Drug Administration Adverse Event Reporting System (FAERs). These show some evidence of increased risks of pulmonary thrombosis, though not pulmonary embolism or venous thrombosis. Observational studies suggest in the general population and non-RA controls there are one to four thromboembolic events per 1000 patient years. In RA, thromboembolic risks increase to three to seven per 1000 patient years. The impact of biologics and disease-modifying anti-rheumatic drugs (DMARDs) on disease risk appears minimal, and the number of thromboembolic events is between four and eight per 1000 patient years. In the short term, full details of thromboembolic events in trials of JAK inhibitors need to be published. As the numbers of thromboembolic events will be small and patients enrolled in trials are not representative of all RA patients who may receive JAK inhibitors, this information is unlikely to provide definitive answers. Consequently, in the longer term, large observational studies are needed to accurately quantify thromboembolic risks attributable to JAK inhibitors and other drugs used to treat RA, and differentiate these from risks attributable to RA itself and its comorbidities.

  17. The cAMP-dependent protein kinase inhibitor H-89 attenuates the bioluminescence signal produced by Renilla Luciferase.

    Directory of Open Access Journals (Sweden)

    Katie J Herbst

    2009-05-01

    Full Text Available Investigations into the regulation and functional roles of kinases such as cAMP-dependent protein kinase (PKA increasingly rely on cellular assays. Currently, there are a number of bioluminescence-based assays, for example reporter gene assays, that allow the study of the regulation, activity, and functional effects of PKA in the cellular context. Additionally there are continuing efforts to engineer improved biosensors that are capable of detecting real-time PKA signaling dynamics in cells. These cell-based assays are often utilized to test the involvement of PKA-dependent processes by using H-89, a reversible competitive inhibitor of PKA.We present here data to show that H-89, in addition to being a competitive PKA inhibitor, attenuates the bioluminescence signal produced by Renilla luciferase (RLuc variants in a population of cells and also in single cells. Using 10 microM of luciferase substrate and 10 microM H-89, we observed that the signal from RLuc and RLuc8, an eight-point mutation variant of RLuc, in cells was reduced to 50% (+/-15% and 54% (+/-14% of controls exposed to the vehicle alone, respectively. In vitro, we showed that H-89 decreased the RLuc8 bioluminescence signal but did not compete with coelenterazine-h for the RLuc8 active site, and also did not affect the activity of Firefly luciferase. By contrast, another competitive inhibitor of PKA, KT5720, did not affect the activity of RLuc8.The identification and characterization of the adverse effect of H-89 on RLuc signal will help deconvolute data previously generated from RLuc-based assays looking at the functional effects of PKA signaling. In addition, for the current application and future development of bioluminscence assays, KT5720 is identified as a more suitable PKA inhibitor to be used in conjunction with RLuc-based assays. These principal findings also provide an important lesson to fully consider all of the potential effects of experimental conditions on a cell

  18. Partial purification and characterization of a wortmannin-sensitive and insulin-stimulated protein kinase that activates heart 6-phosphofructo-2-kinase.

    OpenAIRE

    Deprez, J; Bertrand, L; Alessi, D R; Krause, U; Hue, L; Rider, M H

    2000-01-01

    A wortmannin-sensitive and insulin-stimulated protein kinase (WISK), which phosphorylates and activates cardiac 6-phosphofructo-2-kinase (PFK-2), was partially purified from perfused rat hearts. Immunoblotting showed that WISK was devoid of protein kinase B (PKB), serum- and glucocorticoid-regulated protein kinase and protein kinase Czeta (PKCzeta). Comparison of the inhibition of WISK, PKCalpha and PKCzeta by different protein kinase inhibitors suggested that WISK was not a member of the PKC...

  19. A phase i study of the cyclin-dependent kinase 4/6 inhibitor ribociclib (LEE011) in patients with advanced solid tumors and lymphomas

    NARCIS (Netherlands)

    Infante, Jeffrey R.; Cassier, Philippe A.; Gerecitano, John F.; Witteveen, Petronella O.; Chugh, Rashmi; Ribrag, Vincent; Chakraborty, Abhijit; Matano, Alessandro; Dobson, Jason R.; Crystal, Adam S.; Parasuraman, Sudha; Shapiro, Geoffrey I.

    2016-01-01

    Purpose: Ribociclib (an oral, highly specific cyclin-dependent kinase 4/6 inhibitor) inhibits tumor growth in preclinical models with intact retinoblastoma protein (Rb+). This first-in-human study investigated the MTD, recommended dose for expansion (RDE), safety, preliminary activity,

  20. Combining the ABL1 kinase inhibitor ponatinib and the histone deacetylase inhibitor vorinostat: a potential treatment for BCR-ABL-positive leukemia.

    Science.gov (United States)

    Okabe, Seiichi; Tauchi, Tetsuzo; Kimura, Shinya; Maekawa, Taira; Kitahara, Toshihiko; Tanaka, Yoko; Ohyashiki, Kazuma

    2014-01-01

    Resistance to imatinib (Gleevec®) in cancer cells is frequently because of acquired point mutations in the kinase domain of BCR-ABL. Ponatinib, also known as AP24534, is an oral multi-targeted tyrosine kinase inhibitor (TKI), and it has been investigated in a pivotal phase 2 clinical trial. The histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid) has been evaluated for its significant clinical activity in hematological malignancies. Thus, treatments combining ABL TKIs with additional drugs may be a promising strategy in the treatment of leukemia. In the current study, we analyzed the efficacy of ponatinib and vorinostat treatment by using BCR-ABL-positive cell lines. Treatment with ponatinib for 72 h inhibited cell growth and induced apoptosis in K562 cells in a dose-dependent manner. We found that ponatinib potently inhibited the growth of Ba/F3 cells ectopically expressing BCR-ABL T315I mutation. Upon BCR-ABL phosphorylation, Crk-L was decreased, and poly (ADP-ribose) polymerase (PARP) was activated in a dose-dependent manner. Combined treatment of Ba/F3 T315I mutant cells with vorinostat and ponatinib resulted in significantly increased cytotoxicity. Additionally, the intracellular signaling of ponatinib and vorinostat was examined. Caspase 3 and PARP activation increased after combination treatment with ponatinib and vorinostat. Moreover, an increase in the phosphorylation levels of γH2A.X was observed. Previously established ponatinib-resistant Ba/F3 cells were also resistant to imatinib, nilotinib, and dasatinib. We investigated the difference in the efficacy of ponatinib and vorinostat by using ponatinib-resistant Ba/F3 cells. Combined treatment of ponatinib-resistant cells with ponatinib and vorinostat caused a significant increase in cytotoxicity. Thus, combined administration of ponatinib and vorinostat may be a powerful strategy against BCR-ABL mutant cells and could enhance the cytotoxic effects of ponatinib in those BCR

  1. Cytokine-induced loss of glucocorticoid function: effect of kinase inhibitors, long-acting β(2-adrenoceptor [corrected] agonist and glucocorticoid receptor ligands.

    Directory of Open Access Journals (Sweden)

    Christopher F Rider

    Full Text Available Acting on the glucocorticoid receptor (NR3C1, glucocorticoids are widely used to treat inflammatory diseases. However, glucocorticoid resistance often leads to suboptimal asthma control. Since glucocorticoid-induced gene expression contributes to glucocorticoid activity, the aim of this study was to use a 2 × glucocorticoid response element (GRE reporter and glucocorticoid-induced gene expression to investigate approaches to combat cytokine-induced glucocorticoid resistance. Pre-treatment with tumor necrosis factor-α (TNF or interleukin-1β inhibited dexamethasone-induced mRNA expression of the putative anti-inflammatory genes RGS2 and TSC22D3, or just TSC22D3, in primary human airway epithelial and smooth muscle cells, respectively. Dexamethasone-induced DUSP1 mRNA was unaffected. In human bronchial epithelial BEAS-2B cells, dexamethasone-induced TSC22D3 and CDKN1C expression (at 6 h was reduced by TNF pre-treatment, whereas DUSP1 and RGS2 mRNAs were unaffected. TNF pre-treatment also reduced dexamethasone-dependent 2×GRE reporter activation. This was partially reversed by PS-1145 and c-jun N-terminal kinase (JNK inhibitor VIII, inhibitors of IKK2 and JNK, respectively. However, neither inhibitor affected TNF-dependent loss of dexamethasone-induced CDKN1C or TSC22D3 mRNA. Similarly, inhibitors of the extracellular signal-regulated kinase, p38, phosphoinositide 3-kinase or protein kinase C pathways failed to attenuate TNF-dependent repression of the 2×GRE reporter. Fluticasone furoate, fluticasone propionate and budesonide were full agonists relative to dexamethasone, while GSK9027, RU24858, des-ciclesonide and GW870086X were partial agonists on the 2×GRE reporter. TNF reduced reporter activity in proportion with agonist efficacy. Full and partial agonists showed various degrees of agonism on RGS2 and TSC22D3 expression, but were equally effective at inducing CDKN1C and DUSP1, and did not affect the repression of CDKN1C or TSC22D3

  2. Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

    International Nuclear Information System (INIS)

    Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

    1986-01-01

    Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca 2+ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting 32 P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated 32 P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor

  3. Prodrugs of herpes simplex thymidine kinase inhibitors.

    Science.gov (United States)

    Yanachkova, Milka; Xu, Wei-Chu; Dvoskin, Sofya; Dix, Edward J; Yanachkov, Ivan B; Focher, Federico; Savi, Lida; Sanchez, M Dulfary; Foster, Timothy P; Wright, George E

    2015-04-01

    Because guanine-based herpes simplex virus thymidine kinase inhibitors are not orally available, we synthesized various 6-deoxy prodrugs of these compounds and evaluated them with regard to solubility in water, oral bioavailability, and efficacy to prevent herpes simplex virus-1 reactivation from latency in a mouse model. Organic synthesis was used to prepare compounds, High Performance Liquid Chromatography (HPLC) to analyze hydrolytic conversion, Mass Spectrometry (MS) to measure oral bioavailability, and mouse latent infection and induced reactivation to evaluate the efficacy of a specific prodrug. Aqueous solubilities of prodrugs were improved, oxidation of prodrugs by animal cytosols occurred in vitro, and oral absorption of the optimal prodrug sacrovir™ (6-deoxy-mCF3PG) in the presence of the aqueous adjuvant Soluplus® and conversion to active compound N(2)-[3-(trifluoromethyl)pheny])guanine (mCF3PG) were accomplished in mice. Treatment of herpes simplex virus-1 latent mice with sacrovir™ in 1% Soluplus in drinking water significantly suppressed herpes simplex virus-1 reactivation and viral genomic replication. Ad libitum oral delivery of sacrovir™ was effective in suppressing herpes simplex virus-1 reactivation in ocularly infected latent mice as measured by the numbers of mice shedding infectious virus at the ocular surface, numbers of trigeminal ganglia positive for infectious virus, number of corneas that had detectable infectious virus, and herpes simplex virus-1 genome copy numbers in trigeminal ganglia following reactivation. These results demonstrate the statistically significant effect of the prodrug on suppressing herpes simplex virus-1 reactivation in vivo. © The Author(s) 2015.

  4. Gold nanoparticles-based electrochemical method for the detection of protein kinase with a peptide-like inhibitor as the bioreceptor

    Directory of Open Access Journals (Sweden)

    Sun K

    2017-03-01

    Full Text Available Kai Sun, Yong Chang, Binbin Zhou, Xiaojin Wang, Lin Liu Henan Province of Key Laboratory of New Optoelectronic Functional Materials, College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, Henan, People’s Republic of China Abstract: This article presents a general method for the detection of protein kinase with a peptide-like kinase inhibitor as the bioreceptor, and it was done by converting gold nanoparticles (AuNPs-based colorimetric assay into sensitive electrochemical analysis. In the colorimetric assay, the kinase-specific aptameric peptide triggered the aggregation of AuNPs in solution. However, the specific binding of peptide to the target protein (kinase inhibited its ability to trigger the assembly of AuNPs. In the electrochemical analysis, peptides immobilized on a gold electrode and presented as solution triggered together the in situ formation of AuNPs-based network architecture on the electrode surface. Nevertheless, the formation of peptide–kinase complex on the electrode surface made the peptide-triggered AuNPs assembly difficult. Electrochemical impedance spectroscopy was used to measure the change in surface property in the binding events. When a ferrocene-labeled peptide (Fc-peptide was used in this design, the network of AuNPs/Fc-peptide produced a good voltammetric signal. The competitive assay allowed for the detection of protein kinase A with a detection limit of 20 mU/mL. This work should be valuable for designing novel optical or electronic biosensors and likely lead to many detection applications. Keywords: electrochemical biosensor, colorimetric assay, gold nanoparticle, aptameric peptide, protein kinase A, signal amplification 

  5. Targeting colorectal cancer cells by a novel sphingosine kinase 1 inhibitor PF-543

    International Nuclear Information System (INIS)

    Ju, TongFa; Gao, DaQuan; Fang, Zheng-yu

    2016-01-01

    In this study, we showed that PF-543, a novel sphingosine kinase 1 (SphK1) inhibitor, exerted potent anti-proliferative and cytotoxic effects against a panel of established (HCT-116, HT-29 and DLD-1) and primary human colorectal cancer (CRC) cells. Its sensitivity was negatively associated with SphK1 expression level in the CRC cells. Surprisingly, PF-543 mainly induced programmed necrosis, but not apoptosis, in the CRC cells. CRC cell necrotic death was detected by lactate dehydrogenase (LDH) release, mitochondrial membrane potential (MMP) collapse and mitochondrial P53-cyclophilin-D (Cyp-D) complexation. Correspondingly, the necrosis inhibitor necrostatin-1 largely attenuated PF-543-induced cytotoxicity against CRC cells. Meanwhile, the Cyp-D inhibitors (sanglifehrin A and cyclosporin A), or shRNA-mediated knockdown of Cyp-D, remarkably alleviated PF-543-induced CRC cell necrotic death. Reversely, over-expression of wild-type Cyp-D in HCT-116 cells significantly increased PF-543's sensitivity. In vivo, PF-543 intravenous injection significantly suppressed HCT-116 xenograft growth in severe combined immunodeficient (SCID) mice, whiling remarkably improving the mice survival. The in vivo activity by PF-543 was largely attenuated when combined with the Cyp-D inhibitor cyclosporin A. Collectively, our results demonstrate that PF-543 exerts potent anti-CRC activity in vitro and in vivo. Mitochondrial programmed necrosis pathway is likely the key mechanism responsible for PF-543's actions in CRC cells. - Highlights: • PF-543 is anti-proliferative and cytotoxic to established and primary CRC cells. • PF-543 induces programmed necrosis, but not apoptosis, in CRC cells. • Modulation of mitochondrial protein cyclophilin-D alters PF-543's sensitivity. • PF-543 inhibits HCT-116 xenograft growth in SCID mice, improving mice survival. • Co-administration of cyclophilin-D inhibitor CsA inhibits PF-543's activity in vivo.

  6. Targeting colorectal cancer cells by a novel sphingosine kinase 1 inhibitor PF-543

    Energy Technology Data Exchange (ETDEWEB)

    Ju, TongFa [Department of Anal-colorectal Surgery, HangZhou First People' s Hospital, HangZhou (China); Gao, DaQuan [Hematological Department, HangZhou First People' s Hospital, HangZhou (China); Fang, Zheng-yu, E-mail: fangzhengyu158@sina.com [Department of Anal-colorectal Surgery, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou (China)

    2016-02-12

    In this study, we showed that PF-543, a novel sphingosine kinase 1 (SphK1) inhibitor, exerted potent anti-proliferative and cytotoxic effects against a panel of established (HCT-116, HT-29 and DLD-1) and primary human colorectal cancer (CRC) cells. Its sensitivity was negatively associated with SphK1 expression level in the CRC cells. Surprisingly, PF-543 mainly induced programmed necrosis, but not apoptosis, in the CRC cells. CRC cell necrotic death was detected by lactate dehydrogenase (LDH) release, mitochondrial membrane potential (MMP) collapse and mitochondrial P53-cyclophilin-D (Cyp-D) complexation. Correspondingly, the necrosis inhibitor necrostatin-1 largely attenuated PF-543-induced cytotoxicity against CRC cells. Meanwhile, the Cyp-D inhibitors (sanglifehrin A and cyclosporin A), or shRNA-mediated knockdown of Cyp-D, remarkably alleviated PF-543-induced CRC cell necrotic death. Reversely, over-expression of wild-type Cyp-D in HCT-116 cells significantly increased PF-543's sensitivity. In vivo, PF-543 intravenous injection significantly suppressed HCT-116 xenograft growth in severe combined immunodeficient (SCID) mice, whiling remarkably improving the mice survival. The in vivo activity by PF-543 was largely attenuated when combined with the Cyp-D inhibitor cyclosporin A. Collectively, our results demonstrate that PF-543 exerts potent anti-CRC activity in vitro and in vivo. Mitochondrial programmed necrosis pathway is likely the key mechanism responsible for PF-543's actions in CRC cells. - Highlights: • PF-543 is anti-proliferative and cytotoxic to established and primary CRC cells. • PF-543 induces programmed necrosis, but not apoptosis, in CRC cells. • Modulation of mitochondrial protein cyclophilin-D alters PF-543's sensitivity. • PF-543 inhibits HCT-116 xenograft growth in SCID mice, improving mice survival. • Co-administration of cyclophilin-D inhibitor CsA inhibits PF-543's activity in vivo.

  7. Plasticity of the PAS domain and a potential role for signal transduction in the histidine kinase DcuS

    NARCIS (Netherlands)

    Etzkorn, M.; Kneuper, H.; Dünnwald, P.; Vijayan, V.; Krämer, J.; Griesinger, C.; Becker, S.; Unden, G.; Baldus, M.

    2008-01-01

    The mechanistic understanding of how membrane-embedded sensor kinases recognize signals and regulate kinase activity is currently limited. Here we report structure-function relationships of the multidomain membrane sensor kinase DcuS using solidstate NMR, structural modeling and mutagenesis.

  8. Significant blockade of multiple receptor tyrosine kinases by MGCD516 (Sitravatinib), a novel small molecule inhibitor, shows potent anti-tumor activity in preclinical models of sarcoma.

    Science.gov (United States)

    Patwardhan, Parag P; Ivy, Kathryn S; Musi, Elgilda; de Stanchina, Elisa; Schwartz, Gary K

    2016-01-26

    Sarcomas are rare but highly aggressive mesenchymal tumors with a median survival of 10-18 months for metastatic disease. Mutation and/or overexpression of many receptor tyrosine kinases (RTKs) including c-Met, PDGFR, c-Kit and IGF1-R drive defective signaling pathways in sarcomas. MGCD516 (Sitravatinib) is a novel small molecule inhibitor targeting multiple RTKs involved in driving sarcoma cell growth. In the present study, we evaluated the efficacy of MGCD516 both in vitro and in mouse xenograft models in vivo. MGCD516 treatment resulted in significant blockade of phosphorylation of potential driver RTKs and induced potent anti-proliferative effects in vitro. Furthermore, MGCD516 treatment of tumor xenografts in vivo resulted in significant suppression of tumor growth. Efficacy of MGCD516 was superior to imatinib and crizotinib, two other well-studied multi-kinase inhibitors with overlapping target specificities, both in vitro and in vivo. This is the first report describing MGCD516 as a potent multi-kinase inhibitor in different models of sarcoma, superior to imatinib and crizotinib. Results from this study showing blockade of multiple driver signaling pathways provides a rationale for further clinical development of MGCD516 for the treatment of patients with soft-tissue sarcoma.

  9. Biological and Molecular Effects of Small Molecule Kinase Inhibitors on Low-Passage Human Colorectal Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Falko Lange

    2014-01-01

    Full Text Available Low-passage cancer cell lines are versatile tools to study tumor cell biology. Here, we have employed four such cell lines, established from primary tumors of colorectal cancer (CRC patients, to evaluate effects of the small molecule kinase inhibitors (SMI vemurafenib, trametinib, perifosine, and regorafenib in an in vitro setting. The mutant BRAF (V600E/V600K inhibitor vemurafenib, but also the MEK1/2 inhibitor trametinib efficiently inhibited DNA synthesis, signaling through ERK1/2 and expression of genes downstream of ERK1/2 in BRAF mutant cells only. In case of the AKT inhibitor perifosine, three cell lines showed a high or intermediate responsiveness to the drug while one cell line was resistant. The multikinase inhibitor regorafenib inhibited proliferation of all CRC lines with similar efficiency and independent of the presence or absence of KRAS, BRAF, PIK3CA, and TP53 mutations. Regorafenib action was associated with broad-range inhibitory effects at the level of gene expression but not with a general inhibition of AKT or MEK/ERK signaling. In vemurafenib-sensitive cells, the antiproliferative effect of vemurafenib was enhanced by the other SMI. Together, our results provide insights into the determinants of SMI efficiencies in CRC cells and encourage the further use of low-passage CRC cell lines as preclinical models.

  10. Expression of phosphorylated raf kinase inhibitor protein (pRKIP) is a predictor of lung cancer survival

    International Nuclear Information System (INIS)

    Huerta-Yepez, Sara; Chia, David; Bonavida, Benjamin; Goodglick, Lee; Yoon, Nam K; Hernandez-Cueto, Angeles; Mah, Vei; Rivera-Pazos, Clara M; Chatterjee, Devasis; Vega, Mario I; Maresh, Erin L; Horvath, Steve

    2011-01-01

    Raf-1 kinase inhibitor protein (RKIP) has been reported to negatively regulate signal kinases of major survival pathways. RKIP activity is modulated in part by phosphorylation on Serine 153 by protein kinase C, which leads to dissociation of RKIP from Raf-1. RKIP expression is low in many human cancers and represents an indicator of poor prognosis and/or induction of metastasis. The prognostic power has typically been based on total RKIP expression and has not considered the significance of phospho-RKIP. The present study examined the expression levels of both RKIP and phospho-RKIP in human lung cancer tissue microarray proteomics technology. Total RKIP and phospho-RKIP expression levels were similar in normal and cancerous tissues. phospho-RKIP levels slightly decreased in metastatic lesions. However, the expression levels of phospho-RKIP, in contrast to total RKIP, displayed significant predictive power for outcome with normal expression of phospho-RKIP predicting a more favorable survival compared to lower levels (P = 0.0118); this was even more pronounced in more senior individuals and in those with early stage lung cancer. This study examines for the first time, the expression profile of RKIP and phospho-RKIP in lung cancer. Significantly, we found that phospho-RKIP was a predictive indicator of survival

  11. Novel VEGFR-2 kinase inhibitor identified by the back-to-front approach

    Science.gov (United States)

    Sanphanya, Kingkan; Phowichit, Suwadee; Wattanapitayakul, Suvara K.; Fokin, Valery V.; Vajragupta, Opa

    2013-01-01

    Novel lead was developed as VEGFR-2 inhibitor by the back-to-front approach. Docking experiment guided that the 3-chloromethylphenylurea motif occupied the back pocket of the VEGFR-2 kinase. The attempt to enhance the binding affinity of 1 was made by expanding structure to access the front pocket using triazole as linker. A library of 1,4-(disubsituted)-1H-1,2,3-triazoles were screened in silico and one lead compound (VH02) was identified with enzymatic IC50 against VEGFR-2 of 0.56 μM. VH02 showed antiangiogenic effect by inhibiting the tube formation of HUVEC cells (EA.hy926) at 0.3 μM which was 13 times lower than its cytotoxic dose. The enzymatic and cellular activities suggested the potential of VH02 as a lead for further optimization. PMID:23562241

  12. Rho kinase inhibitor fasudil mitigates high-cholesterol diet-induced hypercholesterolemia and vascular damage.

    Science.gov (United States)

    Abdali, Nibrass Taher; Yaseen, Awny H; Said, Eman; Ibrahim, Tarek M

    2017-04-01

    The current study was designed to investigate the potential beneficial therapeutic outcome of Rho kinase inhibitor (fasudil) against hypercholesterolemia-induced myocardial and vascular injury in rabbits together with diet modification. Sixteen male rabbits were randomly divided into four groups: normal control group which received standard rabbit chow, hypercholesterolemic control group, and treated groups which received cholesterol-rich rabbit chow (1.5% cholesterol) for 8 weeks. Treated groups received either fasudil (100 mg/kg/day) or rosuvastatin (2.5 mg/kg/day) starting from the ninth week for further 4 weeks with interruption of the cholesterol-rich chow. Biochemical assessment of serum cholesterol, triglyceride, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and myocardial oxidative/antioxidant biomarkers malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH), besides biochemical assessment of serum nitric oxide (NO), creatine kinase (CK), and lactate dehydrogenase (LDH) activities and serum total antioxidant capacity (TAC), was conducted. Serum vascular cell adhesion molecule 1 (VCAM-1) and serum Rho-associated protein kinase 1 (ROCK-1) were also evaluated along with histopathological examination of aorta specimens. Fasudil administration significantly decreased serum cholesterol, triglyceride (TG), and LDL and significantly increased serum HDL, with concomitant decrease in serum CK and LDH activities, NO, and restoration of serum TAC. Myocardial MDA significantly declined; SOD activity and GSH contents were restored. Serum ROCK-1 and VCAM-1 levels significantly declined as well. Vascular improvement was confirmed with histopathological examination, which revealed normal aortic intema with the absence of atheromas. Fasudil has promising anti-atherogenic activity mediated primarily via alleviation of hypercholesterolemia-induced oxidative stress and modulation of inflammatory response.

