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Sample records for specific bace-1 inhibitor

  1. Developing BACE-1 inhibitors for FXS

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    Cara J Westmark

    2013-05-01

    Full Text Available Fragile X syndrome (FXS is a debilitating genetic disorder with no cure and few therapeutic options. Excessive signaling through metabotropic glutamate receptor 5 (mGluR5 in FXS leads to increased translation of numerous synaptic proteins and exaggerated long-term depression (LTD. Two of the overexpressed proteins are amyloid-beta protein precursor (APP and its metabolite amyloid-beta (Aβ, which have been well-studied in Alzheimer’s disease (AD. Here we discus the possibility that pharmaceuticals under study for the modulation of these proteins in AD might be viable therapeutic strategies for FXS. Specifically, a recently identified acetyltransferase (ATase inhibitor that reduces the levels and activity of β-site APP cleaving enzyme (BACE-1 has strong potential to attenuate BACE-1 activity and maintain homeostatic levels APP catabolites in FXS.

  2. Preparation and biological evaluation of conformationally constrained BACE1 inhibitors.

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    Winneroski, Leonard L; Schiffler, Matthew A; Erickson, Jon A; May, Patrick C; Monk, Scott A; Timm, David E; Audia, James E; Beck, James P; Boggs, Leonard N; Borders, Anthony R; Boyer, Robert D; Brier, Richard A; Hudziak, Kevin J; Klimkowski, Valentine J; Garcia Losada, Pablo; Mathes, Brian M; Stout, Stephanie L; Watson, Brian M; Mergott, Dustin J

    2015-07-01

    The BACE1 enzyme is a key target for Alzheimer's disease. During our BACE1 research efforts, fragment screening revealed that bicyclic thiazine 3 had low millimolar activity against BACE1. Analysis of the co-crystal structure of 3 suggested that potency could be increased through extension toward the S3 pocket and through conformational constraint of the thiazine core. Pursuit of S3-binding groups produced low micromolar inhibitor 6, which informed the S3-design for constrained analogs 7 and 8, themselves prepared via independent, multi-step synthetic routes. Biological characterization of BACE inhibitors 6-8 is described. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Target specific proteochemometric model development for BACE1 - protein flexibility and structural water are critical in virtual screening.

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    Manoharan, Prabu; Chennoju, Kiranmai; Ghoshal, Nanda

    2015-07-01

    BACE1 is an attractive target in Alzheimer's disease (AD) treatment. A rational drug design effort for the inhibition of BACE1 is actively pursued by researchers in both academic and pharmaceutical industries. This continued effort led to the steady accumulation of BACE1 crystal structures, co-complexed with different classes of inhibitors. This wealth of information is used in this study to develop target specific proteochemometric models and these models are exploited for predicting the prospective BACE1 inhibitors. The models developed in this study have performed excellently in predicting the computationally generated poses, separately obtained from single and ensemble docking approaches. The simple protein-ligand contact (SPLC) model outperforms other sophisticated high end models, in virtual screening performance, developed during this study. In an attempt to account for BACE1 protein active site flexibility information in predictive models, we included the change in the area of solvent accessible surface and the change in the volume of solvent accessible surface in our models. The ensemble and single receptor docking results obtained from this study indicate that the structural water mediated interactions improve the virtual screening results. Also, these waters are essential for recapitulating bioactive conformation during docking study. The proteochemometric models developed in this study can be used for the prediction of BACE1 inhibitors, during the early stage of AD drug discovery.

  4. Unveiling a novel transient druggable pocket in BACE-1 through molecular simulations: Conformational analysis and binding mode of multisite inhibitors

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    Di Pietro, Ornella; Laughton, Charles A.