  13. Carboplatin binding to histidine

    Energy Technology Data Exchange (ETDEWEB)

    Tanley, Simon W. M. [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom); Diederichs, Kay [University of Konstanz, D-78457 Konstanz (Germany); Kroon-Batenburg, Loes M. J. [Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Levy, Colin [University of Manchester, 131 Princess Street, Manchester M1 7DN (United Kingdom); Schreurs, Antoine M. M. [Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Helliwell, John R., E-mail: john.helliwell@manchester.ac.uk [University of Manchester, Brunswick Street, Manchester M13 9PL (United Kingdom)

    2014-08-29

    An X-ray crystal structure showing the binding of purely carboplatin to histidine in a model protein has finally been obtained. This required extensive crystallization trials and various novel crystal structure analyses. Carboplatin is a second-generation platinum anticancer agent used for the treatment of a variety of cancers. Previous X-ray crystallographic studies of carboplatin binding to histidine (in hen egg-white lysozyme; HEWL) showed the partial conversion of carboplatin to cisplatin owing to the high NaCl concentration used in the crystallization conditions. HEWL co-crystallizations with carboplatin in NaBr conditions have now been carried out to confirm whether carboplatin converts to the bromine form and whether this takes place in a similar way to the partial conversion of carboplatin to cisplatin observed previously in NaCl conditions. Here, it is reported that a partial chemical transformation takes place but to a transplatin form. Thus, to attempt to resolve purely carboplatin binding at histidine, this study utilized co-crystallization of HEWL with carboplatin without NaCl to eliminate the partial chemical conversion of carboplatin. Tetragonal HEWL crystals co-crystallized with carboplatin were successfully obtained in four different conditions, each at a different pH value. The structural results obtained show carboplatin bound to either one or both of the N atoms of His15 of HEWL, and this particular variation was dependent on the concentration of anions in the crystallization mixture and the elapsed time, as well as the pH used. The structural details of the bound carboplatin molecule also differed between them. Overall, the most detailed crystal structure showed the majority of the carboplatin atoms bound to the platinum centre; however, the four-carbon ring structure of the cyclobutanedicarboxylate moiety (CBDC) remained elusive. The potential impact of the results for the administration of carboplatin as an anticancer agent are described.

  14. PI3Kδ inhibitor idelalisib in combination with BTK inhibitor ONO/GS-4059 in diffuse large B cell lymphoma with acquired resistance to PI3Kδ and BTK inhibitors.

    Directory of Open Access Journals (Sweden)

    Anella Yahiaoui

    Full Text Available Activated B-cell-like diffuse large B-cell lymphoma relies on B-cell receptor signaling to drive proliferation and survival. Downstream of the B-cell receptor, the key signaling kinases Bruton's tyrosine kinase and phosphoinositide 3-kinase δ offer opportunities for therapeutic intervention by agents such as ibrutinib, ONO/GS-4059, and idelalisib. Combination therapy with such targeted agents could provide enhanced efficacy due to complimentary mechanisms of action. In this study, we describe both the additive interaction of and resistance mechanisms to idelalisib and ONO/GS-4059 in a model of activated B-cell-like diffuse large B-cell lymphoma. Significant tumor regression was observed with a combination of PI3Kδ and Bruton's tyrosine kinase inhibitors in the mouse TMD8 xenograft. Acquired resistance to idelalisib in the TMD8 cell line occurred by loss of phosphatase and tensin homolog and phosphoinositide 3-kinase pathway upregulation, but not by mutation of PIK3CD. Sensitivity to idelalisib could be restored by combining idelalisib and ONO/GS-4059. Further evaluation of targeted inhibitors revealed that the combination of idelalisib and the phosphoinositide-dependent kinase-1 inhibitor GSK2334470 or the AKT inhibitor MK-2206 could partially overcome resistance. Characterization of acquired Bruton's tyrosine kinase inhibitor resistance revealed a novel tumor necrosis factor alpha induced protein 3 mutation (TNFAIP3 Q143*, which led to a loss of A20 protein, and increased p-IκBα. The combination of idelalisib and ONO/GS-4059 partially restored sensitivity in this resistant line. Additionally, a mutation in Bruton's tyrosine kinase at C481F was identified as a mechanism of resistance. The combination activity observed with idelalisib and ONO/GS-4059, taken together with the ability to overcome resistance, could lead to a new therapeutic option in activated B-cell-like diffuse large B-cell lymphoma. A clinical trial is currently underway to

  15. Development of Poly Lactic/Glycolic Acid (PLGA Microspheres for Controlled Release of Rho-Associated Kinase Inhibitor

    Directory of Open Access Journals (Sweden)

    Sho Koda

    2017-01-01

    Full Text Available Purpose. The purpose of this study was to investigate the feasibility of poly lactic/glycolic acid (PLGA as a drug delivery carrier of Rho kinase (ROCK inhibitor for the treatment of corneal endothelial disease. Method. ROCK inhibitor Y-27632 and PLGA were dissolved in water with or without gelatin (W1, and a double emulsion [(W1/O/W2] was formed with dichloromethane (O and polyvinyl alcohol (W2. Drug release curve was obtained by evaluating the released Y-27632 by using high performance liquid chromatography. PLGA was injected into the anterior chamber or subconjunctiva in rabbit eyes, and ocular complication was evaluated by slitlamp microscope and histological analysis. Results. Y-27632 incorporated PLGA microspheres with different molecular weights, and different composition ratios of lactic acid and glycolic acid were fabricated. A high molecular weight and low content of glycolic acid produced a slower and longer release. The Y-27632 released from PLGA microspheres significantly promoted the cell proliferation of cultured corneal endothelial cells. The injection of PLGA did not induce any evident eye complication. Conclusions. ROCK inhibitor-incorporated PLGA microspheres were fabricated, and the microspheres achieved the sustained release of ROCK inhibitor over 7–10 days in vitro. Our data should encourage researchers to use PLGA microspheres for treating corneal endothelial diseases.

  16. Ibrutinib targets mutant-EGFR kinase with a distinct binding conformation.

    Science.gov (United States)

    Wang, Aoli; Yan, Xiao-E; Wu, Hong; Wang, Wenchao; Hu, Chen; Chen, Cheng; Zhao, Zheng; Zhao, Peng; Li, Xixiang; Wang, Li; Wang, Beilei; Ye, Zi; Wang, Jinhua; Wang, Chu; Zhang, Wei; Gray, Nathanael S; Weisberg, Ellen L; Chen, Liang; Liu, Jing; Yun, Cai-Hong; Liu, Qingsong

    2016-10-25

    Ibrutinib, a clinically approved irreversible BTK kinase inhibitor for Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL) etc, has been reported to be potent against EGFR mutant kinase and currently being evaluated in clinic for Non Small Cell Lung Cancer (NSCLC). Through EGFR wt/mutant engineered isogenic BaF3 cell lines we confirmed the irreversible binding mode of Ibrutinib with EGFR wt/mutant kinase via Cys797. However, comparing to typical irreversible EGFR inhibitor, such as WZ4002, the washing-out experiments revealed a much less efficient covalent binding for Ibrutinib. The biochemical binding affinity examination in the EGFR L858R/T790M kinase revealed that, comparing to more efficient irreversible inhibitor WZ4002 (Kd: 0.074 μM), Ibrutinib exhibited less efficient binding (Kd: 0.18 μM). An X-ray crystal structure of EGFR (T790M) in complex with Ibrutinib exhibited a unique DFG-in/c-Helix-out inactive binding conformation, which partially explained the less efficiency of covalent binding and provided insight for further development of highly efficient irreversible binding inhibitor for the EGFR mutant kinase. These results also imply that, unlike the canonical irreversible inhibitor, sustained effective concentration might be required for Ibrutinib in order to achieve the maximal efficacy in the clinic application against EGFR driven NSCLC.

  17. Reduced intensity conditioning is superior to nonmyeloablative conditioning for older chronic myelogenous leukemia patients undergoing hematopoietic cell transplant during the tyrosine kinase inhibitor era

    DEFF Research Database (Denmark)

    Warlick, Erica; Ahn, Kwang Woo; Pedersen, Tanya L

    2012-01-01

    Tyrosine kinase inhibitors (TKIs) and reduced intensity conditioning (RIC)/nonmyeloablative (NMA) conditioning hematopoietic cell transplants (HCTs) have changed the therapeutic strategy for chronic myelogenous leukemia (CML) patients. We analyzed post-HCT outcomes of 306 CML patients reported to...

  18. Clonal evolution of AML on novel FMS-like tyrosine kinase-3 (FLT3 inhibitor therapy with evolving actionable targets

    Directory of Open Access Journals (Sweden)

    Pashtoon M. Kasi

    2016-01-01

    Full Text Available For acute myeloid leukemia (AML, identification of activating mutations in the FMS-like tyrosine kinase-3 (FLT3 has led to the development of several FLT3-inhibitors. Here we present clinical and next generation sequencing data at the time of progression of a patient on a novel FLT3-inhibitor clinical trial (ASP2215 to show that employing therapeutic interventions with these novel targeted therapies can lead to consequences secondary to selective pressure and clonal evolution of cancer. We describe novel findings alongside data on treatment directed towards actionable aberrations acquired during the process. (Clinical Trial: NCT02014558; registered at: 〈https://clinicaltrials.gov/ct2/show/NCT02014558〉

  19. Crizotinib-Resistant ROS1 Mutations Reveal a Predictive Kinase Inhibitor Sensitivity Model for ROS1- and ALK-Rearranged Lung Cancers.

    Science.gov (United States)

    Facchinetti, Francesco; Loriot, Yohann; Kuo, Mei-Shiue; Mahjoubi, Linda; Lacroix, Ludovic; Planchard, David; Besse, Benjamin; Farace, Françoise; Auger, Nathalie; Remon, Jordi; Scoazec, Jean-Yves; André, Fabrice; Soria, Jean-Charles; Friboulet, Luc

    2016-12-15

    The identification of molecular mechanisms conferring resistance to tyrosine kinase inhibitor (TKI) is a key step to improve therapeutic results for patients with oncogene addiction. Several alterations leading to EGFR and anaplastic lymphoma kinase (ALK) resistance to TKI therapy have been described in non-small cell lung cancer (NSCLC). Only two mutations in the ROS1 kinase domain responsible for crizotinib resistance have been described in patients thus far. A patient suffering from a metastatic NSCLC harboring an ezrin (EZR)-ROS1 fusion gene developed acquired resistance to the ALK/ROS1 inhibitor crizotinib. Molecular analysis (whole-exome sequencing, CGH) and functional studies were undertaken to elucidate the mechanism of resistance. Based on this case, we took advantage of the structural homology of ROS1 and ALK to build a predictive model for drug sensitivity regarding future ROS1 mutations. Sequencing revealed a dual mutation, S1986Y and S1986F, in the ROS1 kinase domain. Functional in vitro studies demonstrated that ROS1 harboring either the S1986Y or the S1986F mutation, while conferring resistance to crizotinib and ceritinib, was inhibited by lorlatinib (PF-06463922). The patient's clinical response confirmed the potency of lorlatinib against S1986Y/F mutations. The ROS1 S1986Y/F and ALK C1156Y mutations are homologous and displayed similar sensitivity patterns to ALK/ROS1 TKIs. We extended this analogy to build a model predicting TKI efficacy against potential ROS1 mutations. Clinical evidence, in vitro validation, and homology-based prediction provide guidance for treatment decision making for patients with ROS1-rearranged NSCLC who progressed on crizotinib. Clin Cancer Res; 22(24); 5983-91. ©2016 AACR. ©2016 American Association for Cancer Research.

  20. Learning curve for the management of tyrosine kinase inhibitors as the first line of treatment for patients with metastatic renal cancer.

    Science.gov (United States)

    Lendínez-Cano, G; Osman García, I; Congregado Ruiz, C B; Conde Sánchez, J M; Medina López, R A

    2018-03-07

    To analyse the learning curve for the management of tyrosine kinase inhibitors as the first line of treatment for patients with metastatic renal cancer. We evaluated 32 consecutive patients treated in our department for metastatic renal cancer with tyrosine kinase inhibitors (pazopanib or sunitinib) as first-line treatment between September 2012 and November 2015. We retrospectively analysed this sample. We measured the time to the withdrawal of the first-line treatment, the time to progression and overall survival using Kaplan-Meier curves. The learning curve was analysed with the cumulative sum (CUSUM) methodology. In our series, the median time to the withdrawal of the first-line treatment was 11 months (95% CI 4.9-17.1). The mean time to progression was 30.4 months (95% CI 22.7-38.1), and the mean overall survival was 34.9 months (95% CI 27.8-42). By applying the CUSUM methodology, we obtained a graph for the CUSUM value of the time to withdrawal of the first-line treatment (CUSUM TW), observing 3 well-differentiated phases: phase 1 or initial learning phase (1-15), phase 2 (16-26) in which the management of the drug progressively improved and phase 3 (27-32) of maximum experience or mastery of the management of these drugs. The number of treated patients needed to achieve the proper management of these patients was estimated at 15. Despite the limitations of the sample size and follow-up time, we estimated (in 15 patients) the number needed to reach the necessary experience in the management of these patients with tyrosine kinase inhibitors. We observed no relationship between the time to the withdrawal of the first-line treatment for any cause and progression. Copyright © 2018 AEU. Publicado por Elsevier España, S.L.U. All rights reserved.

  1. Inhibition of colony-stimulating-factor-1 signaling in vivo with the orally bioavailable cFMS kinase inhibitor GW2580.

    Science.gov (United States)

    Conway, James G; McDonald, Brad; Parham, Janet; Keith, Barry; Rusnak, David W; Shaw, Eva; Jansen, Marilyn; Lin, Peiyuan; Payne, Alan; Crosby, Renae M; Johnson, Jennifer H; Frick, Lloyd; Lin, Min-Hwa Jasmine; Depee, Scott; Tadepalli, Sarva; Votta, Bart; James, Ian; Fuller, Karen; Chambers, Timothy J; Kull, Frederick C; Chamberlain, Stanley D; Hutchins, Jeff T

    2005-11-01

    Colony-stimulating-factor-1 (CSF-1) signaling through cFMS receptor kinase is increased in several diseases. To help investigate the role of cFMS kinase in disease, we identified GW2580, an orally bioavailable inhibitor of cFMS kinase. GW2580 completely inhibited human cFMS kinase in vitro at 0.06 microM and was inactive against 26 other kinases. GW2580 at 1 microM completely inhibited CSF-1-induced growth of mouse M-NFS-60 myeloid cells and human monocytes and completely inhibited bone degradation in cultures of human osteoclasts, rat calvaria, and rat fetal long bone. In contrast, GW2580 did not affect the growth of mouse NS0 lymphoblastoid cells, human endothelial cells, human fibroblasts, or five human tumor cell lines. GW2580 also did not affect lipopolysaccharide (LPS)-induced TNF, IL-6, and prostaglandin E2 production in freshly isolated human monocytes and mouse macrophages. After oral administration, GW2580 blocked the ability of exogenous CSF-1 to increase LPS-induced IL-6 production in mice, inhibited the growth of CSF-1-dependent M-NFS-60 tumor cells in the peritoneal cavity, and diminished the accumulation of macrophages in the peritoneal cavity after thioglycolate injection. Unexpectedly, GW2580 inhibited LPS-induced TNF production in mice, in contrast to effects on monocytes and macrophages in vitro. In conclusion, GW2580's selective inhibition of monocyte growth and bone degradation is consistent with cFMS kinase inhibition. The ability of GW2580 to chronically inhibit CSF-1 signaling through cFMS kinase in normal and tumor cells in vivo makes GW2580 a useful tool in assessing the role of cFMS kinase in normal and disease processes.

  2. A Stabilized Demethoxyviridin Derivative Inhibits PI3 kinase

    Science.gov (United States)

    Yuan, Hushan; Pupo, Monica T.; Blois, Joe; Smith, Adam; Weissleder, Ralph; Clardy, Jon; Josephson, Lee

    2009-01-01

    The viridins like demethoxyviridin (Dmv) and wortmannin (Wm) are nanomolar inhibitors of the PI3 kinases, a family of enzymes that play key roles in a host of regulatory processes. Central to the use of these compounds to investigate the role of PI3 kinase in biological systems, or as scaffolds for drug development, are the interrelated issues of stability, chemical reactivity, and bioactivity as inhibitors of PI3 kinase. We found that Dmv was an even more potent inhibitor of PI3 kinase than Wm. However, Dmv was notably less stable than Wm in PBS, with a half-life of 26 min vs Wm’s half-life of 3470 min. Dmv, like Wm, disappeared in culture media with a half-life of less than 1 min. To overcome Dmv’s instability, it was esterified at the C1 position, and then reacted with glycine at the C20 position. The resulting Dmv derivative, termed SA-DmvC20-Gly had a half-life of 218 min in PBS and 64 min in culture media. SA-DmvC20-Gly underwent an exchange reaction at the C20 position with N-acetyl lysine in a manner similar to a WmC20 derivative, WmC20-Proline. SA-DmvC20-Gly inhibited PI3 kinase with an IC50 of 44 nM, compared to Wm’s IC50 of 12 nM. These results indicate that the stability of Dmv can be manipulated by reactions at the C1 and C20 positions, while substantially maintaining its ability to inhibit PI3 kinase. Our results indicate it may be possible to obtain stabilized Dmv derivatives for use as PI3 kinase inhibitors in biological systems. PMID:19523825

  3. Egress of CD19(+)CD5(+) cells into peripheral blood following treatment with the Bruton tyrosine kinase inhibitor ibrutinib in mantle cell lymphoma patients

    NARCIS (Netherlands)

    Chang, Betty Y.; Francesco, Michelle; de Rooij, Martin F. M.; Magadala, Padmaja; Steggerda, Susanne M.; Huang, Min Mei; Kuil, Annemieke; Herman, Sarah E. M.; Chang, Stella; Pals, Steven T.; Wilson, Wyndham; Wiestner, Adrian; Spaargaren, Marcel; Buggy, Joseph J.; Elias, Laurence

    2013-01-01

    Ibrutinib (PCI-32765) is a highly potent oral Bruton tyrosine kinase (BTK) inhibitor in clinical development for treating B-cell lymphoproliferative diseases. Patients with chronic lymphocytic leukemia (CLL) often show marked, transient increases of circulating CLL cells following ibrutinib

  4. Imidazopyridine-based inhibitors of glycogen synthase kinase 3: synthesis and evaluation of amide isostere replacements of the carboxamide scaffold.

    Science.gov (United States)

    Yngve, Ulrika; Söderman, Peter; Svensson, Mats; Rosqvist, Susanne; Arvidsson, Per I

    2012-11-01

    In this study, we explored the effect of bioisostere replacement in a series of glycogen synthase kinase 3 (GSK3) inhibitors based on the imidazopyridine core. The synthesis and biological evaluation of a number of novel sulfonamide, 1,2,4-oxadiazole, and thiazole derivates as amide bioisosteres, as well as a computational rationalization of the obtained results are reported. Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.

  5. Side-effects of protein kinase inhibitors on ion channels

    Indian Academy of Sciences (India)

    2013-11-06

    Nov 6, 2013 ... with aberrant kinase activity, including cancers, arthritis and cardiovascular disorders. Several strategies .... family, the β-adrenergic receptor kinase (βARK), the ribosomal S6 ..... urinary bladder smooth muscle cells. While no ...

  6. 2-Fluoro-l-histidine

    Directory of Open Access Journals (Sweden)

    Kiran K. Andra

    2010-11-01

    Full Text Available The title compound, C6H8FN3O2, an analog of histidine, shows a reduced side-chain pKa (ca 1. The title structure exhibits a shortening of the bond between the proximal ring N atom and the F-substituted ring C atom, indicating an increase in π-bond character due to an inductive effect of fluorine.

  7. The anthraquinone emodin inhibits the non-exported FIKK kinase from Plasmodium falciparum.

    Science.gov (United States)

    Lin, Benjamin C; Harris, Darcy R; Kirkman, Lucy M D; Perez, Astrid M; Qian, Yiwen; Schermerhorn, Janse T; Hong, Min Y; Winston, Dennis S; Xu, Lingyin; Lieber, Alexander M; Hamilton, Matthew; Brandt, Gabriel S

    2017-12-01

    The FIKK family of kinases is unique to parasites of the Apicomplexan order, which includes all malaria parasites. Plasmodium falciparum, the most virulent form of human malaria, has a family of 19 FIKK kinases, most of which are exported into the host red blood cell during malaria infection. Here, we confirm that FIKK 8 is a non-exported member of the FIKK kinase family. Through expression and purification of the recombinant kinase domain, we establish that emodin is a relatively high-affinity (IC 50 =2μM) inhibitor of PfFk8. Closely related anthraquinones do not inhibit PfFk8, suggesting that the particular substitution pattern of emodin is critical to the inhibitory pharmacophore. This first report of a P. falciparum FIKK kinase inhibitor lays the groundwork for developing specific inhibitors of the various members of the FIKK kinase family in order to probe their physiological function. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Pivotal role of glycogen synthase kinase-3: A therapeutic target for Alzheimer's disease.

    Science.gov (United States)

    Maqbool, Mudasir; Mobashir, Mohammad; Hoda, Nasimul

    2016-01-01

    Neurodegenerative diseases are among the most challenging diseases with poorly known mechanism of cause and paucity of complete cure. Out of all the neurodegenerative diseases, Alzheimer's disease is the most devastating and loosening of thinking and judging ability disease that occurs in the old age people. Many hypotheses came forth in order to explain its causes. In this review, we have enlightened Glycogen Synthase Kinase-3 which has been considered as a concrete cause for Alzheimer's disease. Plaques and Tangles (abnormal structures) are the basic suspects in damaging and killing of nerve cells wherein Glycogen Synthase Kinase-3 has a key role in the formation of these fatal accumulations. Various Glycogen Synthase Kinase-3 inhibitors have been reported to reduce the amount of amyloid-beta as well as the tau hyperphosphorylation in both neuronal and nonneuronal cells. Additionally, Glycogen Synthase Kinase-3 inhibitors have been reported to enhance the adult hippocampal neurogenesis in vivo as well as in vitro. Keeping the chemotype of the reported Glycogen Synthase Kinase-3 inhibitors in consideration, they may be grouped into natural inhibitors, inorganic metal ions, organo-synthetic, and peptide like inhibitors. On the basis of their mode of binding to the constituent enzyme, they may also be grouped as ATP, nonATP, and allosteric binding sites competitive inhibitors. ATP competitive inhibitors were known earlier inhibitors but they lack efficient selectivity. This led to find the new ways for the enzyme inhibition. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  9. [Syk inhibitors].

    Science.gov (United States)

    Kimura, Yukihiro; Chihara, Kazuyasu; Takeuchi, Kenji; Sada, Kiyonao

    2013-07-01

    Non-receptor type of protein-tyrosine kinase Syk (spleen tyrosine kinase) was isolated in the University of Fukui in 1991. Syk is known to be essential for the various physiological functions, especially in hematopoietic lineage cells. Moreover, ectopic expression of Syk by epigenetic changes is reported to cause retinoblastoma. Recently, novel Syk inhibitors were developed and its usefulness has been evaluated in the treatment of allergic rhinitis, rheumatoid arthritis, and idiopathic thrombocytopenic purpura. In this review, we will summarize the history, structure, and function of Syk, and then describe the novel Syk inhibitors and their current status. Furthermore, we will introduce our findings of the adaptor protein 3BP2 (c-Abl SH3 domain-binding protein-2), as a novel target of Syk.

  10. Can the tyrosine kinase inhibitors trigger metabolic encephalopathy in cirrhotic patients?

    Science.gov (United States)

    Brandi, Giovanni; de Rosa, Francesco; Calzà, Laura; Girolamo, Stefania Di; Tufoni, Manuel; Ricci, Carmen Serena; Cirignotta, Fabio; Caraceni, Paolo; Biasco, Guido

    2013-03-01

    Sorafenib is the standard treatment of advanced hepatocarcinoma (HCC) in cirrhotic patients with preserved liver function. It shares many adverse effects with other tyrosine-kinase (TK) inhibitors and antiangiogenic drugs. TK inhibitors could have a direct toxicity on CNS, both by interfering with TK-related pathways and by inhibiting angiogenesis. The aim of this study was to investigate whether sorafenib administration can be associated to metabolic encephalopathy in patients with cirrhosis. We retrospectively reviewed medical records of all cirrhotic patients treated with sorafenib for HCC afferent at our Department from January 2009 to December 2011. Among 62 patients, we identified 10 patients with clinically significant cognitive impairment. Seven of these were clearly diagnosed with overt hepatic encephalopathy (HE), one with brain metastases and two with drug-related toxic-metabolic encephalopathy. These last two cases were characterized by severe cognitive impairment, mood alteration and memory deficit. Clinical exam, blood tests and brain CT excluded organic causes of encephalopathy and precipitating factors of HE. Sorafenib discontinuation was associated with complete reversal of the syndrome, which recurred on drug re-administration in one case. Our study suggests that sorafenib may be a precipitating factor of metabolic encephalopathy in cirrhotic patients with advanced HCC. This neurological syndrome appears to be not responsive to the conventional treatment for HE, but it is fully reversible by drug discontinuation. It can be speculated that the potential direct neuronal action of sorafenib may represent a trigger for the onset of metabolic encephalopathy in a subset of cirrhotic patients. © 2012 John Wiley & Sons A/S.