    2017-01-01

    The critical role of BACE-1 in the formation of neurotoxic ß-amyloid peptides in the brain makes it an attractive target for an efficacious treatment of Alzheimer’s disease. However, the development of clinically useful BACE-1 inhibitors has proven to be extremely challenging. In this study we examine the binding mode of a novel potent inhibitor (compound 1, with IC50 80 nM) designed by synergistic combination of two fragments—huprine and rhein—that individually are endowed with very low activity against BACE-1. Examination of crystal structures reveals no appropriate binding site large enough to accommodate 1. Therefore we have examined the conformational flexibility of BACE-1 through extended molecular dynamics simulations, paying attention to the highly flexible region shaped by loops 8–14, 154–169 and 307–318. The analysis of the protein dynamics, together with studies of pocket druggability, has allowed us to detect the transient formation of a secondary binding site, which contains Arg307 as a key residue for the interaction with small molecules, at the edge of the catalytic cleft. The formation of this druggable “floppy” pocket would enable the binding of multisite inhibitors targeting both catalytic and secondary sites. Molecular dynamics simulations of BACE-1 bound to huprine-rhein hybrid compounds support the feasibility of this hypothesis. The results provide a basis to explain the high inhibitory potency of the two enantiomeric forms of 1, together with the large dependence on the length of the oligomethylenic linker. Furthermore, the multisite hypothesis has allowed us to rationalize the inhibitory potency of a series of tacrine-chromene hybrid compounds, specifically regarding the apparent lack of sensitivity of the inhibition constant to the chemical modifications introduced in the chromene unit. Overall, these findings pave the way for the exploration of novel functionalities in the design of optimized BACE-1 multisite inhibitors

  5. Sebestenoids A-D, BACE1 inhibitors from Cordia sebestena.

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    Dai, Jingqiu; Sorribas, Analia; Yoshida, Wesley Y; Williams, Philip G

    2010-12-01

    Bioassay-guided fractionation of an extract prepared from the fruits of Cordia sebestena led to the isolation of sebestenoids A-D (1-4). Their structures were elucidated on the basis of extensive NMR experiments and mass spectroscopic measurements. Compounds 1-4 exhibited moderate inhibition of the aspartic protease BACE1. Copyright © 2010 Elsevier Ltd. All rights reserved.

  6. Potent and Selective BACE-1 Peptide Inhibitors Lower Brain Aβ Levels Mediated by Brain Shuttle Transport

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    Nadine Ruderisch

    2017-10-01

    Full Text Available Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidβ (Aβ antibodies and secretase inhibitors. However, the blood-brain barrier (BBB limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aβ levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aβ. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance.

  7. Design, Synthesis and Evaluation of 3-(2-Aminoheterocycle-4-benzyloxyphenylbenzamide Derivatives as BACE-1 Inhibitors

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    Lushan Yu

    2013-03-01

    Full Text Available Three series of 3-(2-aminoheterocycle-4-benzyloxyphenylbenzamide derivatives, 2-aminooxazoles, 2-aminothiazoles, and 2-amino-6H-1,3,4-thiadizines were designed, synthesized and evaluated as β-secretase (BACE-1 inhibitors. Preliminary structure-activity relationships revealed that the existence of a 2-amino-6H-1,3,4-thiadizine moiety and α-naphthyl group were favorable for BACE-1 inhibition. Among the synthesized compounds, 5e exhibited the most potent BACE-1 inhibitory activity, with an IC50 value of 9.9 μΜ and it exhibited high brain uptake potential in Madin-Darby anine kidney cell lines (MDCK and a Madin-Darby canine kidney-multidrug resistance 1 (MDCK-MDR1 model.

  8. Design, synthesis and evaluation of 3-(2-aminoheterocycle)-4-benzyloxyphenylbenzamide derivatives as BACE-1 inhibitors.

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    Shangguan, Shihao; Wang, Fei; Liao, Yong; Yu, Haiping; Li, Jia; Huang, Wenhai; Hu, Haihong; Yu, Lushan; Hu, Yongzhou; Sheng, Rong

    2013-03-20

    Three series of 3-(2-aminoheterocycle)-4-benzyloxyphenylbenzamide derivatives, 2-aminooxazoles, 2-aminothiazoles, and 2-amino-6H-1,3,4-thiadizines were designed, synthesized and evaluated as β-secretase (BACE-1) inhibitors. Preliminary structure-activity relationships revealed that the existence of a 2-amino-6H-1,3,4-thiadizine moiety and α-naphthyl group were favorable for BACE-1 inhibition. Among the synthesized compounds, 5e exhibited the most potent BACE-1 inhibitory activity, with an IC50 value of 9.9 μΜ and it exhibited high brain uptake potential in Madin-Darby anine kidney cell lines (MDCK) and a Madin-Darby canine kidney-multidrug resistance 1 (MDCK-MDR1) model.

  9. Conformational Dynamics and Binding Free Energies of Inhibitors of BACE-1: From the Perspective of Protonation Equilibria.

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    M Olivia Kim

    2015-10-01

    Full Text Available BACE-1 is the β-secretase responsible for the initial amyloidogenesis in Alzheimer's disease, catalyzing hydrolytic cleavage of substrate in a pH-sensitive manner. The catalytic mechanism of BACE-1 requires water-mediated proton transfer from aspartyl dyad to the substrate, as well as structural flexibility in the flap region. Thus, the coupling of protonation and conformational equilibria is essential to a full in silico characterization of BACE-1. In this work, we perform constant pH replica exchange molecular dynamics simulations on both apo BACE-1 and five BACE-1-inhibitor complexes to examine the effect of pH on dynamics and inhibitor binding properties of BACE-1. In our simulations, we find that solution pH controls the conformational flexibility of apo BACE-1, whereas bound inhibitors largely limit the motions of the holo enzyme at all levels of pH. The microscopic pKa values of titratable residues in BACE-1 including its aspartyl dyad are computed and compared between apo and inhibitor-bound states. Changes in protonation between the apo and holo forms suggest a thermodynamic linkage between binding of inhibitors and protons localized at the dyad. Utilizing our recently developed computational protocol applying the binding polynomial formalism to the constant pH molecular dynamics (CpHMD framework, we are able to obtain the pH-dependent binding free energy profiles for various BACE-1-inhibitor complexes. Our results highlight the importance of correctly addressing the binding-induced protonation changes in protein-ligand systems where binding accompanies a net proton transfer. This work comprises the first application of our CpHMD-based free energy computational method to protein-ligand complexes and illustrates the value of CpHMD as an all-purpose tool for obtaining pH-dependent dynamics and binding free energies of biological systems.

  10. Piperazine sulfonamide BACE1 inhibitors: Design, synthesis, and in vivo characterization

    Energy Technology Data Exchange (ETDEWEB)

    Cumming, Jared; Babu, Suresh; Huang, Ying; Carrol, Carolyn; Chen, Xia; Favreau, Leonard; Greenlee, William; Guo, Tao; Kennedy, Matthew; Kuvelkar, Reshma; Le, Thuy; Li, Guoqing; McHugh, Nansie; Orth, Peter; Ozgur, Lynne; Parker, Eric; Saionz, Kurt; Stamford, Andrew; Strickland, Corey; Tadesse, Dawit; Voigta, Johannes; Zhang, Lili; Zhang, Qi (Ligand); (Merck)

    2010-08-17

    With collaboration between chemistry, X-ray crystallography, and molecular modeling, we designed and synthesized a series of novel piperazine sulfonamide BACE1 inhibitors. Iterative exploration of the non-prime side and S2{prime} sub-pocket of the enzyme culminated in identification of an analog that potently lowers peripheral A{beta}{sub 40} in transgenic mice with a single subcutaneous dose.

  11. Fragment-based virtual screening approach and molecular dynamics simulation studies for identification of BACE1 inhibitor leads.