  11. Discovery and characterization of LY2784544, a small-molecule tyrosine kinase inhibitor of JAK2V617F

    International Nuclear Information System (INIS)

    Ma, L; Clayton, J R; Walgren, R A; Zhao, B; Evans, R J; Smith, M C; Heinz-Taheny, K M; Kreklau, E L; Bloem, L; Pitou, C; Shen, W; Strelow, J M; Halstead, C; Rempala, M E; Parthasarathy, S; Gillig, J R; Heinz, L J; Pei, H; Wang, Y; Stancato, L F; Dowless, M S; Iversen, P W; Burkholder, T P

    2013-01-01

    Owing to the prevalence of the JAK2V617F mutation in myeloproliferative neoplasms (MPNs), its constitutive activity, and ability to recapitulate the MPN phenotype in mouse models, JAK2V617F kinase is an attractive therapeutic target. We report the discovery and initial characterization of the orally bioavailable imidazopyridazine, LY2784544, a potent, selective and ATP-competitive inhibitor of janus kinase 2 (JAK2) tyrosine kinase. LY2784544 was discovered and characterized using a JAK2-inhibition screening assay in tandem with biochemical and cell-based assays. LY2784544 in vitro selectivity for JAK2 was found to be equal or superior to known JAK2 inhibitors. Further studies showed that LY2784544 effectively inhibited JAK2V617F-driven signaling and cell proliferation in Ba/F3 cells (IC 50 =20 and 55 nM, respectively). In comparison, LY2784544 was much less potent at inhibiting interleukin-3-stimulated wild-type JAK2-mediated signaling and cell proliferation (IC 50 =1183 and 1309 nM, respectively). In vivo, LY2784544 effectively inhibited STAT5 phosphorylation in Ba/F3-JAK2V617F-GFP (green fluorescent protein) ascitic tumor cells (TED 50 =12.7 mg/kg) and significantly reduced (P<0.05) Ba/F3-JAK2V617F-GFP tumor burden in the JAK2V617F-induced MPN model (TED 50 =13.7 mg/kg, twice daily). In contrast, LY2784544 showed no effect on erythroid progenitors, reticulocytes or platelets. These data suggest that LY2784544 has potential for development as a targeted agent against JAK2V617F and may have properties that allow suppression of JAK2V617F-induced MPN pathogenesis while minimizing effects on hematopoietic progenitor cells

  12. A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation

    International Nuclear Information System (INIS)

    Park, So Jung; Park, Young Jun; Shin, Ji Hyun; Kim, Eun Sung; Hwang, Jung Jin; Jin, Dong-Hoon; Kim, Jin Cheon; Cho, Dong-Hyung

    2011-01-01

    Highlights: → We screened and identified Tyrphostin A9, a receptor tyrosine kinase inhibitor as a strong mitochondria fission inducer. → Tyrphostin A9 treatment promotes mitochondria dysfunction and contributes to cytotoxicity in cancer cells. → Tyrphostin A9 induces apoptotic cell death through a Drp1-mediated pathway. → Our studies suggest that Tyrphostin A9 induces mitochondria fragmentation and apoptotic cell death via Drp1 dependently. -- Abstract: Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactive chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death.

  13. Isomeric mono-, di-, and tri-bromobenzo-1H-triazoles as inhibitors of human protein kinase CK2α.

    Directory of Open Access Journals (Sweden)

    Romualda Wąsik

    Full Text Available To further clarify the role of the individual bromine atoms of 4,5,6,7-tetrabromotriazole (TBBt, a relatively selective inhibitor of protein kinase CK2, we have examined the inhibition (IC(50 of human CK2α by the two mono-, the four di-, and the two tri- bromobenzotriazoles relative to that of TBBt. Halogenation of the central vicinal C(5/C(6 atoms proved to be a key factor in enhancing inhibitory activity, in that 5,6-di-Br(2Bt and 4,5,6-Br(3Bt were almost as effective inhibitors as TBBt, notwithstanding their marked differences in pK(a for dissociation of the triazole proton. The decrease in pK(a on halogenation of the peripheral C(4/C(7 atoms virtually nullifies the gain due to hydrophobic interactions, and does not lead to a decrease in IC(50. Molecular modeling of structures of complexes of the ligands with the enzyme, as well as QSAR analysis, pointed to a balance of hydrophobic and electrostatic interactions as a discriminator of inhibitory activity. The role of halogen bonding remains debatable, as originally noted for the crystal structure of TBBt with CK2α (pdb1j91. Finally we direct attention to the promising applicability of our series of well-defined halogenated benzotriazoles to studies on inhibition of kinases other than CK2.

  14. Novel p38α MAP kinase inhibitors identified from yoctoReactor DNA-encoded small molecule library

    DEFF Research Database (Denmark)

    Petersen, L. K.; Blakskjær, P.; Chaikuad, A.

    2016-01-01

    A highly specific and potent (7 nM cellular IC50) inhibitor of p38α kinase was identified directly from a 12.6 million membered DNA-encoded small molecule library. This was achieved using the high fidelity yoctoReactor technology (yR) for preparing the DNA-encoded library, and a homogeneous...... interactions. Moreover, the crystal structure showed, that although buried in the p38α active site, the original DNA attachment point of the compound was accessible through a channel created by the distorted P-loop conformation. This study demonstrates the usability of DNA-encoded library technologies...

  15. Histidine in Continuum Electrostatics Protonation State Calculations

    Science.gov (United States)

    Couch, Vernon; Stuchebruckhov, Alexei

    2014-01-01

    A modification to the standard continuum electrostatics approach to calculate protein pKas which allows for the decoupling of histidine tautomers within a two state model is presented. Histidine with four intrinsically coupled protonation states cannot be easily incorporated into a two state formalism because the interaction between the two protonatable sites of the imidazole ring is not purely electrostatic. The presented treatment, based on a single approximation of the interrelation between histidine’s charge states, allows for a natural separation of the two protonatable sites associated with the imidazole ring as well as the inclusion of all protonation states within the calculation. PMID:22072521

  16. Differential action of small molecule HER kinase inhibitors on receptor heterodimerization: therapeutic implications.

    Science.gov (United States)

    Sánchez-Martín, M; Pandiella, A

    2012-07-01

    Deregulation of ErbB/HER receptor tyrosine kinases has been linked to several types of cancer. The mechanism of activation of these receptors includes establishment of receptor dimers. Here, we have analyzed the action of different small molecule HER tyrosine kinase inhibitors (TKIs) on HER receptor dimerization. Breast cancer cell lines were treated with distinct TKIs and the formation of HER2-HER3 dimers was analyzed by coimmunoprecipitation and western blot or by Förster resonance energy transfer assays. Antibody-dependent cellular cytotoxicity was analyzed by measuring the release of lactate dehydrogenase and cell viability. Lapatinib and neratinib interfered with ligand-induced dimerization of HER receptors; while pelitinib, gefitinib, canertinib or erlotinib did not. Moreover, lapatinib and neratinib were able to disrupt previously formed receptor dimers. Structural analyses allowed the elucidation of the mechanism by which some TKIs prevent the formation of HER receptor dimers, while others do not. Experiments aimed at defining the functional importance of dimerization indicated that TKIs that impeded dimerization prevented down-regulation of HER2 receptors, and favored the action of trastuzumab. We postulate that TKIs that prevent dimerization and down-regulation of HER2 may augment the antitumoral action of trastuzumab, and this mechanism of action should be considered in the treatment of HER2 positive tumors which combine TKIs with antireceptor antibodies. Copyright © 2011 UICC.

  17. Protein-induced geometric constraints and charge transfer in bacteriochlorophyll-histidine complexes in LH2.

    Science.gov (United States)

    Wawrzyniak, Piotr K; Alia, A; Schaap, Roland G; Heemskerk, Mattijs M; de Groot, Huub J M; Buda, Francesco

    2008-12-14

    Bacteriochlorophyll-histidine complexes are ubiquitous in nature and are essential structural motifs supporting the conversion of solar energy into chemically useful compounds in a wide range of photosynthesis processes. A systematic density functional theory study of the NMR chemical shifts for histidine and for bacteriochlorophyll-a-histidine complexes in the light-harvesting complex II (LH2) is performed using the BLYP functional in combination with the 6-311++G(d,p) basis set. The computed chemical shift patterns are consistent with available experimental data for positive and neutral(tau) (N(tau) protonated) crystalline histidines. The results for the bacteriochlorophyll-a-histidine complexes in LH2 provide evidence that the protein environment is stabilizing the histidine close to the Mg ion, thereby inducing a large charge transfer of approximately 0.5 electronic equivalent. Due to this protein-induced geometric constraint, the Mg-coordinated histidine in LH2 appears to be in a frustrated state very different from the formal neutral(pi) (N(pi) protonated) form. This finding could be important for the understanding of basic functional mechanisms involved in tuning the electronic properties and exciton coupling in LH2.

  18. [Ionization energies and infrared spectra studies of histidine using density functional theory].

    Science.gov (United States)

    Hu, Qiong; Wang, Guo-Ying; Liu, Gang; Ou, Jia-Ming; Wang, Rui-Li

    2010-05-01

    Histidines provide axial ligands to the primary electron donors in photosynthetic reaction centers (RCs) and play an important role in the protein environments of these donors. In this paper the authors present a systematic study of ionization energies and vibrational properties of histidine using hybrid density functional theory (DFT). All calculations were undertaken by using B3LYP method in combination with four basis sets: 6-31G(d), 6-31G(df, p), 6-31+G(d) and 6-311+G(2d, 2p) with the aim to investigate how the basis sets influence the calculation results. To investigate solvent effects and gain a detailed understanding of marker bands of histidine, the ionization energies of histidine and the vibrational frequencies of histidine which are unlabeled and 13C, 15N, and 2H labeled in the gas phase, CCl4, protein environment, THF and water solution, which span a wide range of dielectric constant, were also calculated. Our results showed that: (1) The main geometry parameters of histidine were impacted by basis sets and mediums, and C2-N3 and N3-C4 bond of imidazole ring of histidine side chain display the maximum bond lengths in the gas phase; (2) single point energies and frequencies calculated were decreased while ionization energies increased with the increasing level of basis sets and diffuse function applied in the same solvent; (3) with the same computational method, the higher the dielectric constant of the solvent used, the lower the ionization energy and vibrational frequency and the higher the intensity obtained. In addition, calculated ionization energy in the gas phase and marker bands of histidine as well as frequency shift upon 13C and 15N labeling at the computationally more expensive 6-311+G(2d, 2p) level are in good agreement with experimental observations available in literatures. All calculations indicated that the results calculated by using higher level basis set with diffuse function were more accurate and closer to the experimental value. In

  19. Tyrosine kinases in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Kobayashi Akiko

    2011-08-01

    Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

  20. Splenomegaly in myelofibrosis—new options for therapy and the therapeutic potential of Janus kinase 2 inhibitors

    Directory of Open Access Journals (Sweden)

    Randhawa Jasleen

    2012-08-01

    Full Text Available Abstract Splenomegaly is a common sign of primary myelofibrosis (PMF, post-polycythemia vera myelofibrosis (post-PV MF, and post-essential thrombocythemia myelofibrosis (post-ET MF that is associated with bothersome symptoms, which have a significant negative impact on patients’ quality of life. It may also be present in patients with advanced polycythemia vera (PV or essential thrombocythemia (ET. Until recently, none of the therapies used to treat MF were particularly effective in reducing splenomegaly. The discovery of an activating Janus kinase 2 (JAK2 activating mutation (JAK2V617F that is present in almost all patients with PV and in about 50-60 % of patients with ET and PMF led to the initiation of several trials investigating the clinical effectiveness of various JAK2 (or JAK1/JAK2 inhibitors for the treatment of patients with ET, PV, and MF. Some of these trials have documented significant clinical benefit of JAK inhibitors, particularly in terms of regression of splenomegaly. In November 2011, the US Food and Drug Administration approved the use of the JAK1- and JAK2-selective inhibitor ruxolitinib for the treatment of patients with intermediate or high-risk myelofibrosis, including PMF, post-PV MF, and post-ET MF. This review discusses current therapeutic options for splenomegaly associated with primary or secondary MF and the treatment potential of the JAK inhibitors in this setting.

  1. The rational design of a novel potent analogue of the 5’-AMP-activated protein kinase inhibitor compound C with improved selectivity and cellular activity

    Science.gov (United States)

    Machrouhi, Fouzia; Ouhamou, Nouara; Laderoute, Keith; Calaoagan, Joy; Bukhtiyarova, Marina; Ehrlich, Paula J.; Klon, Anthony E.

    2010-01-01

    We have designed and synthesized analogues of compound C, a non-specific inhibitor of 5’-AMP-activated protein kinase (AMPK), using a computational fragment-based drug design (FBDD) approach. Synthesizing only twenty-seven analogues yielded a compound that was equipotent to compound C in the inhibition of the human AMPK (hAMPK) α2 subunit in the heterotrimeric complex in vitro, exhibited significantly improved selectivity against a subset of relevant kinases, and demonstrated enhanced cellular inhibition of AMPK. PMID:20932747

  2. The target landscape of clinical kinase drugs.

    Science.gov (United States)

    Klaeger, Susan; Heinzlmeir, Stephanie; Wilhelm, Mathias; Polzer, Harald; Vick, Binje; Koenig, Paul-Albert; Reinecke, Maria; Ruprecht, Benjamin; Petzoldt, Svenja; Meng, Chen; Zecha, Jana; Reiter, Katrin; Qiao, Huichao; Helm, Dominic; Koch, Heiner; Schoof, Melanie; Canevari, Giulia; Casale, Elena; Depaolini, Stefania Re; Feuchtinger, Annette; Wu, Zhixiang; Schmidt, Tobias; Rueckert, Lars; Becker, Wilhelm; Huenges, Jan; Garz, Anne-Kathrin; Gohlke, Bjoern-Oliver; Zolg, Daniel Paul; Kayser, Gian; Vooder, Tonu; Preissner, Robert; Hahne, Hannes; Tõnisson, Neeme; Kramer, Karl; Götze, Katharina; Bassermann, Florian; Schlegl, Judith; Ehrlich, Hans-Christian; Aiche, Stephan; Walch, Axel; Greif, Philipp A; Schneider, Sabine; Felder, Eduard Rudolf; Ruland, Juergen; Médard, Guillaume; Jeremias, Irmela; Spiekermann, Karsten; Kuster, Bernhard

    2017-12-01

    Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  3. Role of Reversible Histidine Coordination in Hydroxylamine Reduction by Plant Hemoglobins (Phytoglobins).

    Science.gov (United States)

    Athwal, Navjot Singh; Alagurajan, Jagannathan; Andreotti, Amy H; Hargrove, Mark S

    2016-10-18

    Reduction of hydroxylamine to ammonium by phytoglobin, a plant hexacoordinate hemoglobin, is much faster than that of other hexacoordinate hemoglobins or pentacoordinate hemoglobins such as myoglobin, leghemoglobin, and red blood cell hemoglobin. The reason for differences in reactivity is not known but could be intermolecular electron transfer between protein molecules in support of the required two-electron reduction, hydroxylamine binding, or active site architecture favoring the reaction. Experiments were conducted with phytoglobins from rice, tomato, and soybean along with human neuroglobin and soybean leghemoglobin that reveal hydroxylamine binding as the rate-limiting step. For hexacoordinate hemoglobins, binding is limited by the dissociation rate constant for the distal histidine, while leghemoglobin is limited by an intrinsically low affinity for hydroxylamine. When the distal histidine is removed from rice phytoglobin, a hydroxylamine-bound intermediate is formed and the reaction rate is diminished, indicating that the distal histidine imidazole side chain is critical for the reaction, albeit not for electron transfer but rather for direct interaction with the substrate. Together, these results demonstrate that phytoglobins are superior at hydroxylamine reduction because they have distal histidine coordination affinity constants near 1, and facile rate constants for binding and dissociation of the histidine side chain. Hexacoordinate hemoglobins such as neuroglobin are limited by tighter histidine coordination that blocks hydroxylamine binding, and pentacoordinate hemoglobins have intrinsically lower hydroxylamine affinities.

  4. The HDAC inhibitor SB939 overcomes resistance to BCR-ABL kinase Inhibitors conferred by the BIM deletion polymorphism in chronic myeloid leukemia.

    Directory of Open Access Journals (Sweden)

    Muhammad Rauzan

    Full Text Available Chronic myeloid leukemia (CML treatment has been improved by tyrosine kinase inhibitors (TKIs such as imatinib mesylate (IM but various factors can cause TKI resistance in patients with CML. One factor which contributes to TKI resistance is a germline intronic deletion polymorphism in the BCL2-like 11 (BIM gene which impairs the expression of pro-apoptotic splice isoforms of BIM. SB939 (pracinostat is a hydroxamic acid based HDAC inhibitor with favorable pharmacokinetic, physicochemical and pharmaceutical properties, and we investigated if this drug could overcome BIM deletion polymorphism-induced TKI resistance. We found that SB939 corrects BIM pre-mRNA splicing in CML cells with the BIM deletion polymorphism, and induces apoptotic cell death in CML cell lines and primary cells with the BIM deletion polymorphism. More importantly, SB939 both decreases the viability of CML cell lines and primary CML progenitors with the BIM deletion and restores TKI-sensitivity. Our results demonstrate that SB939 overcomes BIM deletion polymorphism-induced TKI resistance, and suggest that SB939 may be useful in treating CML patients with BIM deletion-associated TKI resistance.

  5. Reconstruction of the Chemotaxis Receptor-Kinase Assembly

    International Nuclear Information System (INIS)

    Park, S.; Borbat, P.; Gonzalez-Bonet, G.; Bhatnagar, J.; Pollard, A.; Freed, J.; Bilwes, A.; Crane, B.

    2006-01-01

    In bacterial chemotaxis, an assembly of transmembrane receptors, the CheA histidine kinase and the adaptor protein CheW processes environmental stimuli to regulate motility. The structure of a Thermotoga maritima receptor cytoplasmic domain defines CheA interaction regions and metal ion-coordinating charge centers that undergo chemical modification to tune receptor response. Dimeric CheA-CheW, defined by crystallography and pulsed ESR, positions two CheWs to form a cleft that is lined with residues important for receptor interactions and sized to clamp one receptor dimer. CheW residues involved in kinase activation map to interfaces that orient the CheW clamps. CheA regulatory domains associate in crystals through conserved hydrophobic surfaces. Such CheA self-contacts align the CheW receptor clamps for binding receptor tips. Linking layers of ternary complexes with close-packed receptors generates a lattice with reasonable component ratios, cooperative interactions among receptors and accessible sites for modification enzymes

  6. Structure-Guided Strategy for the Development of Potent Bivalent ERK Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Lechtenberg, Bernhard C. [Cancer; Mace, Peter D. [Cancer; Sessions, E. Hampton [Sanford Burnham Prebys Medical Discovery Institute at Lake Nona, Orlando, Florida 32827, United States; Williamson, Robert [Sanford Burnham Prebys Medical Discovery Institute at Lake Nona, Orlando, Florida 32827, United States; Stalder, Romain [Sanford Burnham Prebys Medical Discovery Institute at Lake Nona, Orlando, Florida 32827, United States; Wallez, Yann [Cancer; Roth, Gregory P. [Sanford Burnham Prebys Medical Discovery Institute at Lake Nona, Orlando, Florida 32827, United States; Riedl, Stefan J. [Cancer; Pasquale, Elena B. [Cancer; Pathology

    2017-06-13

    ERK is the effector kinase of the RAS-RAF-MEK-ERK signaling cascade, which promotes cell transformation and malignancy in many cancers and is thus a major drug target in oncology. Kinase inhibitors targeting RAF or MEK are already used for the treatment of certain cancers, such as melanoma. Although the initial response to these drugs can be dramatic, development of drug resistance is a major challenge, even with combination therapies targeting both RAF and MEK. Importantly, most resistance mechanisms still rely on activation of the downstream effector kinase ERK, making it a promising target for drug development efforts. Here, we report the design and structural/functional characterization of a set of bivalent ERK inhibitors that combine a small molecule inhibitor that binds to the ATP-binding pocket with a peptide that selectively binds to an ERK protein interaction surface, the D-site recruitment site (DRS). Our studies show that the lead bivalent inhibitor, SBP3, has markedly improved potency compared to the small molecule inhibitor alone. Unexpectedly, we found that SBP3 also binds to several ERK-related kinases that contain a DRS, highlighting the importance of experimentally verifying the predicted specificity of bivalent inhibitors. However, SBP3 does not target any other kinases belonging to the same CMGC branch of the kinome. Additionally, our modular click chemistry inhibitor design facilitates the generation of different combinations of small molecule inhibitors with ERK-targeting peptides.

  7. Comparative Molecular Dynamics Simulations of Mitogen-Activated Protein Kinase-Activated Protein Kinase 5

    Directory of Open Access Journals (Sweden)

    Inger Lindin

    2014-03-01

    Full Text Available The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD simulations of: (1 MK5 alone; (2 MK5 in complex with an inhibitor; and (3 MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding.

  8. Liquid chromatography-tandem mass spectrometric assay for the quantitative determination of the tyrosine kinase inhibitor quizartinib in mouse plasma using salting-out liquid-liquid extraction

    NARCIS (Netherlands)

    Retmana, Irene A; Wang, Jing; Schinkel, Alfred H; Schellens, Jan H M; Beijnen, Jos H; Sparidans, Rolf W

    2017-01-01

    A bioanalytical assay for quizartinib -a potent, and selective FLT3 tyrosine kinase inhibitor- in mouse plasma was developed and validated. Salting-out assisted liquid-liquid extraction (SALLE), using acetonitrile and magnesium sulfate, was selected as sample pretreatment with deuterated quizartinib

  9. Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids: evidence for histidine-mediated membrane fusion at acidic pH.

    Science.gov (United States)

    Kumar, V V; Pichon, C; Refregiers, M; Guerin, B; Midoux, P; Chaudhuri, A

    2003-08-01

    Presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers, such as polylysine, has previously been demonstrated to significantly enhance their in vitro gene delivery efficiencies. Towards harnessing improved transfection property through covalent grafting of endosome-disrupting single histidine functionality in the molecular structure of cationic lipids, herein, we report on the design, the synthesis and the transfection efficiency of two novel nonglycerol-based histidylated cationic amphiphiles. We found that L-histidine-(N,N-di-n-hexadecylamine)ethylamide (lipid 1) and L-histidine-(N,N-di-n-hexadecylamine,-N-methyl)ethylamide (lipid 2) in combination with cholesterol gave efficient transfections into various cell lines. The transfection efficiency of Chol/lipid 1 lipoplexes into HepG2 cells was two order of magnitude higher than that of FuGENE(TM)6 and DC-Chol lipoplexes, whereas it was similar into A549, 293T7 and HeLa cells. A better efficiency was obtained with Chol/lipid 2 lipoplexes when using the cytosolic luciferase expression vector (pT7Luc) under the control of the bacterial T7 promoter. Membrane fusion activity measurements using fluorescence resonance energy transfer (FRET) technique showed that the histidine head-groups of Chol/lipid 1 liposomes mediated membrane fusion in the pH range 5-7. In addition, the transgene expression results using the T7Luc expression vector convincingly support the endosome-disrupting role of the presently described mono-histidylated cationic transfection lipids and the release of DNA into the cytosol. We conclude that covalent grafting of a single histidine amino acid residue to suitable twin-chain hydrophobic compounds is able to impart remarkable transfection properties on the resulting mono-histidylated cationic amphiphile, presumably via the endosome-disrupting characteristics of the histidine functionalities.

  10. Synthesis and in Vitro and in Vivo Evaluation of Phosphoinositide-3-kinase Inhibitors.

    Science.gov (United States)

    Burger, Matthew T; Knapp, Mark; Wagman, Allan; Ni, Zhi-Jie; Hendrickson, Thomas; Atallah, Gordana; Zhang, Yanchen; Frazier, Kelly; Verhagen, Joelle; Pfister, Keith; Ng, Simon; Smith, Aaron; Bartulis, Sarah; Merrit, Hanne; Weismann, Marion; Xin, Xiaohua; Haznedar, Joshua; Voliva, Charles F; Iwanowicz, Ed; Pecchi, Sabina

    2011-01-13

    Phospoinositide-3-kinases (PI3K) are important oncology targets due to the deregulation of this signaling pathway in a wide variety of human cancers. A series of 2-morpholino, 4-substituted, 6-(3-hydroxyphenyl) pyrimidines have been reported as potent inhibitors of PI3Ks. Herein, we describe the structure-guided optimization of these pyrimidines with a focus on replacing the phenol moiety, while maintaining potent target inhibition and improving in vivo properties. A series of 2-morpholino, 4-substituted, 6-heterocyclic pyrimidines, which potently inhibit PI3K, were discovered. Within this series a compound, 17, was identified with suitable pharmacokinetic (PK) properties, which allowed for the establishment of a PI3K PK/pharmacodynamic-efficacy relationship as determined by in vivo inhibition of AKT(Ser473) phosphorylation and tumor growth inhibition in a mouse A2780 tumor xenograft model.