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    Manoharan, Prabu; Ghoshal, Nanda

    2018-05-01

    Traditional structure-based virtual screening method to identify drug-like small molecules for BACE1 is so far unsuccessful. Location of BACE1, poor Blood Brain Barrier permeability and P-glycoprotein (Pgp) susceptibility of the inhibitors make it even more difficult. Fragment-based drug design method is suitable for efficient optimization of initial hit molecules for target like BACE1. We have developed a fragment-based virtual screening approach to identify/optimize the fragment molecules as a starting point. This method combines the shape, electrostatic, and pharmacophoric features of known fragment molecules, bound to protein conjugate crystal structure, and aims to identify both chemically and energetically feasible small fragment ligands that bind to BACE1 active site. The two top-ranked fragment hits were subjected for a 53 ns MD simulation. Principle component analysis and free energy landscape analysis reveal that the new ligands show the characteristic features of established BACE1 inhibitors. The potent method employed in this study may serve for the development of potential lead molecules for BACE1-directed Alzheimer's disease therapeutics.

  12. Creation of Novel Cores for β-Secretase (BACE-1) Inhibitors: A Multiparameter Lead Generation Strategy.

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    Viklund, Jenny; Kolmodin, Karin; Nordvall, Gunnar; Swahn, Britt-Marie; Svensson, Mats; Gravenfors, Ylva; Rahm, Fredrik

    2014-04-10

    In order to find optimal core structures as starting points for lead optimization, a multiparameter lead generation workflow was designed with the goal of finding BACE-1 inhibitors as a treatment for Alzheimer's disease. De novo design of core fragments was connected with three predictive in silico models addressing target affinity, permeability, and hERG activity, in order to guide synthesis. Taking advantage of an additive SAR, the prioritized cores were decorated with a few, well-characterized substituents from known BACE-1 inhibitors in order to allow for core-to-core comparisons. Prediction methods and analyses of how physicochemical properties of the core structures correlate to in vitro data are described. The syntheses and in vitro data of the test compounds are reported in a separate paper by Ginman et al. [J. Med. Chem. 2013, 56, 4181-4205]. The affinity predictions are described in detail by Roos et al. [J. Chem. Inf. 2014, DOI: 10.1021/ci400374z].

  13. From BACE1 Inhibitor to Multifunctionality of Tryptoline and Tryptamine Triazole Derivatives for Alzheimer’s Disease

    OpenAIRE

    Jiaranaikulwanitch, Jutamas; Govitrapong, Piyarat; Fokin, Valery V.; Vajragupta, Opa

    2012-01-01

    Efforts to discover new drugs for Alzheimer’s disease emphasizing multiple targets was conducted seeking to inhibit amyloid oligomer formation and to prevent radical formation. The tryptoline and tryptamine cores of BACE1 inhibitors previously identified by virtual screening were modified in silico for additional modes of action. These core structures were readily linked to different side chains using 1,2,3-triazole rings as bridges by copper catalyzed azide-alkyne cycloa...

  14. Discovery of imidazopyridines containing isoindoline-1,3-dione framework as a new class of BACE1 inhibitors: Design, synthesis and SAR analysis.

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    Azimi, Sara; Zonouzi, Afsaneh; Firuzi, Omidreza; Iraji, Aida; Saeedi, Mina; Mahdavi, Mohammad; Edraki, Najmeh

    2017-09-29

    Alzheimer's disease is characterized by chronic neurodegeneration leading to dementia. The main cause of neurodegeneration is considered to be the accumulation of amyloid-β. Inhibiting BACE1 is a well-studied approach to lower the burden of amyloid-β aggregates. We designed a series of imidazopyridines-based compounds bearing phthalimide moieties as inhibitors of BACE1. The compounds 8a-o were synthesized by the Groebke-Blackburn-Bienaymé three-component reaction of heteroaromatic amidines, aldehydes and isocyanides. Evaluating the BACE1 inhibitory effects of the synthesized compounds revealed that introducing an aminocyclohexyl moiety in the imidazopyridine core resulted in a significant improvement in its BACE1 inhibitory potential. In this regard, compound 8e was the most potent against BACE1 with an IC 50 value of 2.84 (±0.95) μM. Molecular docking revealed that the nitrogen atom of imidazopyridines and the oxygen atom of the phenoxypropyl linker were involved in hydrogen bound interactions with Asp228 and Asp32 of BACE1 active site, respectively. The phthalimide moiety oriented toward the flap pocket and interacted with phe108, lle110, Trp115, Ile118 through van der Waal's and hydrophobic interactions. These findings demonstrate that imidazopyridines-based compounds bearing phthalimide moiety have the potential to decrease amyloid-β levels and ameliorate the symptoms of Alzheimer's disease. Copyright © 2017. Published by Elsevier Masson SAS.

  15. From BACE1 Inhibitor to Multifunctionality of Tryptoline and Tryptamine Triazole Derivatives for Alzheimer’s Disease

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    Jutamas Jiaranaikulwanitch

    2012-07-01

    Full Text Available Efforts to discover new drugs for Alzheimer’s disease emphasizing multiple targets was conducted seeking to inhibit amyloid oligomer formation and to prevent radical formation. The tryptoline and tryptamine cores of BACE1 inhibitors previously identified by virtual screening were modified in silico for additional modes of action. These core structures were readily linked to different side chains using 1,2,3-triazole rings as bridges by copper catalyzed azide-alkyne cycloaddition reactions. Three compounds among the sixteen designed compounds exerted multifunctional activities including β-secretase inhibitory action, anti-amyloid aggregation, metal chelating and antioxidant effects at micromolar levels. the neuroprotective effects of the multifunctional compounds 6h, 12c and 12h on Aβ1-42 induced neuronal cell death at 1 μM were significantly greater than those of the potent single target compound, BACE1 inhibitor IV and were comparable to curcumin. The observed synergistic effect resulting from the reduction of the Aβ1-42 neurotoxicity cascade substantiates the validity of our multifunctional strategy in drug discovery for Alzheimer’s disease.

  16. Bace1 activity impairs neuronal glucose metabolism: rescue by beta-hydroxybutyrate and lipoic acid

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    John A Findlay

    2015-10-01

    Full Text Available Glucose hypometabolism and impaired mitochondrial function in neurons have been suggested to play early and perhaps causative roles in Alzheimer’s disease (AD pathogenesis. Activity of the aspartic acid protease, beta-site amyloid precursor protein (APP cleaving enzyme 1 (BACE1, responsible for beta amyloid peptide generation, has recently been demonstrated to modify glucose metabolism. We therefore examined, using a human neuroblastoma (SH-SY5Y cell line, whether increased BACE1 activity is responsible for a reduction in cellular glucose metabolism. Overexpression of active BACE1, but not a protease-dead mutant BACE1, protein in SH-SY5Y cells reduced glucose oxidation and the basal oxygen consumption rate, which was associated with a compensatory increase in glycolysis. Increased BACE1 activity had no effect on the mitochondrial electron transfer process but was found to diminish substrate delivery to the mitochondria by inhibition of key mitochondrial decarboxylation reaction enzymes. This BACE1 activity-dependent deficit in glucose oxidation was alleviated by the presence of beta hydroxybutyrate or α-lipoic acid. Consequently our data indicate that raised cellular BACE1 activity drives reduced glucose oxidation in a human neuronal cell line through impairments in the activity of specific tricarboxylic acid cycle enzymes. Because this bioenergetic deficit is recoverable by neutraceutical compounds we suggest that such agents, perhaps in conjunction with BACE1 inhibitors, may be an effective therapeutic strategy in the early-stage management or treatment of AD.

  17. Investigation of naphthofuran moiety as potential dual inhibitor against BACE-1 and GSK-3β: molecular dynamics simulations, binding energy, and network analysis to identify first-in-class dual inhibitors against Alzheimer's disease.