  11. Focal Adhesion Kinase Inhibitors in Combination with Erlotinib Demonstrate Enhanced Anti-Tumor Activity in Non-Small Cell Lung Cancer.

    Directory of Open Access Journals (Sweden)

    Grant A Howe

    Full Text Available Blockade of epidermal growth factor receptor (EGFR activity has been a primary therapeutic target for non-small cell lung cancers (NSCLC. As patients with wild-type EGFR have demonstrated only modest benefit from EGFR tyrosine kinase inhibitors (TKIs, there is a need for additional therapeutic approaches in patients with wild-type EGFR. As a key component of downstream integrin signalling and known receptor cross-talk with EGFR, we hypothesized that targeting focal adhesion kinase (FAK activity, which has also been shown to correlate with aggressive stage in NSCLC, would lead to enhanced activity of EGFR TKIs. As such, EGFR TKI-resistant NSCLC cells (A549, H1299, H1975 were treated with the EGFR TKI erlotinib and FAK inhibitors (PF-573,228 or PF-562,271 both as single agents and in combination. We determined cell viability, apoptosis and 3-dimensional growth in vitro and assessed tumor growth in vivo. Treatment of EGFR TKI-resistant NSCLC cells with FAK inhibitor alone effectively inhibited cell viability in all cell lines tested; however, its use in combination with the EGFR TKI erlotinib was more effective at reducing cell viability than either treatment alone when tested in both 2- and 3-dimensional assays in vitro, with enhanced benefit seen in A549 cells. This increased efficacy may be due in part to the observed inhibition of Akt phosphorylation when the drugs were used in combination, where again A549 cells demonstrated the most inhibition following treatment with the drug combination. Combining erlotinib with FAK inhibitor was also potent in vivo as evidenced by reduced tumor growth in the A549 mouse xenograft model. We further ascertained that the enhanced sensitivity was irrespective of the LKB1 mutational status. In summary, we demonstrate the effectiveness of combining erlotinib and FAK inhibitors for use in known EGFR wild-type, EGFR TKI resistant cells, with the potential that a subset of cell types, which includes A549, could be

  12. Survey of tyrosine kinase signaling reveals ROS kinase fusions in human cholangiocarcinoma.

    Directory of Open Access Journals (Sweden)

    Ting-Lei Gu

    Full Text Available Cholangiocarcinoma, also known as bile duct cancer, is the second most common primary hepatic carcinoma with a median survival of less than 2 years. The molecular mechanisms underlying the development of this disease are not clear. To survey activated tyrosine kinases signaling in cholangiocarcinoma, we employed immunoaffinity profiling coupled to mass spectrometry and identified DDR1, EPHA2, EGFR, and ROS tyrosine kinases, along with over 1,000 tyrosine phosphorylation sites from about 750 different proteins in primary cholangiocarcinoma patients. Furthermore, we confirmed the presence of ROS kinase fusions in 8.7% (2 out of 23 of cholangiocarcinoma patients. Expression of the ROS fusions in 3T3 cells confers transforming ability both in vitro and in vivo, and is responsive to its kinase inhibitor. Our data demonstrate that ROS kinase is a promising candidate for a therapeutic target and for a diagnostic molecular marker in cholangiocarcinoma. The identification of ROS tyrosine kinase fusions in cholangiocarcinoma, along with the presence of other ROS kinase fusions in lung cancer and glioblastoma, suggests that a more broadly based screen for activated ROS kinase in cancer is warranted.

  13. Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.

    Science.gov (United States)

    Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

    2003-08-01

    DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.

  14. Identification of a Glycogen Synthase Kinase-3[beta] Inhibitor that Attenuates Hyperactivity in CLOCK Mutant Mice

    Energy Technology Data Exchange (ETDEWEB)

    Kozikowski, Alan P.; Gunosewoyo, Hendra; Guo, Songpo; Gaisina, Irina N.; Walter, Richard L.; Ketcherside, Ariel; McClung, Colleen A.; Mesecar, Andrew D.; Caldarone, Barbara (Psychogenics); (Purdue); (UIC); (UTSMC)

    2012-05-02

    Bipolar disorder is characterized by a cycle of mania and depression, which affects approximately 5 million people in the United States. Current treatment regimes include the so-called 'mood-stabilizing drugs', such as lithium and valproate that are relatively dated drugs with various known side effects. Glycogen synthase kinase-3{beta} (GSK-3{beta}) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3{beta}. We designed a series of second generation benzofuran-3-yl-(indol-3-yl)maleimides containing a piperidine ring that possess IC{sub 50} values in the range of 4 to 680 nM against human GSK-3{beta}. One of these compounds exhibits reasonable kinase selectivity and promising preliminary absorption, distribution, metabolism, and excretion (ADME) data. The administration of this compound at doses of 10 to 25 mg kg{sup -1} resulted in the attenuation of hyperactivity in amphetamine/chlordiazepoxide-induced manic-like mice together with enhancement of prepulse inhibition, similar to the effects found for valproate (400 mg kg{sup -1}) and the antipsychotic haloperidol (1 mg kg{sup -1}). We also tested this compound in mice carrying a mutation in the central transcriptional activator of molecular rhythms, the CLOCK gene, and found that the same compound attenuates locomotor hyperactivity in response to novelty. This study further demonstrates the use of inhibitors of GSK-3{beta} in the treatment of manic episodes of bipolar/mood disorders, thus further validating GSK-3{beta} as a relevant therapeutic target in the identification of new therapies for bipolar patients.

  15. Histidine-lysine peptides as carriers of nucleic acids.

    Science.gov (United States)

    Leng, Qixin; Goldgeier, Lisa; Zhu, Jingsong; Cambell, Patricia; Ambulos, Nicholas; Mixson, A James

    2007-03-01

    With their biodegradability and diversity of permutations, peptides have significant potential as carriers of nucleic acids. This review will focus on the sequence and branching patterns of peptide carriers composed primarily of histidines and lysines. While lysines within peptides are important for binding to the negatively charged phosphates, histidines are critical for endosomal lysis enabling nucleic acids to reach the cytosol. Histidine-lysine (HK) polymers by either covalent or ionic bonds with liposomes augment transfection compared to liposome carriers alone. More recently, we have examined peptides as sole carriers of nucleic acids because of their intrinsic advantages compared to the bipartite HK/liposome carriers. With a protocol change and addition of a histidine-rich tail, HK peptides as sole carriers were more effective than liposomes alone in several cell lines. While four-branched polymers with a primary repeating sequence pattern of -HHK- were more effective as carriers of plasmids, eight-branched polymers with a sequence pattern of -HHHK- were more effective as carriers of siRNA. Compared to polyethylenimine, HK carriers of siRNA and plasmids had reduced toxicity. When injected intravenously, HK polymers in complex with plasmids encoding antiangiogenic proteins significantly decreased tumor growth. Furthermore, modification of HK polymers with polyethylene glycol and vascular-specific ligands increased specificity of the polyplex to the tumor by more than 40-fold. Together with further development and insight on the structure of HK polyplexes, HK peptides may prove to be useful as carriers of different forms of nucleic acids both in vitro and in vivo.

  16. The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility

    Science.gov (United States)

    Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I.; Hantschel, Oliver

    2014-11-01

    The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

  17. Syk inhibitors.

    Science.gov (United States)

    Chihara, Kazuyasu; Kimura, Yukihiro; Honjo, Chisato; Takeuchi, Kenji; Sada, Kiyonao

    2013-01-01

    Non-receptor type of protein-tyrosine kinase Syk (spleen tyrosine kinase) was isolated in University of Fukui in 1991. Syk is most highly expressed by haemopoietic cells and known to play crucial roles in the signal transduction through various immunoreceptors of the adaptive immune response. However, recent reports demonstrate that Syk also mediates other biological functions, such as innate immune response, osteoclast maturation, platelet activation and cellular adhesion. Moreover, ectopic expression of Syk by epigenetic changes is reported to cause retinoblastoma. Because of its critical roles on the cellular functions, the development of Syk inhibitors for clinical use has been desired. Although many candidate compounds were produced, none of them had progressed to clinical trials. However, novel Syk inhibitors were finally developed and its usefulness has been evaluated in the treatment of allergic rhinitis, rheumatoid arthritis and idiopathic thrombocytopenic purpura. In this review, we will summarize the history, structure and function of Syk, and then the novel Syk inhibitors and their current status. In addition, we will introduce our research focused on the functions of Syk on Dectin-1-mediated mast cell activation.

  18. Mechanism of the pH-induced conformational change in the sensor domain of the DraK Histidine kinase via the E83, E105, and E107 residues.

    Science.gov (United States)

    Yeo, Kwon Joo; Hong, Young-Soo; Jee, Jun-Goo; Lee, Jae Kyoung; Kim, Hyo Jeong; Park, Jin-Wan; Kim, Eun-Hee; Hwang, Eunha; Kim, Sang-Yoon; Lee, Eun-Gyeong; Kwon, Ohsuk; Cheong, Hae-Kap

    2014-01-01

    The DraR/DraK two-component system was found to be involved in the differential regulation of antibiotic biosynthesis in a medium-dependent manner; however, its function and signaling and sensing mechanisms remain unclear. Here, we describe the solution structure of the extracellular sensor domain of DraK and suggest a mechanism for the pH-dependent conformational change of the protein. The structure contains a mixed alpha-beta fold, adopting a fold similar to the ubiquitous sensor domain of histidine kinase. A biophysical study demonstrates that the E83, E105, and E107 residues have abnormally high pKa values and that they drive the pH-dependent conformational change for the extracellular sensor domain of DraK. We found that a triple mutant (E83L/E105L/E107A) is pH independent and mimics the low pH structure. An in vivo study showed that DraK is essential for the recovery of the pH of Streptomyces coelicolor growth medium after acid shock. Our findings suggest that the DraR/DraK two-component system plays an important role in the pH regulation of S. coelicolor growth medium. This study provides a foundation for the regulation and the production of secondary metabolites in Streptomyces.

  19. 3D QSAR models built on structure-based alignments of Abl tyrosine kinase inhibitors.

    Science.gov (United States)

    Falchi, Federico; Manetti, Fabrizio; Carraro, Fabio; Naldini, Antonella; Maga, Giovanni; Crespan, Emmanuele; Schenone, Silvia; Bruno, Olga; Brullo, Chiara; Botta, Maurizio

    2009-06-01

    Quality QSAR: A combination of docking calculations and a statistical approach toward Abl inhibitors resulted in a 3D QSAR model, the analysis of which led to the identification of ligand portions important for affinity. New compounds designed on the basis of the model were found to have very good affinity for the target, providing further validation of the model itself.The X-ray crystallographic coordinates of the Abl tyrosine kinase domain in its active, inactive, and Src-like inactive conformations were used as targets to simulate the binding mode of a large series of pyrazolo[3,4-d]pyrimidines (known Abl inhibitors) by means of GOLD software. Receptor-based alignments provided by molecular docking calculations were submitted to a GRID-GOLPE protocol to generate 3D QSAR models. Analysis of the results showed that the models based on the inactive and Src-like inactive conformations had very poor statistical parameters, whereas the sole model based on the active conformation of Abl was characterized by significant internal and external predictive ability. Subsequent analysis of GOLPE PLS pseudo-coefficient contour plots of this model gave us a better understanding of the relationships between structure and affinity, providing suggestions for the next optimization process. On the basis of these results, new compounds were designed according to the hydrophobic and hydrogen bond donor and acceptor contours, and were found to have improved enzymatic and cellular activity with respect to parent compounds. Additional biological assays confirmed the important role of the selected compounds as inhibitors of cell proliferation in leukemia cells.

  20. Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

    Science.gov (United States)

    Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

    1999-03-05

    Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

  1. A novel quantitative kinase assay using bacterial surface display and flow cytometry.

    Directory of Open Access Journals (Sweden)

    Sónia Troeira Henriques

    Full Text Available The inhibition of tyrosine kinases is a successful approach for the treatment of cancers and the discovery of kinase inhibitor drugs is the focus of numerous academic and pharmaceutical laboratories. With this goal in mind, several strategies have been developed to measure kinase activity and to screen novel tyrosine kinase inhibitors. Nevertheless, a general non-radioactive and inexpensive approach, easy to implement and adapt to a range of applications, is still missing. Herein, using Bcr-Abl tyrosine kinase, an oncogenic target and a model protein for cancer studies, we describe a novel cost-effective high-throughput screening kinase assay. In this approach, named the BacKin assay, substrates displayed on a Bacterial cell surface are incubated with Kinase and their phosphorylation is examined and quantified by flow cytometry. This approach has several advantages over existing approaches, as using bacteria (i.e. Escherichia coli to display peptide substrates provides a self renewing solid support that does not require laborious chemical strategies. Here we show that the BacKin approach can be used for kinetic and mechanistic studies, as well as a platform to characterize and identify small-molecule or peptide-based kinase inhibitors with potential applications in drug development.

  2. Hydrogen-deuterium exchange in imidazole as a tool for studying histidine phosphorylation.

    Science.gov (United States)

    Cebo, Małgorzata; Kielmas, Martyna; Adamczyk, Justyna; Cebrat, Marek; Szewczuk, Zbigniew; Stefanowicz, Piotr

    2014-12-01

    Isotope exchange at the histidine C2 atom of imidazole in D2O solution is well known to occur at a significantly slower rate than the exchange of amide protons. Analysis of the kinetics of this isotope-exchange reaction is proposed herein as a method of detecting histidine phosphorylation. This modification of His-containing peptides is challenging to pinpoint because of its instability under acidic conditions as well as during CID-MS analysis. In this work, we investigated the effect of phosphorylation of the histidine side chain in peptides on deuterium-hydrogen exchange (DHX) in the imidazole. The results demonstrate that phosphorylation dramatically slows the rate of the DHX reaction. This phenomenon can be applied to detect phosphorylation of peptides at the histidine residue (e.g., in enzymatic digests). We also found that the influence of the peptide sequence on the exchange kinetics is relatively small. A CID fragmentation experiment revealed that there was no detectable hydrogen scrambling in peptides deuterated at C2 of the imidazole ring. Therefore, MS/MS can be used to directly identify the locations of deuterium ions incorporated into peptides containing multiple histidine moieties.

  3. Tyrosine kinase receptor inhibitor-targeted combined chemotherapy for metastatic bladder cancer

    Directory of Open Access Journals (Sweden)

    Chia-Lun Wu

    2012-04-01

    Full Text Available Overexpression of hypoxia-inducible factor-1 alpha is noted during the invasive and metastatic process of transitional cell carcinoma. It will upregulate vascular endothelial growth factor (VEGF and drive proliferation, invasiveness, metastasis, and antiapoptotic ability of cancer cells. We proposed that tyrosine kinase receptor inhibitor, sunitinib malate—(Sutent; Pfizer Inc., Taiwan, combined with chemotherapeutic drug may present synergistic cytotoxic enhancement to transitional cell carcinoma cells with subsequent inhibition of their cellular behaviors, including proliferation, invasiveness, and metastatic activity. The contents of VEGF-A in mouse bladder tumor cells (MBT-2 and culture medium were detected by quantification-polymerase chain reaction and Western blot individually. The inhibitory concentrations of various chemotherapeutic drugs, sunitinib, and their combination treatment in MBT-2 were determined by 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT assay. Microchamber transmembrane migration assay was applied in evaluation of the inhibitory effects of different dosages of sunitinib and combination treatment on tumor cells. The cell cycle and apoptosis were analyzed after combination therapy by flow cytometry. Variation in apoptotic pathway was elucidated by Western blot using specific antibodies with cleaved PARP and caspase-3. Metastatic animal model mimicked by tail vein injection of MBT-2 cells was used to evaluate the treatment efficiency in tumor weight and survival rate. The mRNA and protein level of VEGF-A in MBT-2 cells increased by 70% at 48 hours interval under hypoxia stress condition. In MTT assay, MBT-2 cells had shown the highest sensitivity to epirubicin. Sunitinib combined with epirubicin had shown a synergistic cytotoxic effect to MBT-2 cells. Sunitinib and its combination with epirubicin showed significant inhibition on MBT-2 cells migration in microchambers. G2/M phase arrest and

  4. Convergent Akt activation drives acquired EGFR inhibitor resistance in lung cancer

    DEFF Research Database (Denmark)

    Jacobsen, Kirstine; Bertran-Alamillo, Jordi; Molina, Miguel Angel

    2017-01-01

    Non-small-cell lung cancer patients with activating epidermal growth factor receptor (EGFR) mutations typically benefit from EGFR tyrosine kinase inhibitor treatment. However, virtually all patients succumb to acquired EGFR tyrosine kinase inhibitor resistance that occurs via diverse mechanisms....

  5. Molecular Imaging of the ATM Kinase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Terence M. [Department of Radiation Oncology, Ohio State University, Columbus, Ohio (United States); Nyati, Shyam [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Ross, Brian D. [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Rehemtulla, Alnawaz, E-mail: alnawaz@umich.edu [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States)

    2013-08-01

    Purpose: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks. ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and noninvasively measures ATM kinase activity in living cells and subjects. Methods and Materials: Using the split luciferase technology, we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence. Results: Treatment of ATMR-expressing cells with ATM inhibitors resulted in a dose-dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, whereas a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and small interfering (si)RNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models that correlated in a time-dependent fashion with changes in Chk2 activity. Conclusions: We describe the development and validation of a novel, specific, noninvasive bioluminescent reporter that enables monitoring of ATM activity in real time, in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in preclinical models.

  6. L-histidine inhibits biofilm formation and FLO11-associated phenotypes in Saccharomyces cerevisiae flor yeasts.

    Science.gov (United States)

    Bou Zeidan, Marc; Zara, Giacomo; Viti, Carlo; Decorosi, Francesca; Mannazzu, Ilaria; Budroni, Marilena; Giovannetti, Luciana; Zara, Severino

    2014-01-01

    Flor yeasts of Saccharomyces cerevisiae have an innate diversity of Flo11p which codes for a highly hydrophobic and anionic cell-wall glycoprotein with a fundamental role in biofilm formation. In this study, 380 nitrogen compounds were administered to three S. cerevisiae flor strains handling Flo11p alleles with different expression levels. S. cerevisiae strain S288c was used as the reference strain as it cannot produce Flo11p. The flor strains generally metabolized amino acids and dipeptides as the sole nitrogen source, although with some exceptions regarding L-histidine and histidine containing dipeptides. L-histidine completely inhibited growth and its effect on viability was inversely related to Flo11p expression. Accordingly, L-histidine did not affect the viability of the Δflo11 and S288c strains. Also, L-histidine dramatically decreased air-liquid biofilm formation and adhesion to polystyrene of the flor yeasts with no effect on the transcription level of the Flo11p gene. Moreover, L-histidine modified the chitin and glycans content on the cell-wall of flor yeasts. These findings reveal a novel biological activity of L-histidine in controlling the multicellular behavior of yeasts [corrected].

  7. Design of Aminobenzothiazole Inhibitors of Rho Kinases 1 and 2 by Using Protein Kinase A as a Structure Surrogate.

    Science.gov (United States)

    Judge, Russell A; Vasudevan, Anil; Scott, Victoria E; Simler, Gricelda H; Pratt, Steve D; Namovic, Marian T; Putman, C Brent; Aguirre, Ana; Stoll, Vincent S; Mamo, Mulugeta; Swann, Steven I; Cassar, Steven C; Faltynek, Connie R; Kage, Karen L; Boyce-Rustay, Janel M; Hobson, Adrian D

    2018-03-16

    We describe the design, synthesis, and structure-activity relationships (SARs) of a series of 2-aminobenzothiazole inhibitors of Rho kinases (ROCKs) 1 and 2, which were optimized to low nanomolar potencies by use of protein kinase A (PKA) as a structure surrogate to guide compound design. A subset of these molecules also showed robust activity in a cell-based myosin phosphatase assay and in a mechanical hyperalgesia in vivo pain model. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. A new trend in pathogenetic treatment of rheumatoid arthritis: tofacitinib, the first inhibitor of Janus kinase

    Directory of Open Access Journals (Sweden)

    Dmitry Evgenyevich Karateev

    2014-01-01

    Full Text Available The Problem of effective treatment of rheumatoid arthritis (RA has not been solved yet. In patients with an inadequate response to the synthetic disease-modifying antirheumatic drugs (DMARDs, TNF inhibitors and other genetically engineered (GE drugs may give an incomplete effect or be totally ineffective in 20–40% of cases. The search for new therapeutic options is very active in many directions, including modulation of intracellular signaling pathways. The most impressive clinical results in RA patients have been achieved for Janus kinase (JAK inhibitor, tofacitinib, – Tofa (CP-690,550, a low molecular weight drug for oral administration. Unlike the conventional synthetic DMARD, Tofa belongs to targeted therapy drugs; its pharmacological effects largely resemble those of GE drugs (blockade of the biological effects of cytokines. In particular, some features of its mechanism of action (e.g., interleukin 6 inhibition make it similar to tocilizumab. In all trials, Tofa proved to exhibit high clinical efficacy and satisfactory safety. Tofa was shown to be significantly more effective than methotrexate and placebo (in monotherapy, as well as in combination with DMARDs in RA patients with an inadequate response to standard DMARDs and TNFa inhibitors (regarding response to treatment, individual parameters of disease activity, achievement of low disease activity and remission, functional impairment. Furthermore, Tofa can inhibit joint damage progression.

  9. Crystal Structures of Trypanosoma cruzi UDP-Galactopyranose Mutase Implicate Flexibility of the Histidine Loop in Enzyme Activation

    Energy Technology Data Exchange (ETDEWEB)

    Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle; Sobrado, Pablo; Tanner, John J. (Virginia Tech); (UMC)

    2012-11-01

    Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitor design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3 {angstrom} movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45% identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10-50, primarily by decreasing k{sub cat}. Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens.

  10. Crystal structures of Trypanosoma cruzi UDP-galactopyranose mutase implicate flexibility of the histidine loop in enzyme activation.

    Science.gov (United States)

    Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle; Sobrado, Pablo; Tanner, John J

    2012-06-19

    Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitor design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3 Å movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45% identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10-50, primarily by decreasing k(cat). Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens.

  11. Discovery of amido-benzisoxazoles as potent c-Kit inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Kunz, Roxanne K.; Rumfelt, Shannon; Chen, Ning; Zhang, Dawei; Tasker, Andrew S.; Bürli, Roland; Hungate, Randall; Yu, Violeta; Nguyen, Yen; Whittington, Douglas A.; Meagher, Kristin L.; Plant, Matthew; Tudor, Yanyan; Schrag, Michael; Xu, Yang; Ng, Gordon Y.; Hu, Essa (Amgen)

    2010-01-12

    Deregulation of the receptor tyrosine kinase c-Kit is associated with an increasing number of human diseases, including certain cancers and mast cell diseases. Interference of c-Kit signaling with multi-kinase inhibitors has been shown clinically to successfully treat gastrointestinal stromal tumors and mastocytosis. Targeted therapy of c-Kit activity may provide therapeutic advantages against off-target effects for non-oncology applications. A new structural class of c-Kit inhibitors is described, including in vitro c-Kit potency, kinase selectivity, and the observed binding mode.

  12. HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck

    Directory of Open Access Journals (Sweden)

    Pene-Dumitrescu Teodora

    2012-03-01

    Full Text Available Abstract Background Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association. Results To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency. Conclusions These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting.

  13. Targeting oncoprotein stability overcomes drug resistance caused by FLT3 kinase domain mutations.

    Directory of Open Access Journals (Sweden)

    Chuanjiang Yu

    Full Text Available FLT3 is the most frequently mutated kinase in acute myeloid leukemia (AML. Internal tandem duplications (ITDs in the juxta-membrane region constitute the majority of activating FLT3 mutations. Several FLT3 kinase inhibitors were developed and tested in the clinic with significant success. However, recent studies have reported the development of secondary drug resistance in patients treated with FLT3 inhibitors. Since FLT3-ITD is an HSP90 client kinase, we here explored if targeting the stability of drug-resistant FLT3 mutant protein could be a potential therapeutic option. We observed that HSP90 inhibitor treatment resulted in the degradation of inhibitor-resistant FLT3-ITD mutants and selectively induced toxicity in cells expressing FLT3-ITD mutants. Thus, HSP90 inhibitors provide a potential therapeutic choice to overcome secondary drug resistance following TKI treatment in FLT3-ITD positive AML.

  14. In Silico Exploration of 1,7-Diazacarbazole Analogs as Checkpoint Kinase 1 Inhibitors by Using 3D QSAR, Molecular Docking Study, and Molecular Dynamics Simulations

    Directory of Open Access Journals (Sweden)

    Xiaodong Gao

    2016-05-01

    Full Text Available Checkpoint kinase 1 (Chk1 is an important serine/threonine kinase with a self-protection function. The combination of Chk1 inhibitors and anti-cancer drugs can enhance the selectivity of tumor therapy. In this work, a set of 1,7-diazacarbazole analogs were identified as potent Chk1 inhibitors through a series of computer-aided drug design processes, including three-dimensional quantitative structure–activity relationship (3D-QSAR modeling, molecular docking, and molecular dynamics simulations. The optimal QSAR models showed significant cross-validated correlation q2 values (0.531, 0.726, fitted correlation r2 coefficients (higher than 0.90, and standard error of prediction (less than 0.250. These results suggested that the developed models possess good predictive ability. Moreover, molecular docking and molecular dynamics simulations were applied to highlight the important interactions between the ligand and the Chk1 receptor protein. This study shows that hydrogen bonding and electrostatic forces are key interactions that confer bioactivity.

  15. Icotinib (BPI-2009H), a novel EGFR tyrosine kinase inhibitor, displays potent efficacy in preclinical studies.

    Science.gov (United States)

    Tan, Fenlai; Shen, Xiaoyan; Wang, Dechang; Xie, Guojian; Zhang, Xiaodong; Ding, Lieming; Hu, Yunyan; He, Wei; Wang, Yanping; Wang, Yinxiang

    2012-05-01

    Icotinib, one of the leading compounds selected from our compound library, was found to be a potent and specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with an IC(50) of 5 nM. When profiled with 88 kinases, Icotinib only showed meaningful inhibitory activity to EGFR and its mutants. Icotinib blocked EGFR-mediated intracellular tyrosine phosphorylation (IC(50)=45 nM) in the human epidermoid carcinoma A431 cell line and inhibits tumor cell proliferation. In vivo studies demonstrated that Icotinib exhibited potent dose-dependent antitumor effects in nude mice carrying a variety of human tumor-derived xenografts. The drug was well tolerated at doses up to 120 mg/kg/day in mice without mortality or significant body weight loss during the treatment. A head to head randomized, double blind phase III trial using Gefitinib as an active control for patients with advanced non-small cell lung cancer (NSCLC) was finished recently (Trial registration ID: NCT01040780). The data shows that Icotinib was non-inferior to Gefitinib in terms of median progression free survival (PFS) and safety superior favor to Icotinib compared to Gefitinib. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  16. Downsizing Treatment with Tyrosine Kinase Inhibitors in Patients with Advanced Gastrointestinal Stromal Tumors Improved Resectability

    Science.gov (United States)

    Sjölund, Katarina; Andersson, Anna; Nilsson, Erik; Nilsson, Ola; Ahlman, Håkan

    2010-01-01

    Background Gastrointestinal stromal tumors (GISTs) express the receptor tyrosine kinase KIT. Most GISTs have mutations in the KIT or PDGFRA gene, causing activation of tyrosine kinase. Imatinib, a tyrosine kinase inhibitor (TKI), is the first-line palliative treatment for advanced GISTs. Sunitinib was introduced for patients with mutations not responsive to imatinib. The aim was to compare the survival of patients with high-risk resected GISTs treated with TKI prior to surgery with historical controls and to determine if organ-preserving surgery was facilitated. Methods Ten high-risk GIST-patients had downsizing/adjuvant TKI treatment: nine with imatinib and one with sunitinib. The patients were matched with historical controls (n = 89) treated with surgery alone, from our population-based series (n = 259). Mutational analysis of KIT and PDGFRA was performed in all cases. The progression-free survival was calculated. Results The primary tumors decreased in mean diameter from 20.4 cm to 10.5 cm on downsizing imatinib. Four patients with R0 resection and a period of adjuvant imatinib had no recurrences versus 67% in the historical control group. Four patients with residual liver metastases have stable disease on continuous imatinib treatment after surgery. One patient has undergone reoperation with liver resection. The downsizing treatment led to organ-preserving surgery in nine patients and improved preoperative nutritional status in one patient. Conclusions Downsizing TKI is recommended for patients with bulky tumors with invasion of adjacent organs. Sunitinib can be used for patients in case of imatinib resistance (e.g., wild-type GISTs), underlining the importance of mutational analysis for optimal surgical planning. PMID:20512492

  17. Bombyx mori cyclin-dependent kinase inhibitor is involved in regulation of the silkworm cell cycle.

    Science.gov (United States)

    Tang, X-F; Zhou, X-L; Zhang, Q; Chen, P; Lu, C; Pan, M-H

    2018-06-01

    Cyclin-dependent kinase inhibitors (CKIs) are negative regulators of the cell cycle. They can bind to cyclin-dependent kinase (CDK)-cyclin complexes and inhibit CDK activities. We identified a single homologous gene of the CDK interacting protein/kinase inhibitory protein (Cip/Kip) family, BmCKI, in the silkworm, Bombyx mori. The gene transcribes two splice variants: a 654-bp-long BmCKI-L (the longer splice variant) encoding a protein with 217 amino acids and a 579-bp-long BmCKI-S (the shorter splice variant) encoding a protein with 192 amino acids. BmCKI-L and BmCKI-S contain the Cip/Kip family conserved cyclin-binding domain and the CDK-binding domain. They are localized in the nucleus and have an unconventional bipartite nuclear localization signal at amino acid residues 181-210. Overexpression of BmCKI-L or BmCKI-S affected cell cycle progression; the cell cycle was arrested in the first gap phase of cell cycle (G1). RNA interference of BmCKI-L or BmCKI-S led to cells accumulating in the second gap phase and the mitotic phase of cell cycle (G2/M). Both BmCKI-L and BmCKI-S are involved in cell cycle regulation and probably have similar effects. The transgenic silkworm with BmCKI-L overexpression (BmCKI-L-OE), exhibited embryonic lethal, larva developmental retardation and lethal phenotypes. These results suggest that BmCKI-L might regulate the growth and development of silkworm. These findings clarify the function of CKIs and increase our understanding of cell cycle regulation in the silkworm. © 2018 The Royal Entomological Society.

  18. Combined therapeutic potential of nuclear receptors with receptor tyrosine kinase inhibitors in lung cancer

    International Nuclear Information System (INIS)

    Wairagu, Peninah M.; Park, Kwang Hwa; Kim, Jihye; Choi, Jong-Whan; Kim, Hyun-Won; Yeh, Byung-Il; Jung, Soon-Hee; Yong, Suk-Joong; Jeong, Yangsik

    2014-01-01

    Highlights: • The 48 NR genes and 48 biological anti-cancer targets are profiled in paired-cells. • Growth inhibition by NR ligands or TKIs is target receptor level-dependent. • T0901317 with gefitinib/PHA665752 shows additive growth inhibition in lung cells. - Abstract: Cancer heterogeneity is a big hurdle in achieving complete cancer treatment, which has led to the emergence of combinational therapy. In this study, we investigated the potential use of nuclear receptor (NR) ligands for combinational therapy with other anti-cancer drugs. We first profiled all 48 NRs and 48 biological anti-cancer targets in four pairs of lung cell lines, where each pair was obtained from the same patient. Two sets of cell lines were normal and the corresponding tumor cell lines while the other two sets consisted of primary versus metastatic tumor cell lines. Analysis of the expression profile revealed 11 NRs and 15 cancer targets from the two pairs of normal versus tumor cell lines, and 9 NRs and 9 cancer targets from the primary versus metastatic tumor cell lines had distinct expression patterns in each category. Finally, the evaluation of nuclear receptor ligand T0901317 for liver X receptor (LXR) demonstrated its combined therapeutic potential with tyrosine kinase inhibitors. The combined treatment of cMET inhibitor PHA665752 or EGFR inhibitor gefitinib with T0901317 showed additive growth inhibition in both H2073 and H1993 cells. Mechanistically, the combined treatment suppressed cell cycle progression by inhibiting cyclinD1 and cyclinB expression. Taken together, this study provides insight into the potential use of NR ligands in combined therapeutics with other biological anti-cancer drugs

  19. A Specific Inhibitor of TGF-β Receptor Kinase, SB-431542, as a Potent Antitumor Agent for Human Cancers

    Directory of Open Access Journals (Sweden)

    Sunil K. Halder

    2005-05-01

    Full Text Available Small molecule inhibitors of signaling pathways have proven to be extremely useful for the development of therapeutic strategies for human cancers. Blocking the tumor-promoting effects of transforming growth factor-β (TGF-β in advanced stage carcinogenesis provides a potentially interesting drug target for therapeutic intervention. Although very few TGF-β receptor kinase inhibitors (TRKI are now emerging in preclinical studies, nothing is known about how these inhibitors might regulate the tumor-suppressive or tumor-promoting effects of TGF-β, or when these inhibitors might be useful for treatment during cancer progression. We have investigated the potential of TRKI in new therapeutic approaches in preclinical models. Here, we demonstrate that the TRKI, SB-431542, inhibits TGF-β-induced transcription, gene expression, apoptosis, and growth suppression. We have observed that SB-431542 attenuates the tumor-promoting effects of TGF-β, including TGF-β-induced EMT, cell motility, migration and invasion, and vascular endothelial growth factor secretion in human cancer cell lines. Interestingly, SB-431542 induces anchorage independent growth of cells that are growth-inhibited by TGF-β, whereas it reduces colony formation by cells that are growth-promoted by TGF-β. However, SB-431542 has no effect on a cell line that failed to respond to TGF-β. This represents a novel potential application of these inhibitors as therapeutic agents for human cancers with the goal of blocking tumor invasion, angiogenesis, and metastasis, when tumors are refractory to TGF-β-induced tumor-suppressor functions but responsive to tumor-promoting effects of TGF-β.

  20. The Bruton tyrosine kinase inhibitor PCI-32765 thwarts chronic lymphocytic leukemia cell survival and tissue homing in vitro and in vivo

    Science.gov (United States)

    Ponader, Sabine; Chen, Shih-Shih; Buggy, Joseph J.; Balakrishnan, Kumudha; Gandhi, Varsha; Wierda, William G.; Keating, Michael J.; O'Brien, Susan; Chiorazzi, Nicholas

    2012-01-01

    B-cell receptor (BCR) signaling is a critical pathway in the pathogenesis of several B-cell malignancies, including chronic lymphocytic leukemia (CLL), and can be targeted by inhibitors of BCR-associated kinases, such as Bruton tyrosine kinase (Btk). PCI-32765, a selective, irreversible Btk inhibitor, is a novel, molecularly targeted agent for patients with B-cell malignancies, and is particularly active in patients with CLL. In this study, we analyzed the mechanism of action of PCI-32765 in CLL, using in vitro and in vivo models, and performed correlative studies on specimens from patients receiving therapy with PCI-32765. PCI-32765 significantly inhibited CLL cell survival, DNA synthesis, and migration in response to tissue homing chemokines (CXCL12, CXCL13). PCI-32765 also down-regulated secretion of BCR-dependent chemokines (CCL3, CCL4) by the CLL cells, both in vitro and in vivo. In an adoptive transfer TCL1 mouse model of CLL, PCI-32765 affected disease progression. In this model, PCI-32765 caused a transient early lymphocytosis, and profoundly inhibited CLL progression, as assessed by weight, development, and extent of hepatospenomegaly, and survival. Our data demonstrate that PCI-32765 effectively inhibits CLL cell migration and survival, possibly explaining some of the characteristic clinical activity of this new targeted agent. PMID:22180443

  1. Photochemically induced dynamic nuclear polarization NMR study of yeast and horse muscle phosphoglycerate kinase

    International Nuclear Information System (INIS)

    Scheffler, J.E.; Cohn, M.

    1986-01-01

    A photochemically induced dynamic nuclear polarization (photo-CIDNP) study of yeast and horse muscle phosphoglycerate kinase with flavin dyes was undertaken to identify the histidine, tryptophan, and tyrosine resonances in the aromatic region of the simplified 1 H NMR spectra of these enzymes and to investigate the effect of substrates on the resonances observable by CIDNP. Identification of the CIDNP-enhanced resonances with respect to the type of amino acid residue has been achieved since only tyrosine yields emission peaks and the dye 8-aminoriboflavin enhances tryptophan but not histidine. By use of the known amino acid sequences and structures derived from X-ray crystallographic studies of the enzymes from the two species, assignment of the specific residues in the protein sequences giving rise to the CIDNP spectra was partially achieved. In addition, flavin dye accessibility was used to probe any changes in enzyme structure induced by substrate binding. The accessibility of a tyrosine to photoexcited flavin is reduced in the presence of MgATP. Since the tyrosine residues are located some distance from the MgATP binding site of the catalytic center, it is proposed either that this change is due to a distant conformational change or that a second metal-ATP site inferred from other studies lies close to one of the tyrosines. Horse muscle phosphoglycerate kinase exhibits seven resonances by CIDNP NMR. The addition of 3-phosphoglycerate and MgATP results in the appearance of two additional resonances in the CIDNP spectrum due to a histidine residue that is inaccessible to flavin in both the enzyme alone and its binary complex with 3-phosphoglycerate. The CIDNP spectra are consistent with the suggestions that binding of 3-phosphoglycerate alone is insufficient to effect domain movement and that binding of both substrates are required for conversion of the horse muscle enzyme to its catalytically active form

  2. Identification of potential inhibitors based on compound proposal contest: Tyrosine-protein kinase Yes as a target.

    Science.gov (United States)

    Chiba, Shuntaro; Ikeda, Kazuyoshi; Ishida, Takashi; Gromiha, M Michael; Taguchi, Y-H; Iwadate, Mitsuo; Umeyama, Hideaki; Hsin, Kun-Yi; Kitano, Hiroaki; Yamamoto, Kazuki; Sugaya, Nobuyoshi; Kato, Koya; Okuno, Tatsuya; Chikenji, George; Mochizuki, Masahiro; Yasuo, Nobuaki; Yoshino, Ryunosuke; Yanagisawa, Keisuke; Ban, Tomohiro; Teramoto, Reiji; Ramakrishnan, Chandrasekaran; Thangakani, A Mary; Velmurugan, D; Prathipati, Philip; Ito, Junichi; Tsuchiya, Yuko; Mizuguchi, Kenji; Honma, Teruki; Hirokawa, Takatsugu; Akiyama, Yutaka; Sekijima, Masakazu

    2015-11-26

    A search of broader range of chemical space is important for drug discovery. Different methods of computer-aided drug discovery (CADD) are known to propose compounds in different chemical spaces as hit molecules for the same target protein. This study aimed at using multiple CADD methods through open innovation to achieve a level of hit molecule diversity that is not achievable with any particular single method. We held a compound proposal contest, in which multiple research groups participated and predicted inhibitors of tyrosine-protein kinase Yes. This showed whether collective knowledge based on individual approaches helped to obtain hit compounds from a broad range of chemical space and whether the contest-based approach was effective.

  3. A novel dual inhibitor of microtubule and Bruton's tyrosine kinase inhibits survival of multiple myeloma and osteoclastogenesis.

    Science.gov (United States)

    Pandey, Manoj K; Gowda, Krishne; Sung, Shen-Shu; Abraham, Thomas; Budak-Alpdogan, Tulin; Talamo, Giampolo; Dovat, Sinisa; Amin, Shantu

    2017-09-01

    Bruton's tyrosine kinase (BTK) regulates many vital signaling pathways and plays a critical role in cell proliferation, survival, migration, and resistance. Previously, we reported that a small molecule, KS99, is an inhibitor of tubulin polymerization. In the present study, we explored whether KS99 is a dual inhibitor of BTK and tubulin polymerization. Although it is known that BTK is required for clonogenic growth and resistance, and microtubules are essential for cancer cell growth, dual targeting of these two components has not been explored previously. Through docking studies, we predicted that KS99 interacts directly with the catalytic domain of BTK and inhibits phosphorylation at the Y223 residue and kinase activities. Treatment of KS99 reduces the cell viability of multiple myeloma (MM) and CD138 + cells, with an IC 50 of between 0.5 and 1.0 μmol/L. We found that KS99 is able to induce apoptosis in MM cells in a caspase-dependent manner. KS99 suppressed the receptor activator of NF-κB ligand (RANKL)-induced differentiation of macrophages to osteoclasts in a dose-dependent manner and, importantly, inhibited the expression of cytokines associated with bone loss. Finally, we found that KS99 inhibits the in vivo tumor growth of MM cells through the inhibition of BTK and tubulin. Overall, our results show that dual inhibition of BTK and tubulin polymerization by KS99 is a viable option in MM treatment, particularly in the inhibition of refraction and relapse. Copyright © 2017 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  4. Developmental effects of the protein kinase inhibitor kenpaullone on the sea urchin embryo.

    Science.gov (United States)

    Anello, Letizia; Cavalieri, Vincenzo; Di Bernardo, Maria

    2018-01-01

    The selection and validation of bioactive compounds require multiple approaches, including in-depth analyses of their biological activity in a whole-animal context. We exploited the sea urchin embryo in a rapid, medium-scale range screening to test the effects of the small synthetic kinase inhibitor kenpaullone. We show that sea urchin embryos specifically respond to this molecule depending on both dose and timing of administration. Phenotypic effects of kenpaullone are not immediately visible, since this molecule affects neither the fertilization nor the spatial arrangement of blastomeres at early developmental stages. Nevertheless, kenpaullone exposure from the beginning of embryogenesis profoundly perturbs specification, detachment from the epithelium, and migration of the primary mesenchyme cells, thus affecting the whole embryonic epithelial mesenchymal transition process. Our results reaffirm the sea urchin embryo as an excellent and sensitive in vivo system, which provides straightforward and rapid response to external stimuli. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Intracellular Retention of ABL Kinase Inhibitors Determines Commitment to Apoptosis in CML Cells

    Science.gov (United States)

    Dziadosz, Marek; Schnöder, Tina; Heidel, Florian; Schemionek, Mirle; Melo, Junia V.; Kindler, Thomas; Müller-Tidow, Carsten; Koschmieder, Steffen; Fischer, Thomas

    2012-01-01

    Clinical development of imatinib in CML established continuous target inhibition as a paradigm for successful tyrosine kinase inhibitor (TKI) therapy. However, recent reports suggested that transient potent target inhibition of BCR-ABL by high-dose TKI (HD-TKI) pulse-exposure is sufficient to irreversibly commit cells to apoptosis. Here, we report a novel mechanism of prolonged intracellular TKI activity upon HD-TKI pulse-exposure (imatinib, dasatinib) in BCR-ABL-positive cells. Comprehensive mechanistic exploration revealed dramatic intracellular accumulation of TKIs which closely correlated with induction of apoptosis. Cells were rescued from apoptosis upon HD-TKI pulse either by repetitive drug wash-out or by overexpression of ABC-family drug transporters. Inhibition of ABCB1 restored sensitivity to HD-TKI pulse-exposure. Thus, our data provide evidence that intracellular drug retention crucially determines biological activity of imatinib and dasatinib. These studies may refine our current thinking on critical requirements of TKI dose and duration of target inhibition for biological activity of TKIs. PMID:22815843

  6. Alterations in brain Protein Kinase A activity and reversal of morphine tolerance by two fragments of native Protein Kinase A inhibitor peptide (PKI).

    Science.gov (United States)

    Dalton, George D; Smith, Forrest L; Smith, Paul A; Dewey, William L

    2005-04-01

    Two peptide fragments of native Protein Kinase A inhibitor (PKI), PKI-(6-22)-amide and PKI-(Myr-14-22)-amide, significantly reversed low-level morphine antinociceptive tolerance in mice. The inhibition of Protein Kinase A (PKA) activity by both peptide fragments was then measured in specific brain regions (thalamus, periaqueductal gray (PAG), and medulla) and in lumbar spinal cord (LSC), which in previous studies have been shown to play a role in morphine-induced analgesia. In drug naive animals, cytosolic PKA activity was greater than particulate PKA activity in each region, while cytosolic and particulate PKA activities were greater in thalamus and PAG compared to medulla and LSC. The addition of both peptides to homogenates from each region completely abolished cytosolic and particulate PKA activities in vitro. Following injection into the lateral ventricle of the brain of drug naive mice and morphine-tolerant mice, both peptides inhibited PKA activity in the cytosolic, but not the particulate fraction of LSC. In addition, cytosolic and particulate PKA activities were inhibited by both peptides in thalamus. These results demonstrate that the inhibition of PKA reverses morphine tolerance. Moreover, the inhibition of PKA activity in specific brain regions and LSC from morphine-tolerant mice by PKI analogs administered i.c.v. is evidence that PKA plays a role in morphine tolerance.

  7. Influence of histidine on zinc transport into rat brain

    International Nuclear Information System (INIS)

    Takeda, Atsushi; Suzuki, Mai; Okada, Shoji; Oku, Naoto

    2000-01-01

    The brain of rats injected intravenously with 65 Zn-His or 65 ZnCl 2 was subjected to autoradiography to study the role of histidine on zinc transport into the brain. One hour after injection, the radioactivity from 65 Zn-His was largely concentrated in the choroid plexus in the ventricles. Six days after injection, the radioactivity from 65 Zn-His was relatively concentrated in the hippocampal CA3 and dentate gyrus and the amygdala. The relative distribution of 65 Zn-His in the brain was similar to that of 65 ZnCl 2 group at both 1 h and 6 days, suggesting that histidine may participate in zinc uptake in the brain. On the other hand, the clearance of the 65 Zn-His group from the blood was higher than that of the 65 ZnCl 2 group. Brain uptake of the former was lower than that of the latter both 1 h and 6 days after injection. These results suggest that zinc uptake in the brain is influenced by histidine levels in the bloodstream. (author)

  8. Three generations of epidermal growth factor receptor tyrosine kinase inhibitors developed to revolutionize the therapy of lung cancer

    Directory of Open Access Journals (Sweden)

    Zhang H

    2016-11-01

    Full Text Available Haijun Zhang Department of Oncology, Zhongda Hospital, Medical School, Southeast University, Nanjing, People’s Republic of China Abstract: Lung cancer, ~80%–85% of which is non-small-cell lung cancer (NSCLC, is the leading cause of cancer-related mortality worldwide. Sensitizing mutations in epidermal growth factor receptor (EGFR gene (EGFRm+, such as exon 19 deletions and exon 21 L858R point mutations, are the most important drivers in NSCLC patients. In this respect, small-molecule EGFR tyrosine kinase inhibitors (TKIs have been designed and developed, which launched the era of targeted, personalized and precise medicine for lung cancer. Patients with EGFRm+ could achieve good responses to the treatment with the first-generation EGFR TKIs, such as erlotinib and gefitinib. However, most patients develop acquired drug resistance mostly driven by the T790M mutation occurring within exon 20. Although the second-generation EGFR TKIs, such as afatinib, dacomitinib and neratinib, demonstrated promising activity against T790M in preclinical models, they have failed to overcome resistance in patients due to dose-limiting toxicity. Recently, the third-generation EGFR TKIs have shown to be effective against cell lines and murine models harboring T790M mutations while sparing wild-type EGFR, which represents a promising breakthrough approach in overcoming T790M-mediated resistance in NSCLC patients. This article provides a comprehensive review of the therapy revolution for NSCLC with three generations of EGFR TKIs. Keywords: lung cancer, epidermal growth factor receptor, tyrosine kinase inhibitors, T790M mutation

  9. Bruton tyrosine kinase inhibitor ibrutinib (PCI-32765) has significant activity in patients with relapsed/refractory B-cell malignancies.

    Science.gov (United States)

    Advani, Ranjana H; Buggy, Joseph J; Sharman, Jeff P; Smith, Sonali M; Boyd, Thomas E; Grant, Barbara; Kolibaba, Kathryn S; Furman, Richard R; Rodriguez, Sara; Chang, Betty Y; Sukbuntherng, Juthamas; Izumi, Raquel; Hamdy, Ahmed; Hedrick, Eric; Fowler, Nathan H

    2013-01-01

    Survival and progression of mature B-cell malignancies depend on signals from the B-cell antigen receptor, and Bruton tyrosine kinase (BTK) is a critical signaling kinase in this pathway. We evaluated ibrutinib (PCI-32765), a small-molecule irreversible inhibitor of BTK, in patients with B-cell malignancies. Patients with relapsed or refractory B-cell lymphoma and chronic lymphocytic leukemia received escalating oral doses of ibrutinib. Two schedules were evaluated: one, 28 days on, 7 days off; and two, once-daily continuous dosing. Occupancy of BTK by ibrutinib in peripheral blood was monitored using a fluorescent affinity probe. Dose escalation proceeded until either the maximum-tolerated dose (MTD) was achieved or, in the absence of MTD, until three dose levels above full BTK occupancy by ibrutinib. Response was evaluated every two cycles. Fifty-six patients with a variety of B-cell malignancies were treated over seven cohorts. Most adverse events were grade 1 and 2 in severity and self-limited. Dose-limiting events were not observed, even with prolonged dosing. Full occupancy of the BTK active site occurred at 2.5 mg/kg per day, and dose escalation continued to 12.5 mg/kg per day without reaching MTD. Pharmacokinetic data indicated rapid absorption and elimination, yet BTK occupancy was maintained for at least 24 hours, consistent with the irreversible mechanism. Objective response rate in 50 evaluable patients was 60%, including complete response of 16%. Median progression-free survival in all patients was 13.6 months. Ibrutinib, a novel BTK-targeting inhibitor, is well tolerated, with substantial activity across B-cell histologies.

  10. Targeting the sugar metabolism of tumors with a first-in-class 6-phosphofructo-2-kinase (PFKFB4) inhibitor.

    Science.gov (United States)

    Chesney, Jason; Clark, Jennifer; Lanceta, Lilibeth; Trent, John O; Telang, Sucheta

    2015-07-20

    Human tumors exhibit increased glucose uptake and metabolism as a result of high demand for ATP and anabolic substrates and this metabolotype is a negative prognostic indicator for survival. Recent studies have demonstrated that cancer cells from several tissue origins and genetic backgrounds require the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4), a regulatory enzyme that synthesizes an allosteric activator of glycolysis, fructose-2,6-bisphosphate. We report the discovery of a first-in-class PFKFB4 inhibitor, 5-(n-(8-methoxy-4-quinolyl)amino)pentyl nitrate (5MPN), using structure-based virtual computational screening. We find that 5MPN is a selective inhibitor of PFKFB4 that suppresses the glycolysis and proliferation of multiple human cancer cell lines but not non-transformed epithelial cells in vitro. Importantly, 5MPN has high oral bioavailability and per os administration of a non-toxic dose of 5MPN suppresses the glucose metabolism and growth of tumors in mice.

  11. Small-molecule inhibitors of Ataxia Telangiectasia and Rad3 related kinase (ATR) sensitize lymphoma cells to UVA radiation

    DEFF Research Database (Denmark)

    Biskup, Edyta; Naym, David Gram; Gniadecki, Robert

    2016-01-01

    inhibited by small molecule antagonists VE-821, VE-822 or Chir-124, or by small interfering RNAs (siRNAs). Cell cycle and viability were assessed by flow cytometry. RESULTS: Small molecule inhibitors of ATR and Chk1 potently sensitized all cell lines to PUVA and, importantly, also to UVA, which by itself...... did not cause apoptotic response. VE-821/2 blocked ATR pathway activation and released the cells from the G2/M block caused by UVA and PUVA, but did not affect apoptosis caused by other chemotherapeutics (etoposide, gemcitabine, doxorubicine) or by hydrogen peroxide. Knockdown of ATR and Chk1 with si......RNA also blocked the ATR pathway and released the cells from G2/M block but did not sensitize the cells to UVA as observed with the small molecule inhibitors. The latter suggested that the synergism between VE-821/2 or Chir-124 and UVA was not solely caused by specific blocking of ATR kinase but also ATR...

  12. Protein kinase Cepsilon is important for migration of neuroblastoma cells

    International Nuclear Information System (INIS)

    Stensman, Helena; Larsson, Christer

    2008-01-01

    Migration is important for the metastatic capacity and thus for the malignancy of cancer cells. There is limited knowledge on regulatory factors that promote the migration of neuroblastoma cells. This study investigates the hypothesis that protein kinase C (PKC) isoforms regulate neuroblastoma cell motility. PKC isoforms were downregulated with siRNA or modulated with activators and inhibitors. Migration was analyzed with scratch and transwell assays. Protein phosphorylation and expression levels were measured with Western blot. Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced migration of SK-N-BE(2)C neuroblastoma cells. Treatment with the general protein kinase C (PKC) inhibitor GF109203X and the inhibitor of classical isoforms Gö6976 inhibited migration while an inhibitor of PKCβ isoforms did not have an effect. Downregulation of PKCε, but not of PKCα or PKCδ, with siRNA led to a suppression of both basal and TPA-stimulated migration. Experiments using PD98059 and LY294002, inhibitors of the Erk and phosphatidylinositol 3-kinase (PI3K) pathways, respectively, showed that PI3K is not necessary for TPA-induced migration. The Erk pathway might be involved in TPA-induced migration but not in migration driven by PKCε. TPA induced phosphorylation of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) which was suppressed by the PKC inhibitors. Treatment with siRNA oligonucleotides against different PKC isoforms before stimulation with TPA did not influence the phosphorylation of MARCKS. PKCε is important for migration of SK-N-BE(2)C neuroblastoma cells. Neither the Erk pathway nor MARCKS are critical downstream targets of PKCε but they may be involved in TPA-mediated migration

  13. Insights into the Inhibition of the p90 Ribosomal S6 Kinase (RSK) by the Flavonol Glycoside SL0101 from the 1.5 Å Crystal Structure of the N-Terminal Domain of RSK2 with Bound Inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Utepbergenov, Darkhan; Derewenda, Urszula; Olekhnovich, Natalya; Szukalska, Gabriela; Banerjee, Budhaditya; Hilinski, Michael K.; Lannigan, Deborah A.; Stukenberg, P. Todd; Derewenda, Zygmunt S. (Lodz - Poland); (UV)

    2012-09-11

    The p90 ribosomal S6 family of kinases (RSK) are potential drug targets, due to their involvement in cancer and other pathologies. There are currently only two known selective inhibitors of RSK, but the basis for selectivity is not known. One of these inhibitors is a naturally occurring kaempferol-a-l-diacetylrhamnoside, SL0101. Here, we report the crystal structure of the complex of the N-terminal kinase domain of the RSK2 isoform with SL0101 at 1.5 {angstrom} resolution. The refined atomic model reveals unprecedented structural reorganization of the protein moiety, as compared to the nucleotide-bound form. The entire N-lobe, the hinge region, and the aD-helix undergo dramatic conformational changes resulting in a rearrangement of the nucleotide binding site with concomitant formation of a highly hydrophobic pocket spatially suited to accommodate SL0101. These unexpected results will be invaluable in further optimization of the SL0101 scaffold as a promising lead for a novel class of kinase inhibitors.

  14. Efficacy and gastrointestinal tolerability of ML3403, a selective inhibitor of p38 MAP kinase and CBS-3595, a dual inhibitor of p38 MAP kinase and phosphodiesterase 4 in CFA-induced arthritis in rats.

    Science.gov (United States)

    Koch, Diana A; Silva, Rodrigo B M; de Souza, Alessandra H; Leite, Carlos E; Nicoletti, Natália F; Campos, Maria M; Laufer, Stefan; Morrone, Fernanda B

    2014-03-01

    Mitogen-activated protein kinase (MAPK) p38 inhibitors have entered the clinical phase, although many of them have failed due to high toxicity and lack of efficacy. In the present study we compared the effects of the selective p38 inhibitor ML3403 and the dual p38-PDE4 inhibitor CBS-3595, on inflammatory and nociceptive parameters in a model of polyarthritis in rats. Male Wistar rats (180-200 g) were used for the complete Freund's adjuvant (CFA)-induced arthritis model and they were evaluated at 14-21 days. We also analysed the effects of these pharmacological tools on liver and gastrointestinal toxicity and on cytokine levels. Repeated CBS-3595 (3 mg/kg) or ML3403 (10 mg/kg) administration produced significant anti-inflammatory actions in the chronic arthritis model induced by CFA. CBS-3595 and ML3403 treatment also markedly reduced the production of the proinflammatory cytokine IL-6 in the paw tissue, whereas it widely increased the levels of the anti-inflammatory cytokine IL-10. Moreover, CBS-3595 produced partial anti-allodynic effects in the CFA model at 4 and 8 days after treatment. Notably, ML3403 and CBS-3595 did not show marked signs of hepatoxicity, as supported by unaltered histological observations in the liver sections. Finally, both compounds were safe in the gastrointestinal tract, according to evaluation of intestinal biopsies. CBS-3595 displayed a superior profile regarding its anti-inflammatory effects. Thus p38 MAPK/PDE4 blocking might well constitute a relevant strategy for the treatment of RA.

  15. GenBank blastx search result: AK288454 [KOME

    Lifescience Database Archive (English)

    Full Text Available protein, putative response regulator of the competence regulon ComE1, putative histidine kinase of the competence regulon ComD1, comp...in, putative histidine kinase of the competence regulon ComD2, putative histidine kinase of the competence... regulon ComD3, putative response regulator of the competence regulon ComE2, putati...etence stimulating protein precursor, hypothetical protein, putative immunity prote

  16. Design, Synthesis, and Pharmacological Evaluation of Novel Multisubstituted Pyridin-3-amine Derivatives as Multitargeted Protein Kinase Inhibitors for the Treatment of Non-Small Cell Lung Cancer.

    Science.gov (United States)

    Zhu, Wei; Chen, Hui; Wang, Yulan; Wang, Jiang; Peng, Xia; Chen, Xianjie; Gao, Yinglei; Li, Chunpu; He, Yulong; Ai, Jing; Geng, Meiyu; Zheng, Mingyue; Liu, Hong

    2017-07-27

    A novel series of pyridin-3-amine derivatives were designed, synthesized, and evaluated as multitargeted protein kinase inhibitors for the treatment of non-small cell lung cancer (NSCLC). Hit 1 was first disclosed by in silico screening against fibroblast growth factor receptors (FGFR), which was subsequently validated by in vitro experiments. The structure-activity relationship (SAR) of its analogues was then explored to afford novel FGFR inhibitors 2a-2p and 3a-3q. Among them, 3m showed potent inhibition against FGFR1, 2, and 3. Interestingly, compound 3m not only inhibited various phosphorylation and downstream signaling across different oncogenic forms in FGFR-overactivated cancer cells but also showed nanomolar level inhibition against several other NSCLC-related oncogene kinases, including RET, EGFR, EGFR/T790M/L858R, DDR2, and ALK. Finally, in vivo pharmacology evaluations of 3m showed significant antitumor activity (TGI = 66.1%) in NCI-H1581 NSCLC xenografts with a good pharmacokinetic profile.

  17. Mechanism of the pH-induced conformational change in the sensor domain of the DraK Histidine kinase via the E83, E105, and E107 residues.

    Directory of Open Access Journals (Sweden)

    Kwon Joo Yeo

    Full Text Available The DraR/DraK two-component system was found to be involved in the differential regulation of antibiotic biosynthesis in a medium-dependent manner; however, its function and signaling and sensing mechanisms remain unclear. Here, we describe the solution structure of the extracellular sensor domain of DraK and suggest a mechanism for the pH-dependent conformational change of the protein. The structure contains a mixed alpha-beta fold, adopting a fold similar to the ubiquitous sensor domain of histidine kinase. A biophysical study demonstrates that the E83, E105, and E107 residues have abnormally high pKa values and that they drive the pH-dependent conformational change for the extracellular sensor domain of DraK. We found that a triple mutant (E83L/E105L/E107A is pH independent and mimics the low pH structure. An in vivo study showed that DraK is essential for the recovery of the pH of Streptomyces coelicolor growth medium after acid shock. Our findings suggest that the DraR/DraK two-component system plays an important role in the pH regulation of S. coelicolor growth medium. This study provides a foundation for the regulation and the production of secondary metabolites in Streptomyces.

  18. Screening of the two-component-system histidine kinases of Listeria monocytogenes EGD-e. LiaS is needed for growth under heat, acid, alkali, osmotic, ethanol and oxidative stresses.

    Science.gov (United States)

    Pöntinen, Anna; Lindström, Miia; Skurnik, Mikael; Korkeala, Hannu

    2017-08-01

    To study the role of each two-component system (TCS) histidine kinase (HK) in stress tolerance of Listeria monocytogenes EGD-e, we monitored the growth of individual HK deletion mutant strains under heat (42.5 °C), acid (pH 5.6), alkali (pH 9.4), osmotic (6% NaCl), ethanol (3.5 vol%), and oxidative (5 mM H 2 O 2 ) stresses. The growth of ΔliaS (Δlmo1021) strain was impaired under each stress, with the most notable decrease under heat and osmotic stresses. The ΔvirS (Δlmo1741) strain showed nearly completely restricted growth at high temperature and impaired growth in ethanol. The growth of ΔagrC (Δlmo0050) strain was impaired under osmotic stress and slightly under oxidative stress. We successfully complemented the HK mutations using a novel allelic exchange based approach. This approach avoided the copy-number problems associated with in trans complementation from a plasmid. The mutant phenotypes were restored to the wild-type level in the complemented strains. This study reveals novel knowledge on the HKs needed for growth of L. monocytogenes EGD-e under abovementioned stress conditions, with LiaS playing multiple roles in stress tolerance of L. monocytogenes EGD-e. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Systematic review of infectious events with the Bruton tyrosine kinase inhibitor ibrutinib in the treatment of hematologic malignancies.

    Science.gov (United States)

    Tillman, Benjamin F; Pauff, James M; Satyanarayana, Gowri; Talbott, Mahsa; Warner, Jeremy L

    2018-04-01

    Ibrutinib is an irreversible inhibitor of Bruton tyrosine kinase (BTK) in B lymphocytes as well as other kinases including interleukin-2-inducible T-cell kinase (ITK) in CD4+ Th2 regulatory T cells. Increased infections have been observed in patients taking ibrutinib. The overall incidence has not been systematically evaluated. The published literature and conference abstracts of prospective clinical trials using ibrutinib in hematologic malignancies were identified and reviewed using PubMed, Google Scholar, and HemOnc.org per PRISMA guidelines. Infectious events with a focus on pneumonia were collated per the Common Terminology Criteria for Adverse Events Version 4.03 grading. Infectious complications are common, occurring in 56% of patients taking single-agent ibrutinib and 52% of those on combination therapy. Approximately one in 5 patients developed pneumonia, which was the major contributor to a 2% rate of death from infections. Many of the cases of pneumonia were due to opportunistic pathogens. Ibrutinib use requires prudent consideration of the impacts on host immunity. We identified a high rate of serious adverse infectious events within prospective clinical trials. Data suggest a role of both BTK and ITK inhibition for the increased events. There was considerable variability in the reporting of adverse events between trials, journals, and conference reports. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Design, Synthesis and Biological Evaluation of Benzohydrazide Derivatives Containing Dihydropyrazoles as Potential EGFR Kinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Hai-Chao Wang

    2016-08-01

    Full Text Available A series of novel benzohydrazide derivatives containing dihydropyrazoles have been synthesized as potential epidermal growth factor receptor (EGFR kinase inhibitors and their biological activities as potential antiproliferative agents have been evaluated. Among these compounds, compound H20 exhibited the most potent antiproliferative activity against four cancer cell line variants (A549, MCF-7, HeLa, HepG2 with IC50 values of 0.46, 0.29, 0.15 and 0.21 μM respectively, which showed the most potent EGFR inhibition activities (IC50 = 0.08 μM for EGFR. Molecular modeling simulation studies were performed in order to predict the biological activity and activity relationship (SAR of these benzohydrazide derivatives. These results suggested that compound H20 may be a promising anticancer agent.

  1. Structural Characterization of LRRK2 Inhibitors

    NARCIS (Netherlands)

    Gilsbach, Bernd K; Messias, Ana C; Ito, Genta; Sattler, Michael; Alessi, Dario R; Wittinghofer, Alfred; Kortholt, Arjan

    2015-01-01

    Kinase inhibition is considered to be an important therapeutic target for LRRK2 mediated Parkinson's disease (PD). Many LRRK2 kinase inhibitors have been reported but have yet to be optimized in order to qualify as drug candidates for the treatment of the disease. In order to start a

  2. Expression and Purification of PI3 Kinase {alpha} and Development of an ATP Depletion and an AlphaScreen PI3 Kinase Activity Assay

    DEFF Research Database (Denmark)

    Boldyreff, Brigitte; Rasmussen, Tine L; Jensen, Hans H

    2008-01-01

    Phosphoinositide-3-kinases are important targets for drug development because many proteins in the PI3 kinase signaling pathway are mutated, hyperactivated, or overexpressed in human cancers. Here, the authors coexpressed the human class Ia PI3 kinase p110alpha catalytic domain with an N-terminal....... In parallel, a second assay format using the AlphaScreen technology was optimized to measure PI3 kinase activity. Both assay formats used should be suitable for high-throughput screening for the identification of PI3 kinase inhibitors. (Journal of Biomolecular Screening XXXX:xx-xx)....

  3. Structure-activity relationship study of oxindole-based inhibitors of cyclin-dependent kinases based on least-squares support vector machines

    International Nuclear Information System (INIS)

    Li Jiazhong; Liu Huanxiang; Yao Xiaojun; Liu Mancang; Hu Zhide; Fan Botao

    2007-01-01

    The least-squares support vector machines (LS-SVMs), as an effective modified algorithm of support vector machine, was used to build structure-activity relationship (SAR) models to classify the oxindole-based inhibitors of cyclin-dependent kinases (CDKs) based on their activity. Each compound was depicted by the structural descriptors that encode constitutional, topological, geometrical, electrostatic and quantum-chemical features. The forward-step-wise linear discriminate analysis method was used to search the descriptor space and select the structural descriptors responsible for activity. The linear discriminant analysis (LDA) and nonlinear LS-SVMs method were employed to build classification models, and the best results were obtained by the LS-SVMs method with prediction accuracy of 100% on the test set and 90.91% for CDK1 and CDK2, respectively, as well as that of LDA models 95.45% and 86.36%. This paper provides an effective method to screen CDKs inhibitors

  4. Helicobacter pylori CagA Inhibits PAR1-MARK Family Kinases by Mimicking Host Substrates

    Energy Technology Data Exchange (ETDEWEB)

    Nesic, D.; Miller, M; Quinkert, Z; Stein, M; Chait, B; Stebbins, C

    2010-01-01

    The CagA protein of Helicobacter pylori interacts with numerous cellular factors and is associated with increased virulence and risk of gastric carcinoma. We present here the cocrystal structure of a subdomain of CagA with the human kinase PAR1b/MARK2, revealing that a CagA peptide mimics substrates of this kinase family, resembling eukaryotic protein kinase inhibitors. Mutagenesis of conserved residues central to this interaction renders CagA inactive as an inhibitor of MARK2.

  5. Ergothioneine, histidine, and two naturally occurring histidine dipeptides as radioprotectors against gamma-irradiation inactivation of bacteriophages T4 and P22

    International Nuclear Information System (INIS)

    Hartman, P.E.; Hartman, Z.; Citardi, M.J.

    1988-01-01

    Bacteriophages P22, T4+, and T4os (osmotic shock-resistant mutant with altered capsids) were diluted in 0.85% NaCl and exposed to gamma irradiation (2.79 Gy/min) at room temperature (24 degrees C). T4+ was more sensitive to inactivation than was P22, and the T4os mutant was even more sensitive than T4+. Catalase exhibited a strong protective effect and superoxide dismutase a weaker protection, indicating that H 2 O 2 or some product derived therefrom was predominant in causing inactivation of plaque formation. Low but significant (0.1-0.3 mM) reduced glutathione (GSH) enhanced phage inactivation, but a higher (1 mM) GSH concentration protected. A similar effect was found for the polyamine, spermidine. In contrast, 0.1 mM L-ergothioneine (2-thiol-L-histidine betaine) exhibited strong protection and 1 mM afforded essentially complete protection. L-Ergothioneine is present in millimolar concentrations in some fungi and is conserved up to millimolar concentrations in critical tissues when consumed by man. L-Histidine and two histidine-containing dipeptides, carnosine and anserine, protected at a concentration of 1 mM, a level at which they are present in striated muscles of various animals

  6. Structural Analysis of DFG-in and DFG-out Dual Src-Abl Inhibitors Sharing a Common Vinyl Purine Template

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Tianjun; Commodore, Lois; Huang, Wei-Sheng; Wang, Yihan; Sawyer, Tomi K.; Shakespeare, William C.; Clackson, Tim; Zhu, Xiaotian; Dalgarno, David C. (ARIAD)

    2010-09-30

    Bcr-Abl is the oncogenic protein tyrosine kinase responsible for chronic myeloid leukemia (CML). Treatment of the disease with imatinib (Gleevec) often results in drug resistance via kinase mutations at the advanced phases of the disease, which has necessitated the development of new mutation-resistant inhibitors, notably against the T315I gatekeeper mutation. As part of our efforts to discover such mutation resistant Abl inhibitors, we have focused on optimizing purine template kinase inhibitors, leading to the discovery of potent DFG-in and DFG-out series of Abl inhibitors that are also potent Src inhibitors. Here we present crystal structures of Abl bound by two such inhibitors, based on a common N9-arenyl purine, and that represent both DFG-in and -out binding modes. In each structure the purine template is bound deeply in the adenine pocket and the novel vinyl linker forms a non-classical hydrogen bond to the gatekeeper residue, Thr315. Specific template substitutions promote either a DFG-in or -out binding mode, with the kinase binding site adjusting to optimize molecular recognition. Bcr-Abl T315I mutant kinase is resistant to all currently marketed Abl inhibitors, and is the focus of intense drug discovery efforts. Notably, our DFG-out inhibitor, AP24163, exhibits modest activity against this mutant, illustrating that this kinase mutant can be inhibited by DFG-out class inhibitors. Furthermore our DFG-out inhibitor exhibits dual Src-Abl activity, absent from the prototypical DFG-out inhibitor, imatinib as well as its analog, nilotinib. The data presented here provides structural guidance for the further design of novel potent DFG-out class inhibitors against Src, Abl and Abl T315I mutant kinases.

  7. A chimeric cyclic interferon-α2b peptide induces apoptosis by sequential activation of phosphatidylinositol 3-kinase, protein kinase Cδ and p38 MAP kinase.

    Science.gov (United States)

    Blank, V C; Bertucci, L; Furmento, V A; Peña, C; Marino, V J; Roguin, L P

    2013-06-10

    We have previously demonstrated that tyrosine phosphorylation of STAT1/3 and p38 mitogen-activated protein kinase (p38 MAPK) activation are involved in the apoptotic response triggered by a chimeric cyclic peptide of the interferon-α2b (IFN-α2b) in WISH cells. Since the peptide also induced serine phosphorylation of STAT proteins, in the present study we examined the kinase involved in serine STAT1 phosphorylation and the signaling effectors acting upstream such activation. We first found that p38 MAPK is involved in serine STAT1 phosphorylation, since a reduction of phophoserine-STAT1 levels was evident after incubating WISH cells with cyclic peptide in the presence of a p38 pharmacological inhibitor or a dominant-negative p38 mutant. Next, we demonstrated that the peptide induced activation of protein kinase Cδ (PKCδ). Based on this finding, the role of this kinase was then evaluated. After incubating WISH cells with a PKCδ inhibitor or after decreasing PKCδ expression levels by RNA interference, both peptide-induced serine STAT1 and p38 phosphorylation levels were significantly decreased, indicating that PKCδ functions as an upstream regulator of p38. We also showed that PKCδ and p38 activation stimulated by the peptide was inhibited by a specific pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) or by a dominant-negative p85 PI3K-regulatory subunit, suggesting that PI3K is upstream in the signaling cascade. In addition, the role of PI3K and PKCδ in cyclic peptide-induced apoptosis was examined. Both signaling effectors were found to regulate the antiproliferative activity and the apoptotic response triggered by the cyclic peptide in WISH cells. In conclusion, we herein demonstrated that STAT1 serine phosphorylation is mediated by the sequential activation of PI3K, PKCδ and p38 MAPK. This signaling cascade contributes to the antitumor effect induced by the chimeric IFN-α2b cyclic peptide in WISH cells. Copyright © 2013 Elsevier Inc

  8. Exploration of charge states of balanol analogues acting as ATP-competitive inhibitors in kinases.

    Science.gov (United States)

    Hardianto, Ari; Yusuf, Muhammad; Liu, Fei; Ranganathan, Shoba

    2017-12-28

    (-)-Balanol is an ATP mimic that inhibits protein kinase C (PKC) isozymes and cAMP-dependent protein kinase (PKA) with limited selectivity. While PKA is a tumour promoter, PKC isozymes act as tumour promoters or suppressors, depending on the cancer type. In particular, PKCε is frequently implicated in cancer promotion, making it a potential target for anticancer drugs. To improve isozyme selectivity of balanol, exhaustive structural and activity relationship (SAR) studies have been performed in the last two decades, but with limited success. More recently, fluorination on balanol has shown improved selectivity for PKCε, although the fluorine effect is not yet clearly understood. Understanding the origin to this fluorine-based selectivity will be valuable for designing better balanol-based ATP mimicking inhibitors. Computational approaches such as molecular dynamics (MD) simulations can decipher the fluorine effect, provided that correct charges have been assigned to a ligand. Balanol analogues have multiple ionisable functional groups and the effect of fluorine substitutions on the exact charge state of each analogue bound to PKA and to PKCε needs to be thoroughly investigated in order to design highly selective inhibitors for therapeutic applications. We explored the charge states of novel fluorinated balanol analogues using MD simulations. For different potential charge states of these analogues, Molecular Mechanics Generalized Born Surface Area (MMGBSA) binding energy values were computed. This study suggests that balanol and the most potent fluorinated analogue (5S fluorine substitution on the azepane ring), have charges on the azepane ring (N1), and the phenolic (C6''OH) and the carboxylate (C15''O 2 H) groups on the benzophenone moiety, when bound to PKCε as well as PKA. To the best our knowledge, this is the first study showing that the phenolate group is charged in balanol and its analogues binding to the ATP site of PKCε. Correct charge

  9. Identification of quercitrin as an inhibitor of the p90 S6 ribosomal kinase (RSK): structure of its complex with the N-terminal domain of RSK2 at 1.8 Å resolution

    Energy Technology Data Exchange (ETDEWEB)

    Derewenda, Urszula; Artamonov, Mykhaylo; Szukalska, Gabriela; Utepbergenov, Darkhan; Olekhnovich, Natalya [University of Virginia, Charlottesville, VA 22908-0736 (United States); Parikh, Hardik I.; Kellogg, Glen E. [Virginia Commonwealth University, Richmond, VA 23298-0540 (United States); Somlyo, Avril V.; Derewenda, Zygmunt S., E-mail: zsd4n@virginia.edu [University of Virginia, Charlottesville, VA 22908-0736 (United States)

    2013-02-01

    The crystal structure of quercitrin, a naturally occurring flavonol glycoside, has been determined in a complex with the N-terminal kinase domain of murine RSK2. The structure revealed that quercitrin inhibits the RSK2 kinase in the same fashion as another known inhibitor, SL0101. Members of the RSK family of kinases constitute attractive targets for drug design, but a lack of structural information regarding the mechanism of selective inhibitors impedes progress in this field. The crystal structure of the N-terminal kinase domain (residues 45–346) of mouse RSK2, or RSK2{sup NTKD}, has recently been described in complex with one of only two known selective inhibitors, a rare naturally occurring flavonol glycoside, kaempferol 3-O-(3′′,4′′-di-O-acetyl-α-l-rhamnopyranoside), known as SL0101. Based on this structure, it was hypothesized that quercitrin (quercetin 3-O-α-l-rhamnopyranoside), a related but ubiquitous and inexpensive compound, might also act as an RSK inhibitor. Here, it is demonstrated that quercitrin binds to RSK2{sup NTKD} with a dissociation constant (K{sub d}) of 5.8 µM as determined by isothermal titration calorimetry, and a crystal structure of the binary complex at 1.8 Å resolution is reported. The crystal structure reveals a very similar mode of binding to that recently reported for SL0101. Closer inspection shows a number of small but significant differences that explain the slightly higher K{sub d} for quercitrin compared with SL0101. It is also shown that quercitrin can effectively substitute for SL0101 in a biological assay, in which it significantly suppresses the contractile force in rabbit pulmonary artery smooth muscle in response to Ca{sup 2+}.

  10. Identification of quercitrin as an inhibitor of the p90 S6 ribosomal kinase (RSK): structure of its complex with the N-terminal domain of RSK2 at 1.8 Å resolution

    International Nuclear Information System (INIS)

    Derewenda, Urszula; Artamonov, Mykhaylo; Szukalska, Gabriela; Utepbergenov, Darkhan; Olekhnovich, Natalya; Parikh, Hardik I.; Kellogg, Glen E.; Somlyo, Avril V.; Derewenda, Zygmunt S.

    2013-01-01

    The crystal structure of quercitrin, a naturally occurring flavonol glycoside, has been determined in a complex with the N-terminal kinase domain of murine RSK2. The structure revealed that quercitrin inhibits the RSK2 kinase in the same fashion as another known inhibitor, SL0101. Members of the RSK family of kinases constitute attractive targets for drug design, but a lack of structural information regarding the mechanism of selective inhibitors impedes progress in this field. The crystal structure of the N-terminal kinase domain (residues 45–346) of mouse RSK2, or RSK2 NTKD , has recently been described in complex with one of only two known selective inhibitors, a rare naturally occurring flavonol glycoside, kaempferol 3-O-(3′′,4′′-di-O-acetyl-α-l-rhamnopyranoside), known as SL0101. Based on this structure, it was hypothesized that quercitrin (quercetin 3-O-α-l-rhamnopyranoside), a related but ubiquitous and inexpensive compound, might also act as an RSK inhibitor. Here, it is demonstrated that quercitrin binds to RSK2 NTKD with a dissociation constant (K d ) of 5.8 µM as determined by isothermal titration calorimetry, and a crystal structure of the binary complex at 1.8 Å resolution is reported. The crystal structure reveals a very similar mode of binding to that recently reported for SL0101. Closer inspection shows a number of small but significant differences that explain the slightly higher K d for quercitrin compared with SL0101. It is also shown that quercitrin can effectively substitute for SL0101 in a biological assay, in which it significantly suppresses the contractile force in rabbit pulmonary artery smooth muscle in response to Ca 2+

  11. Aurora A kinase RNAi and small molecule inhibition of Aurora kinases with VE-465 induce apoptotic death in multiple myeloma cells.

    Science.gov (United States)

    Evans, Robert; Naber, Claudia; Steffler, Tara; Checkland, Tamara; Keats, Jonathan; Maxwell, Christopher; Perry, Troy; Chau, Heidi; Belch, Andrew; Pilarski, Linda; Reiman, Tony

    2008-03-01

    The expression of RHAMM and other centrosome-associated genes are known to correlate with the extent of centrosome amplification in multiple myeloma, and with poor prognosis. RHAMM has a significant interaction with TPX2, a protein which regulates the localization and action of Aurora A kinase (AURKA) at the spindle poles. AURKA is known to be a central determinant of centrosome and spindle function and is a target for cancer therapy. Given these observations, we investigated the role of Aurora kinases as therapeutic targets in myeloma. Here we report that AURKA is expressed ubiquitously in myeloma, to varying degrees, in both cell lines and patients' bone marrow plasma cells. siRNA targeting AURKA induces apoptotic cell death in myeloma cell lines. The Aurora kinase inhibitor VE-465 also induces apoptosis and death in myeloma cell lines and primary myeloma plasma cells. The combination of VE-465 and dexamethasone improves cell killing compared with the use of either agent alone, even in cells resistant to the single agents. The phenotype of myeloma cells treated with VE-465 is consistent with published reports on the effects of Aurora kinase inhibition. Aurora kinase inhibitors should be pursued as potential treatments for myeloma.

  12. LDL cholesterol counteracts the antitumour effect of tyrosine kinase inhibitors against renal cell carcinoma.

    Science.gov (United States)

    Naito, Sei; Makhov, Peter; Astsaturov, Igor; Golovine, Konstantin; Tulin, Alexei; Kutikov, Alexander; Uzzo, Robert G; Kolenko, Vladimir M

    2017-04-25

    Treatment with tyrosine kinase inhibitors (TKIs) significantly improves survival of patients with renal cell carcinoma (RCC). However, about one-quarter of the RCC patients are primarily refractory to treatment with TKIs. We examined viability of RCC and endothelial cells treated with low-density lipoprotein (LDL) and/or TKIs. Next, we validated the potential role of PI3K/AKT signalling in LDL-mediated TKI resistance. Finally, we examined the effect of a high-fat/high-cholesterol diet on the response of RCC xenograft tumours to sunitinib. The addition of LDL cholesterol increases activation of PI3K/AKT signalling and compromises the antitumour efficacy of TKIs against RCC and endothelial cells. Furthermore, RCC xenograft tumours resist TKIs in mice fed a high-fat/high-cholesterol diet. The ability of renal tumours to maintain their cholesterol homoeostasis may be a critical component of TKI resistance in RCC patients.

  13. Overcoming imatinib resistance using Src inhibitor CGP76030, Abl inhibitor nilotinib and Abl/Lyn inhibitor INNO-406 in newly established K562 variants with BCR-ABL gene amplification.

    Science.gov (United States)

    Morinaga, Koji; Yamauchi, Takahiro; Kimura, Shinya; Maekawa, Taira; Ueda, Takanori

    2008-06-01

    Because imatinib (IM) resistance in chronic myeloid leukemia is primarily caused by the re-establishment of Abl kinase, new inhibitors may be efficacious. We evaluated 3 new agents against 2 new K562 variants, IM-R1 and IM-R2 cells, which were developed having 7- and 27-fold greater IM resistance, respectively, than the parental K562 cells. Both variants possessed BCR-ABL gene amplification along with elevated levels of its transcript and protein. Greater BCR-ABL gene amplification was observed in IM-R2 cells than in IM-R1 cells, which was consistent with the higher mRNA and protein levels of Bcr-Abl, and ultimately correlated with the greater IM resistance in IM-R2 cells. No mutation in the Abl kinase domain was detected in either variant. Despite the absence of Lyn overexpression, the Src kinase inhibitor CGP76030 showed positive cooperability with IM in inhibiting cell growth of not only K562 cells but also these 2 variants. This might be because of the augmented inhibition of Erk1/2 phosphorylation. The new Abl kinase inhibitor nilotinib was 10-fold more potent than IM in inhibiting the growth of K562 cells. Nilotinib inhibited the growth of IM-R1 and IM-R2 cells as potently as K562 cells. The combination of nilotinib with CGP76030 showed little additivity, because the potency of nilotinib masked the efficacy of CGP76030. The new dual Abl/Lyn inhibitor INNO-406 (formerly NS-187) was slightly more potent than nilotinib in inhibiting the growth of all 3 cell lines. Because BCR-ABL gene amplification occurs in blast crisis, these inhibitors might overcome IM resistance in such patients' leukemia. (c) 2008 Wiley-Liss, Inc.

  14. Discovery of novel 2-(4-aryl-2-methylpiperazin-1-yl)-pyrimidin-4-ones as glycogen synthase kinase-3β inhibitors.

    Science.gov (United States)

    Kohara, Toshiyuki; Nakayama, Kazuki; Watanabe, Kazutoshi; Kusaka, Shin-Ichi; Sakai, Daiki; Tanaka, Hiroshi; Fukunaga, Kenji; Sunada, Shinji; Nabeno, Mika; Saito, Ken-Ichi; Eguchi, Jun-Ichi; Mori, Akiko; Tanaka, Shinji; Bessho, Tomoko; Takiguchi-Hayashi, Keiko; Horikawa, Takashi

    2017-08-15

    We herein describe the results of further evolution of glycogen synthase kinase (GSK)-3β inhibitors from our promising compounds containing a 3-methylmorpholine moiety. Transformation of the morpholine moiety into a piperazine moiety resulted in potent GSK-3β inhibitors. SAR studies focused on the nitrogen atom of the piperazine moiety revealed that a phenyl group afforded potent inhibitory activity toward GSK-3β. Docking studies indicated that the phenyl group on the piperazine nitrogen atom and the methyl group on the piperazine make cation-π and CH-π interactions with GSK-3β respectively. 4-Methoxyphenyl analogue 29 showed most potent inhibitory activity toward GSK-3β with good in vitro and in vivo pharmacokinetic profiles, and 29 demonstrated a significant decrease in tau phosphorylation after oral administration in mice. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Kinase detection with gallium nitride based high electron mobility transistors.

    Science.gov (United States)

    Makowski, Matthew S; Bryan, Isaac; Sitar, Zlatko; Arellano, Consuelo; Xie, Jinqiao; Collazo, Ramon; Ivanisevic, Albena

    2013-07-01

    A label-free kinase detection system was fabricated by the adsorption of gold nanoparticles functionalized with kinase inhibitor onto AlGaN/GaN high electron mobility transistors (HEMTs). The HEMTs were operated near threshold voltage due to the greatest sensitivity in this operational region. The Au NP/HEMT biosensor system electrically detected 1 pM SRC kinase in ionic solutions. These results are pertinent to drug development applications associated with kinase sensing.

  16. The mTOR kinase inhibitor Everolimus decreases S6 kinase phosphorylation but fails to reduce mutant huntingtin levels in brain and is not neuroprotective in the R6/2 mouse model of Huntington's disease

    Directory of Open Access Journals (Sweden)

    Frentzel Stefan

    2010-06-01

    Full Text Available Abstract Background Huntington's disease (HD is a progressive neurodegenerative disorder caused by a CAG repeat expansion within the huntingtin gene. Mutant huntingtin protein misfolds and accumulates within neurons where it mediates its toxic effects. Promoting mutant huntingtin clearance by activating macroautophagy is one approach for treating Huntington's disease (HD. In this study, we evaluated the mTOR kinase inhibitor and macroautophagy promoting drug everolimus in the R6/2 mouse model of HD. Results Everolimus decreased phosphorylation of the mTOR target protein S6 kinase indicating brain penetration. However, everolimus did not activate brain macroautophagy as measured by LC3B Western blot analysis. Everolimus protected against early declines in motor performance; however, we found no evidence for neuroprotection as determined by brain pathology. In muscle but not brain, everolimus significantly decreased soluble mutant huntingtin levels. Conclusions Our data suggests that beneficial behavioral effects of everolimus in R6/2 mice result primarily from effects on muscle. Even though everolimus significantly modulated its target brain S6 kinase, this did not decrease mutant huntingtin levels or provide neuroprotection.

  17. Growth inhibitory effects of the dual ErbB1/ErbB2 tyrosine kinase inhibitor PKI-166 on human prostate cancer xenografts.

    Science.gov (United States)

    Mellinghoff, Ingo K; Tran, Chris; Sawyers, Charles L

    2002-09-15

    Experiments with human prostate cancer cell lines have shown that forced overexpression of the ErbB2-receptor tyrosine kinase (RTK) promotes androgen-independent growth and increases androgen receptor-transcriptional activity in a ligand-independent fashion. To investigate the relationship between ErbB-RTK signaling and androgen in genetically unmanipulated human prostate cancer, we performed biochemical and biological studies with the dual ErbB1/ErbB2 RTK inhibitor PKI-166 using human prostate cancer xenograft models with isogenic sublines reflecting the transition from androgen-dependent to androgen-independent growth. In the presence of low androgen concentrations, PKI-166 showed profound growth-inhibitory effects on tumor growth, which could be partially reversed by androgen add-back. At physiological androgen concentrations, androgen withdrawal greatly enhanced the ability of PKI-166 to retard tumor growth. The level of extracellular signal-regulated kinase activation correlated with the response to PKI-166 treatment, whereas the expression levels of ErbB1 and ErbB2 did not. These results suggest that ErbB1/ErbB2 RTKs play an important role in the biology of androgen-independent prostate cancer and provide a rationale for clinical evaluation of inhibitors targeted to this pathway.

  18. Intramolecular interactions stabilizing compact conformations of the intrinsically disordered kinase-inhibitor domain of Sic1: a molecular dynamics investigation.

    Directory of Open Access Journals (Sweden)

    Matteo eLambrughi

    2012-11-01

    Full Text Available Cyclin-dependent kinase inhibitors (CKIs are key regulatory proteins of the eukaryotic cell cycle, which modulate cyclin-dependent kinase (Cdk activity. CKIs perform their inhibitory effect by the formation of ternary complexes with a target kinase and its cognate cyclin. These regulators generally belong to the class of intrinsically disordered proteins (IDPs, which lack a well-defined and organized three-dimensional structure in their free state, undergoing folding upon binding to specific partners. Unbound IDPs are not merely random-coil structures, but can present intrinsically folded structural units (IFSUs and collapsed conformations. These structural features can be relevant to protein function in vivo.The yeast CKI Sic1 is a 284-amino acid IDP that binds to Cdk1 in complex with the Clb5,6 cyclins, preventing phosphorylation of G1 substrates and, therefore, entrance to the S phase. Sic1 degradation, triggered by multiple phosphorylation events, promotes cell-cycle progression. Previous experimental studies pointed out a propensity of Sic1 and its isolated domains to populate both extended and compact conformations. The present contribution provides models of the compact conformations of the Sic1 kinase-inhibitory domain (KID by all-atom molecular-dynamics simulations in explicit solvent and in the absence of interactors. The results are integrated by spectroscopic and spectrometric data. Helical IFSUs are identified, along with networks of intramolecular interactions. The results identify a group of hub residues and electrostatic interactions which are likely to be involved in the stabilization of globular states.

  19. A current and comprehensive review of cyclin-dependent kinase inhibitors for the treatment of metastatic breast cancer.

    Science.gov (United States)

    Bilgin, Burak; Sendur, Mehmet A N; Şener Dede, Didem; Akıncı, Muhammed Bülent; Yalçın, Bülent

    2017-09-01

    Resistance to endocrine treatment generally occurs over time, especially in the metastatic stage. In this paper, we aimed to review the mechanisms of cyclin-dependent kinase (CDK) 4/6 inhibition and clinical usage of new agents in the light of recent literature updates. A literature search was carried out using PubMed, Medline and ASCO and ESMO annual-meeting abstracts by using the following search keywords; "palbociclib", "abemaciclib", "ribociclib", "cyclin-dependent kinase inhibitors" and "CDK 4/6" in metastatic breast cancer (MBC). The last search was on 10 June 2017. CDKs and cyclins are two molecules that have a key role in cell cycle progression. Today, there are three highly selective CDK4/6 inhibitors in clinical development - palbociclib, ribociclib and abemaciclib. Palbociclib and ribociclib were recently approved by the US FDA in combination with letrozole for the treatment of MBC in a first-line setting, as well as palbociclib in combination with fulvestrant for hormone-receptor (HR)-positive MBC that had progressed while on previous endocrine therapy according to the PALOMA-1, MONALEESA-2 and PALOMA-3 trials, respectively. In the recently published randomized phase III MONARCH 2 trial, abemaciclib plus letrozole had longer progression-free survival and higher objective response rates with less serious adverse events in advanced HR-positive breast cancer previously treated with hormonal treatment. CDK4/6 inhibition is a new and promising target for patients with hormone-receptor-positive MBC. Both palbociclib and ribociclib showed significant additive benefit for patients receiving first-line treatment for HR-positive, epidermal growth factor receptor-2-negative advanced breast cancer. Palbociclib and abemaciclib also had significant activity in combination with fulvestrant for patients with MBC that progressed on previous endocrine therapy.

  20. Toward the Validation of Maternal Embryonic Leucine Zipper Kinase: Discovery, Optimization of Highly Potent and Selective Inhibitors, and Preliminary Biology Insight

    Energy Technology Data Exchange (ETDEWEB)

    Touré, B. Barry; Giraldes, John; Smith, Troy; Sprague, Elizabeth R.; Wang, Yaping; Mathieu, Simon; Chen, Zhuoliang; Mishina, Yuji; Feng, Yun; Yan-Neale, Yan; Shakya, Subarna; Chen, Dongshu; Meyer, Matthew; Puleo, David; Brazell, J. Tres; Straub, Christopher; Sage, David; Wright, Kirk; Yuan, Yanqiu; Chen, Xin; Duca, Jose; Kim, Sean; Tian, Li; Martin, Eric; Hurov, Kristen; Shao, Wenlin (Novartis)

    2016-05-26

    MELK kinase has been implicated in playing an important role in tumorigenesis. Our previous studies suggested that MELK is involved in the regulation of cell cycle and its genetic depletion leads to growth inhibition in a subset of high MELK-expressing basal-like breast cancer cell lines. Herein we describe the discovery and optimization of novel MELK inhibitors 8a and 8b that recapitulate the cellular effects observed by short hairpin ribonucleic acid (shRNA)-mediated MELK knockdown in cellular models. We also discovered a novel fluorine-induced hydrophobic collapse that locked the ligand in its bioactive conformation and led to a 20-fold gain in potency. These novel pharmacological inhibitors achieved high exposure in vivo and were well tolerated, which may allow further in vivo evaluation.

  1. Influence of histidine on zinc transport into rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Takeda, Atsushi; Suzuki, Mai; Okada, Shoji; Oku, Naoto [Shizuoka Univ. (Japan). School of Pharmaceutical Sciences

    2000-06-01

    The brain of rats injected intravenously with {sup 65}Zn-His or {sup 65}ZnCl{sub 2} was subjected to autoradiography to study the role of histidine on zinc transport into the brain. One hour after injection, the radioactivity from {sup 65}Zn-His was largely concentrated in the choroid plexus in the ventricles. Six days after injection, the radioactivity from {sup 65}Zn-His was relatively concentrated in the hippocampal CA3 and dentate gyrus and the amygdala. The relative distribution of {sup 65}Zn-His in the brain was similar to that of {sup 65}ZnCl{sub 2} group at both 1 h and 6 days, suggesting that histidine may participate in zinc uptake in the brain. On the other hand, the clearance of the {sup 65}Zn-His group from the blood was higher than that of the {sup 65}ZnCl{sub 2} group. Brain uptake of the former was lower than that of the latter both 1 h and 6 days after injection. These results suggest that zinc uptake in the brain is influenced by histidine levels in the bloodstream. (author)

  2. Theoretical study on the interaction of pyrrolopyrimidine derivatives as LIMK2 inhibitors: insight into structure-based inhibitor design.

    Science.gov (United States)

    Shen, Mingyun; Zhou, Shunye; Li, Youyong; Li, Dan; Hou, Tingjun

    2013-10-01

    LIM kinases (LIMKs), downstream of Rho-associated protein kinases (ROCKs) and p21-activated protein kinases (PAKs), are shown to be promising targets for the treatment of cancers. In this study, the inhibition mechanism of 41 pyrrolopyrimidine derivatives as LIMK2 inhibitors was explored through a series of theoretical approaches. First, a model of LIMK2 was generated through molecular homology modeling, and the studied inhibitors were docked into the binding active site of LIMK2 by the docking protocol, taking into consideration the flexibility of the protein. The binding poses predicted by molecular docking for 17 selected inhibitors with different bioactivities complexed with LIMK2 underwent molecular dynamics (MD) simulations, and the binding free energies for the complexes were predicted by using the molecular mechanics/generalized born surface area (MM/GBSA) method. The predicted binding free energies correlated well with the experimental bioactivities (r(2) = 0.63 or 0.62). Next, the free energy decomposition analysis was utilized to highlight the following key structural features related to biological activity: (1) the important H-bond between Ile408 and pyrrolopyrimidine, (2) the H-bonds between the inhibitors and Asp469 and Gly471 which maintain the stability of the DFG-out conformation, and (3) the hydrophobic interactions between the inhibitors and several key residues (Leu337, Phe342, Ala345, Val358, Lys360, Leu389, Ile408, Leu458 and Leu472). Finally, a variety of LIMK2 inhibitors with a pyrrolopyrimidine scaffold were designed, some of which showed improved potency according to the predictions. Our studies suggest that the use of molecular docking with MD simulations and free energy calculations could be a powerful tool for understanding the binding mechanism of LIMK2 inhibitors and for the design of more potent LIMK2 inhibitors.

  3. Inhibition of ErbB2 by receptor tyrosine kinase inhibitors causes myofibrillar structural damage without cell death in adult rat cardiomyocytes

    International Nuclear Information System (INIS)

    Pentassuglia, Laura; Graf, Michael; Lane, Heidi; Kuramochi, Yukio; Cote, Gregory; Timolati, Francesco; Sawyer, Douglas B.; Zuppinger, Christian; Suter, Thomas M.

    2009-01-01

    Inhibition of ErbB2 (HER2) with monoclonal antibodies, an effective therapy in some forms of breast cancer, is associated with cardiotoxicity, the pathophysiology of which is poorly understood. Recent data suggest, that dual inhibition of ErbB1 (EGFR) and ErbB2 signaling is more efficient in cancer therapy, however, cardiac safety of this therapeutic approach is unknown. We therefore tested an ErbB1-(CGP059326) and an ErbB1/ErbB2-(PKI166) tyrosine kinase inhibitor in an in-vitro system of adult rat ventricular cardiomyocytes and assessed their effects on 1. cell viability, 2. myofibrillar structure, 3. contractile function, and 4. MAPK- and Akt-signaling alone or in combination with Doxorubicin. Neither CGP nor PKI induced cardiomyocyte necrosis or apoptosis. PKI but not CGP caused myofibrillar structural damage that was additive to that induced by Doxorubicin at clinically relevant doses. These changes were associated with an inhibition of excitation-contraction coupling. PKI but not CGP decreased p-Erk1/2, suggesting a role for this MAP-kinase signaling pathway in the maintenance of myofibrils. These data indicate that the ErbB2 signaling pathway is critical for the maintenance of myofibrillar structure and function. Clinical studies using ErbB2-targeted inhibitors for the treatment of cancer should be designed to include careful monitoring for cardiac dysfunction.

  4. Dissociation of branched-chain alpha-keto acid dehydrogenase kinase (BDK) from branched-chain alpha-keto acid dehydrogenase complex (BCKDC) by BDK inhibitors.

    Science.gov (United States)

    Murakami, Taro; Matsuo, Masayuki; Shimizu, Ayako; Shimomura, Yoshiharu

    2005-02-01

    Branched-chain alpha-keto acid dehydrogenase kinase (BDK) phosphorylates and inactivates the branched-chain alpha-keto acid dehydrogenase complex (BCKDC), which is the rate-limiting enzyme in the branched-chain amino acid catabolism. BDK has been believed to be bound to the BCKDC. However, recent our studies demonstrated that protein-protein interaction between BDK and BCKDC is one of the factors to regulate BDK activity. Furthermore, only the bound form of BDK appears to have its activity. In the present study, we examined effects of BDK inhibitors on the amount of BDK bound to the BCKDC using rat liver extracts. The bound form of BDK in the extracts of liver from low protein diet-fed rats was measured by an immunoprecipitation pull down assay with or without BDK inhibitors. Among the BDK inhibitors. alpha-ketoisocaproate, alpha-chloroisocaproate, and a-ketoisovalerate released the BDK from the complex. Furthermore, the releasing effect of these inhibitors on the BDK appeared to depend on their inhibition constants. On the other hand, clofibric acid and thiamine pyrophosphate had no effect on the protein-protein interaction between two enzymes. These results suggest that the dissociation of the BDK from the BCKDC is one of the mechanisms responsible for the action of some inhibitors to BDK.

  5. Differential role of EGF and BFGF in human GBM-TIC proliferation: relationship to EGFR-tyrosine kinase inhibitor sensibility.

    Science.gov (United States)

    Bajetto, A; Porcile, C; Pattarozzi, A; Scotti, L; Aceto, A; Daga, A; Barbieri, F; Florio, T

    2013-01-01

    Glioblastoma multiforme (GBM) is among the most devastating human tumors being rapidly fatal despite aggressive surgery, radiation and chemotherapies. It is characterized by extensive dissemination of tumor cells within the brain that hinders complete surgical resection. GBM tumor initiating-cells (TICs) are a rare subpopulation of cells responsible for tumor development, growth, invasiveness and recurrence after chemotherapy. TICs from human GBM can be selected in vitro using the same conditions permissive for the growth of normal neural cells, of which share some features including marker expression, self-renewal capacity, long-term proliferation, and ability to differentiate into neuronal and glial cells. EGFR overexpression and its constitutive activation is one of the most important signaling alteration identified in GBM, and its pharmacological targeting represents an attractive therapeutic goal. We previously demonstrated that human GBM TICs have different sensitivity to the EGFR kinase inhibitors erlotinib and gefitinib, depending on the differential modulation of downstream signaling cascades. In this work we investigated the mechanisms of resistance to erlotinib in two human GBM TIC cultures, analyzing EGF and bFGF individual contribution to proliferation, clonogenicity, and migration. We demonstrated the presence of a small cell subpopulation whose proliferation is supported by EGF and a larger one mainly dependent on bFGF. Thus, insensitivity to EGFR kinase inhibitors as far as TIC proliferation results from a predominant FGFR activation that hides the inhibitory effects induced on EGFR signaling. Conversely, EGF and bFGF induced cell migration with similar efficacy. In addition, unlike neural stem/progenitors cells, the removal of chondroitin sulphate proteoglycans from cell surface was unable to discern EGF- and bFGF-dependent subpopulations in GBM TICs.

  6. Kinase profiling of liposarcomas using RNAi and drug screening assays identified druggable targets

    Directory of Open Access Journals (Sweden)

    Deepika Kanojia

    2017-11-01

    Full Text Available Abstract Background Liposarcoma, the most common soft tissue tumor, is understudied cancer, and limited progress has been made in the treatment of metastatic disease. The Achilles heel of cancer often is their kinases that are excellent therapeutic targets. However, very limited knowledge exists of therapeutic critical kinase targets in liposarcoma that could be potentially used in disease management. Methods Large RNAi and small-molecule tyrosine kinase inhibitor screens were performed against the proliferative capacity of liposarcoma cell lines of different subtypes. Each small molecule inhibitor was either FDA approved or in a clinical trial. Results Screening assays identified several previously unrecognized targets including PTK2 and KIT in liposarcoma. We also observed that ponatinib, multi-targeted tyrosine kinase inhibitor, was the most effective drug with anti-growth effects against all cell lines. In vitro assays showed that ponatinib inhibited the clonogenic proliferation of liposarcoma, and this anti-growth effect was associated with apoptosis and cell cycle arrest at the G0/G1 phase as well as a decrease in the KIT signaling pathway. In addition, ponatinib inhibited in vivo growth of liposarcoma in a xenograft model. Conclusions Two large-scale kinase screenings identified novel liposarcoma targets and a FDA-approved inhibitor, ponatinib with clear anti-liposarcoma activity highlighting its potential therapy for treatment of this deadly tumor.

  7. Rho-Kinase Inhibition Ameliorates Dasatinib-Induced Endothelial Dysfunction and Pulmonary Hypertension

    Directory of Open Access Journals (Sweden)

    Csilla Fazakas

    2018-05-01

    Full Text Available The multi-kinase inhibitor dasatinib is used for treatment of imatinib-resistant chronic myeloid leukemia, but is prone to induce microvascular dysfunction. In lung this can manifest as capillary leakage with pleural effusion, pulmonary edema or even pulmonary arterial hypertension. To understand how dasatinib causes endothelial dysfunction we examined the effects of clinically relevant concentrations of dasatinib on both human pulmonary arterial macro- and microvascular endothelial cells (ECs. The effects of dasatinib was compared to imatinib and nilotinib, two other clinically used BCR/Abl kinase inhibitors that do not inhibit Src. Real three-dimensional morphology and high resolution stiffness mapping revealed softening of both macro- and microvascular ECs upon dasatinib treatment, which was not observed in response to imatinib. In a dose-dependent manner, dasatinib decreased transendothelial electrical resistance/impedance and caused a permeability increase as well as disruption of tight adherens junctions in both cell types. In isolated perfused and ventilated rat lungs, dasatinib increased mean pulmonary arterial pressure, which was accompanied by a gain in lung weight. The Rho-kinase inhibitor Y27632 partly reversed the dasatinib-induced changes in vitro and ex vivo, presumably by acting downstream of Src. Co-administration of the Rho-kinase inhibitor Y27632 completely blunted the increased pulmonary pressure in response to dasatinib. In conclusion, a dasatinib-induced permeability increase in human pulmonary arterial macro- and microvascular ECs might explain many of the adverse effects of dasatinib in patients. Rho-kinase inhibition might be suitable to ameliorate these effects.

  8. Interleukin-1 beta induced synthesis of protein kinase C-delta and protein kinase C-epsilon in EL4 thymoma cells: possible involvement of phosphatidylinositol 3-kinase.

    Science.gov (United States)

    Varley, C L; Royds, J A; Brown, B L; Dobson, P R

    2001-01-01

    We present evidence here that the proinflammatory cytokine, interleukin-1 beta (IL-1 beta) stimulates a significant increase in protein kinase C (PKC)-epsilon and PKC-delta protein levels and increases PKC-epsilon, but not PKC-delta, transcripts in EL4 thymoma cells. Incubation of EL4 cells with IL-1 beta induced protein synthesis of PKC-epsilon (6-fold increase) by 7 h and had a biphasic effect on PKC-delta levels with peaks at 4 h (2-fold increase) and 24 h (4-fold increase). At the level of mRNA, PKC-epsilon, but not PKC-delta levels, were induced after incubation of EL4 cells with IL-1 beta. The signalling mechanisms utilized by IL-1 beta to induce the synthesis of these PKC isoforms were investigated. Two phosphatidylinositol (PI) 3-kinase-specific inhibitors, wortmannin and LY294002, inhibited IL-1 beta-induced synthesis of PKC-epsilon. However, the PI 3-kinase inhibitors had little effect on the IL-1 beta-induced synthesis of PKC-delta in these cells. Our results indicate that IL-1 beta induced both PKC-delta and PKC-epsilon expression over different time periods. Furthermore, our evidence suggests that IL-1 beta induction of PKC-epsilon, but not PKC-delta, may occur via the PI 3-kinase pathway. Copyright 2001 S. Karger AG, Basel

  9. Development of the Bruton's tyrosine kinase inhibitor ibrutinib for B cell malignancies.

    Science.gov (United States)

    Gayko, Urte; Fung, Mann; Clow, Fong; Sun, Steven; Faust, Elizabeth; Price, Samiyeh; James, Danelle; Doyle, Margaret; Bari, Samina; Zhuang, Sen Hong

    2015-11-01

    Ibrutinib is a first-in-class oral covalent inhibitor of Bruton's tyrosine kinase that has demonstrated clinical benefit for many patients with B cell malignancies. Positive results in initial trials led the U.S. Food and Drug Administration to grant ibrutinib three breakthrough therapy designations for mantle cell lymphoma (MCL), del17p chronic lymphocytic leukemia (CLL), and Waldenström's macroglobulinemia (WM). Ibrutinib was approved for these three cancers within 14 months of the original U.S. approval. Additionally, ibrutinib is approved for patient subsets with MCL and/or CLL in >45 other countries. Via a unique mechanism of action, ibrutinib inhibits B cell signaling pathways that regulate the survival, proliferation, adhesion, and homing of cancerous cells. This marks a paradigm shift from the conventional cytotoxic chemotherapy approach to treating B cell malignancies. Ibrutinib continues to be evaluated across a range of B cell malignancies, either as single-agent therapy or in combination with other therapies, and continues to transform the lives of these patients. © 2015 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals Inc. on behalf of The New York Academy of Sciences.

  10. Relationships of Dietary Histidine and Obesity in Northern Chinese Adults, an Internet-Based Cross-Sectional Study

    Directory of Open Access Journals (Sweden)

    Yan-Chuan Li

    2016-07-01

    Full Text Available Our previous studies have demonstrated that histidine supplementation significantly ameliorates inflammation and oxidative stress in obese women and high-fat diet-induced obese rats. However, the effects of dietary histidine on general population are not known. The objective of this Internet-based cross-sectional study was to evaluate the associations between dietary histidine and prevalence of overweight/obesity and abdominal obesity in northern Chinese population. A total of 2376 participants were randomly recruited and asked to finish our Internet-based dietary questionnaire for the Chinese (IDQC. Afterwards, 88 overweight/obese participants were randomly selected to explore the possible mechanism. Compared with healthy controls, dietary histidine was significantly lower in overweight (p < 0.05 and obese (p < 0.01 participants of both sexes. Dietary histidine was inversely associated with body mass index (BMI, waist circumference (WC and blood pressure in overall population and stronger associations were observed in women and overweight/obese participants. Higher dietary histidine was associated with lower prevalence of overweight/obesity and abdominal obesity, especially in women. Further studies indicated that higher dietary histidine was associated with lower fasting blood glucose (FBG, homeostasis model assessment of insulin resistance (HOMA-IR, 2-h postprandial glucose (2 h-PG, tumor necrosis factor-α (TNF-α, interleukin-1β (IL-1β, interleukin-6 (IL-6, C-reactive protein (CRP, malonaldehyde (MDA and vaspin and higher glutathione peroxidase (GSH-Px, superoxide dismutase (SOD and adiponectin of overweight/obese individuals of both sexes. In conclusion, higher dietary histidine is inversely associated with energy intake, status of insulin resistance, inflammation and oxidative stress in overweight/obese participants and lower prevalence of overweight/obesity in northern Chinese adults.

  11. Relationships of Dietary Histidine and Obesity in Northern Chinese Adults, an Internet-Based Cross-Sectional Study.

    Science.gov (United States)

    Li, Yan-Chuan; Li, Chun-Long; Qi, Jia-Yue; Huang, Li-Na; Shi, Dan; Du, Shan-Shan; Liu, Li-Yan; Feng, Ren-Nan; Sun, Chang-Hao

    2016-07-11

    Our previous studies have demonstrated that histidine supplementation significantly ameliorates inflammation and oxidative stress in obese women and high-fat diet-induced obese rats. However, the effects of dietary histidine on general population are not known. The objective of this Internet-based cross-sectional study was to evaluate the associations between dietary histidine and prevalence of overweight/obesity and abdominal obesity in northern Chinese population. A total of 2376 participants were randomly recruited and asked to finish our Internet-based dietary questionnaire for the Chinese (IDQC). Afterwards, 88 overweight/obese participants were randomly selected to explore the possible mechanism. Compared with healthy controls, dietary histidine was significantly lower in overweight (p obese (p Dietary histidine was inversely associated with body mass index (BMI), waist circumference (WC) and blood pressure in overall population and stronger associations were observed in women and overweight/obese participants. Higher dietary histidine was associated with lower prevalence of overweight/obesity and abdominal obesity, especially in women. Further studies indicated that higher dietary histidine was associated with lower fasting blood glucose (FBG), homeostasis model assessment of insulin resistance (HOMA-IR), 2-h postprandial glucose (2 h-PG), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), C-reactive protein (CRP), malonaldehyde (MDA) and vaspin and higher glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and adiponectin of overweight/obese individuals of both sexes. In conclusion, higher dietary histidine is inversely associated with energy intake, status of insulin resistance, inflammation and oxidative stress in overweight/obese participants and lower prevalence of overweight/obesity in northern Chinese adults.

  12. Design and synthesis of the first generation of novel potent, selective, and in vivo active (benzothiazol-2-yl)acetonitrile inhibitors of the c-Jun N-terminal kinase.

    Science.gov (United States)

    Gaillard, Pascale; Jeanclaude-Etter, Isabelle; Ardissone, Vittoria; Arkinstall, Steve; Cambet, Yves; Camps, Montserrat; Chabert, Christian; Church, Dennis; Cirillo, Rocco; Gretener, Denise; Halazy, Serge; Nichols, Anthony; Szyndralewiez, Cedric; Vitte, Pierre-Alain; Gotteland, Jean-Pierre

    2005-07-14

    Several lines of evidence support the hypothesis that c-Jun N-terminal kinase (JNKs) plays a critical role in a wide range of diseases including cell death (apoptosis)-related disorders (neurodegenerative diseases, brain, heart, and renal ischemia, epilepsy) and inflammatory disorders (multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases). Screening of our internal compound collection for inhibitors of JNK3 led to the identification of (benzothiazol-2-yl)acetonitrile derivatives as potent and selective JNK1, -2, -3 inhibitors. Starting from initial hit 1 (AS007149), the chemistry and initial structure-activity relationship (SAR) of this novel and unique kinase inhibitor template were explored. Investigation of the SAR rapidly revealed that the benzothiazol-2-ylacetonitrile pyrimidine core was crucial to retain a good level of potency on rat JNK3. Therefore, compound 6 was further optimized by exploring a number of distal combinations in place of the chlorine atom. This led to the observation that the presence of an aromatic group, two carbons away from the aminopyrimidine moiety and bearing substituents conferring hydrogen bond acceptor (HBA) properties, could improve the potency. Further improvements to the biological and biopharmaceutical profile of the most promising compounds were performed, resulting in the discovery of compound 59 (AS601245). The in vitro and in vivo anti-inflammatory potential of this new JNK inhibitor was investigated and found to demonstrate efficacy per oral route in an experimental model of rheumatoid arthritis (RA).

  13. The tricarboxylic acid cycle activity in cultured primary astrocytes is strongly accelerated by the protein tyrosine kinase inhibitor tyrphostin 23

    DEFF Research Database (Denmark)

    Hohnholt, Michaela C; Blumrich, Eva-Maria; Waagepetersen, Helle S

    2017-01-01

    production. In addition, T23-treatment strongly increased the molecular carbon labeling of the TCA cycle intermediates citrate, succinate, fumarate and malate, and significantly increased the incorporation of (13)C-labelling into the amino acids glutamate, glutamine and aspartate. These results clearly......Tyrphostin 23 (T23) is a well-known inhibitor of protein tyrosine kinases and has been considered as potential anti-cancer drug. T23 was recently reported to acutely stimulate the glycolytic flux in primary cultured astrocytes. To investigate whether T23 also affects the tricarboxylic acid (TCA...

  14. Effective intracellular inhibition of the cAMP-dependent protein kinase by microinjection of a modified form of the specific inhibitor peptide PKi in living fibroblasts.

    Science.gov (United States)

    Fernandez, A; Mery, J; Vandromme, M; Basset, M; Cavadore, J C; Lamb, N J

    1991-08-01

    In order to obtain a peptide retaining its biological activity following microinjection into living cells, we have modified a synthetic peptide [PKi(m)(6-24)], derived from the specific inhibitor protein of the cAMP-dependent protein kinase (A-kinase) in two ways: (1) substitution of the arginine at position 18 for a D-arginine; (2) blockade of the side chain on the C-terminal aspartic acid by a cyclohexyl ester group. In an in vitro assay, PKi(m) has retained a specific inhibitory activity against A-kinase as assessed against six other kinases, with similar efficiency to that of the unmodified PKi(5-24) peptide. Microinjection of PKi(m) into living fibroblasts reveals its capacity to prevent the changes in cell morphology and cytoskeleton induced by drugs which activate endogenous A-kinase, whereas the original PKi peptide failed to do so. This inhibition of A-kinase in vivo by PKi(m) lasts between 4 and 6 h after injection. In light of its effective half-life, this modified peptide opens a route for the use of biologically active peptides in vivo, an approach which has been hampered until now by the exceedingly short half-life of peptides inside living cells. By providing a direct means of inhibiting A-kinase activity for sufficiently long periods to observe effects on cellular functions in living cells, PKi(m) represents a powerful tool in studying the potential role of cAMP-dependent phosphorylation in vivo.

  15. Identifying kinase dependency in cancer cells by integrating high-throughput drug screening and kinase inhibition data.

    Science.gov (United States)

    Ryall, Karen A; Shin, Jimin; Yoo, Minjae; Hinz, Trista K; Kim, Jihye; Kang, Jaewoo; Heasley, Lynn E; Tan, Aik Choon

    2015-12-01

    Targeted kinase inhibitors have dramatically improved cancer treatment, but kinase dependency for an individual patient or cancer cell can be challenging to predict. Kinase dependency does not always correspond with gene expression and mutation status. High-throughput drug screens are powerful tools for determining kinase dependency, but drug polypharmacology can make results difficult to interpret. We developed Kinase Addiction Ranker (KAR), an algorithm that integrates high-throughput drug screening data, comprehensive kinase inhibition data and gene expression profiles to identify kinase dependency in cancer cells. We applied KAR to predict kinase dependency of 21 lung cancer cell lines and 151 leukemia patient samples using published datasets. We experimentally validated KAR predictions of FGFR and MTOR dependence in lung cancer cell line H1581, showing synergistic reduction in proliferation after combining ponatinib and AZD8055. KAR can be downloaded as a Python function or a MATLAB script along with example inputs and outputs at: http://tanlab.ucdenver.edu/KAR/. aikchoon.tan@ucdenver.edu. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface*

    Science.gov (United States)

    Wojcik, John; Lamontanara, Allan Joaquim; Grabe, Grzegorz; Koide, Akiko; Akin, Louesa; Gerig, Barbara; Hantschel, Oliver; Koide, Shohei

    2016-01-01

    Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl. PMID:26912659

  17. Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface.

    Science.gov (United States)

    Wojcik, John; Lamontanara, Allan Joaquim; Grabe, Grzegorz; Koide, Akiko; Akin, Louesa; Gerig, Barbara; Hantschel, Oliver; Koide, Shohei

    2016-04-15

    Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Oriented immobilization of His-tagged kinase RIO1 protein on redox active N-(IDA-like)-Cu(II) monolayer deposited on gold electrode—The base of electrochemical biosensor

    International Nuclear Information System (INIS)

    Mielecki, Marcin; Wojtasik, Justyn; Zborowska, Magdalena; Kurzątkowska, Katarzyna; Grzelak, Krystyna; Dehaen, Wim; Radecki, Jerzy; Radecka, Hanna

    2013-01-01

    Highlights: ► The redox active N-(IDA-like)-Cu(II) monolayer is suitable for oriented and stable immobilization of His-tagged kinase Rio1. ► Cu(II) deposited onto the electrode surface play double role: immobilization sites for His-tagged proteins and transduction centres tracking the protein–small molecule interactions. ► The base of biosensor response towards target compound is the change of Rio1 conformation lading to alternation of the permeability of counter ions to Cu(II) redox centres. -- Abstract: The fabrication of electrochemical biosensor consists of the following successive steps: formation of thiol derivative of iminodiacetic acid (IDA-like/N-heterocyclic donor) and N-acetylcysteamine (NAC) self-assembled monolayer on the Au electrode, complexation of Cu(II) by N(IDA-like) attached to the surface of the Au electrode and immobilization of kinase protein Rio1 through N(IDA-like)-Cu(II)-histidine-tag covalent bond formation. Each step of modification was controlled by cyclic voltammetry, electrochemical impedance spectrometry and atomic force microscopy. The interactions between rHis 6 -Rio1 attached to the surface of the electrode and tyrphostin inhibitor (2E)-N-Benzyl-2-cyano-3-(3,4-dihydroxyphenyl)-acrylamide (AG-490) and its analogue (2-cyano-N-(4-methoxyphenyl)-3-(pyridin-3-yl)prop-2-enamide) (CPE), present in aqueous solution were monitored with Osteryoung square wave voltammetry. The basis of the biosensor response was the change in the electrochemical properties of Cu(II) redox centres upon formation of the rHis 6 -Rio1-inhibitor complex. A linear responses with high reproducibility and stability were observed between 0.10 and 0.40 μM of AG-490 as well as of CPE. The interaction between rHis 6 -Rio1 and AG-490 was stronger than the interaction with its analogue CPE. Cu(II) redox current decrease of 37.9 ± 1.6% and 23.3 ± 1.0% were observed in the presence of 0.40 μM of AG-490 and CPE, respectively. The presented biosensor could be

  19. Mangiferin, a novel nuclear factor kappa B-inducing kinase inhibitor, suppresses metastasis and tumor growth in a mouse metastatic melanoma model

    Energy Technology Data Exchange (ETDEWEB)

    Takeda, Tomoya; Tsubaki, Masanobu; Sakamoto, Kotaro; Ichimura, Eri; Enomoto, Aya; Suzuki, Yuri [Division of Pharmacotherapy, Kinki University School of Pharmacy, Kowakae, Higashi-, Osaka (Japan); Itoh, Tatsuki [Department of Food Science and Nutrition, Kinki University School of Agriculture, Nara, Nara (Japan); Imano, Motohiro [Department of Surgery, Kinki University School of Medicine, Osakasayama, Osaka (Japan); Tanabe, Genzoh; Muraoka, Osamu [Laboratory of Pharmaceutical Organic Chemistry, School of Pharmacy, Kinki University, Kowakae, Higashi-, Osaka (Japan); Matsuda, Hideaki [Department of Natural Drugs Resources, Kinki University School of Pharmacy, Kowakae, Higashi-, Osaka (Japan); Satou, Takao [Department of Pathology, Kinki University School of Medicine, Osakasayama, Osaka (Japan); Nishida, Shozo, E-mail: nishida@phar.kindai.ac.jp [Division of Pharmacotherapy, Kinki University School of Pharmacy, Kowakae, Higashi-, Osaka (Japan)

    2016-09-01

    Advanced metastatic melanoma, one of the most aggressive malignancies, is currently without reliable therapy. Therefore, new therapies are urgently needed. Mangiferin is a naturally occurring glucosylxanthone and exerts many beneficial biological activities. However, the effect of mangiferin on metastasis and tumor growth of metastatic melanoma remains unclear. In this study, we evaluated the effect of mangiferin on metastasis and tumor growth in a mouse metastatic melanoma model. We found that mangiferin inhibited spontaneous metastasis and tumor growth. Furthermore, mangiferin suppressed the nuclear translocation of nuclear factor kappa B (NF-κB) and expression of phosphorylated NF-κB-inducing kinase (NIK), inhibitor of kappa B kinase (IKK), and inhibitor of kappa B (IκB) and increases the expression of IκB protein in vivo. In addition, we found that mangiferin inhibited the expression of matrix metalloproteinases (MMPs) and very late antigens (VLAs) in vivo. Mangiferin treatment also increased the expression of cleaved caspase-3, cleaved Poly ADP ribose polymerase-1 (PARP-1), p53 upregulated modulator of apoptosis (PUMA), p53, and phosphorylated p53 proteins, and decreased the expression of Survivin and Bcl-associated X (Bcl-xL) proteins in vivo. These results indicate that mangiferin selectivity suppresses the NF-κB pathway via inhibition of NIK activation, thereby inhibiting metastasis and tumor growth. Importantly, the number of reported NIK selective inhibitors is limited. Taken together, our data suggest that mangiferin may be a potential therapeutic agent with a new mechanism of targeting NIK for the treatment of metastatic melanoma. - Highlights: • Mangiferin prolongs survival in mice by inhibiting metastasis and tumor growth • Mangiferin selectivity suppresses the NF-κB pathway via inhibition of NIK activation • Mangiferin regulates the expression of MMPs, VLAs, and apoptosis regulatory proteins.

  20. Pharmacogenetics of tyrosine kinase inhibitors in gastrointestinal stromal tumor and chronic myeloid leukemia.

    Science.gov (United States)

    Ravegnini, Gloria; Sammarini, Giulia; Angelini, Sabrina; Hrelia, Patrizia

    2016-07-01

    Gastrointestinal stromal tumors (GIST) and chronic myeloid leukemia (CML) are two tumor types deeply different from each other. Despite the differences, these disorders share treatment with tyrosine kinase inhibitor imatinib. Despite the success of imatinib, the response rates vary among different individuals and pharmacogenetics may play an important role in the final clinical outcome. In this review, the authors provide an overview of the pharmacogenetic literature analyzing the role of polymorphisms in both GIST and CML treatment efficacy and toxicity. So far, several polymorphisms influencing the pharmacokinetic determinants of imatinib have been identified. However, the data are not yet conclusive enough to translate pharmacogenetic tests in clinical practice. In this context, the major obstacles to pharmacogenetic test validation are represented by the small sample size of most studies, ethnicity and population admixture as confounding source, and uncertainty related to genetic variants analyzed. In conclusion, a combination of different theoretical approaches, experimental model systems and statistical methods is clearly needed, in order to appreciate pharmacogenetics applied to clinical practice in the near future.