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    Kumar, Akhil; Srivastava, Gaurava; Srivastava, Swati; Verma, Seema; Negi, Arvind S; Sharma, Ashok

    2017-08-01

    BACE-1 and GSK-3β are potential therapeutic drug targets for Alzheimer's disease. Recently, both the targets received attention for designing dual inhibitors for Alzheimer's disease. Until now, only two-scaffold triazinone and curcumin have been reported as BACE-1 and GSK-3β dual inhibitors. Docking, molecular dynamics, clustering, binding energy, and network analysis of triazinone derivatives with BACE-1 and GSK-3β was performed to get molecular insight into the first reported dual inhibitor. Further, we designed and evaluated a naphthofuran series for its ability to inhibit BACE-1 and GSK-3β with the computational approaches. Docking study of naphthofuran series showed a good binding affinity towards both the targets. Molecular dynamics, binding energy, and network analysis were performed to compare their binding with the targets and amino acids responsible for binding. Naphthofuran series derivatives showed good interaction within the active site residues of both of the targets. Hydrogen bond occupancy and binding energy suggested strong binding with the targets. Dual-inhibitor binding was mostly governed by the hydrophobic interactions for both of the targets. Per residue energy decomposition and network analysis identified the key residues involved in the binding and inhibiting BACE-1 and GSK-3β. The results indicated that naphthofuran series derivative 11 may be a promising first-in-class dual inhibitor against BACE-1 and GSK-3β. This naphthofuran series may be further explored to design better dual inhibitors. Graphical abstract Naphthofuran derivative as a dual inhibitor for BACE-1 and GSK-3β.

  18. Combining NMR and X-ray crystallography in fragment-based drug discovery: discovery of highly potent and selective BACE-1 inhibitors.

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    Wyss, Daniel F; Wang, Yu-Sen; Eaton, Hugh L; Strickland, Corey; Voigt, Johannes H; Zhu, Zhaoning; Stamford, Andrew W

    2012-01-01

    Fragment-based drug discovery (FBDD) has become increasingly popular over the last decade. We review here how we have used highly structure-driven fragment-based approaches to complement more traditional lead discovery to tackle high priority targets and those struggling for leads. Combining biomolecular nuclear magnetic resonance (NMR), X-ray crystallography, and molecular modeling with structure-assisted chemistry and innovative biology as an integrated approach for FBDD can solve very difficult problems, as illustrated in this chapter. Here, a successful FBDD campaign is described that has allowed the development of a clinical candidate for BACE-1, a challenging CNS drug target. Crucial to this achievement were the initial identification of a ligand-efficient isothiourea fragment through target-based NMR screening and the determination of its X-ray crystal structure in complex with BACE-1, which revealed an extensive H-bond network with the two active site aspartate residues. This detailed 3D structural information then enabled the design and validation of novel, chemically stable and accessible heterocyclic acylguanidines as aspartic acid protease inhibitor cores. Structure-assisted fragment hit-to-lead optimization yielded iminoheterocyclic BACE-1 inhibitors that possess desirable molecular properties as potential therapeutic agents to test the amyloid hypothesis of Alzheimer's disease in a clinical setting.

  19. Inhibition of BACE1 Activity by a DNA Aptamer in an Alzheimer's Disease Cell Model.

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    Huiyu Liang

    Full Text Available An initial step in amyloid-β (Aβ production includes amyloid precursor protein (APP cleavage via β-Site amyloid precursor protein cleaving enzyme 1 (BACE1. Increased levels of brain Aβ have been implicated in the pathogenesis of Alzheimer's disease (AD. Thus, β-secretase represents a primary target for inhibitor drug development in AD. In this study, aptamers were obtained from combinatorial oligonucleotide libraries using a technology referred to as systematic evolution of ligands by exponential enrichment (SELEX. A purified human BACE1 extracellular domain was used as a target to conduct an in vitro selection process using SELEX. Two DNA aptamers were capable of binding to BACE1 with high affinity and good specificity, with Kd values in the nanomolar range. We subsequently confirmed that one aptamer, A1, exhibited a distinct inhibitory effect on BACE1 activity in an AD cell model. We detected the effects of M17-APPsw cells that stably expressed Swedish mutant APP after aptamer A1 treatment. Aβ40 and Aβ42 concentrations secreted by M17-APPsw cells decreased intracellularly and in culture media. Furthermore, Western blot analysis indicated that sAPPβ expression significantly decreased in the A1 treated versus control groups. These findings support the preliminary feasibility of an aptamer evolved from a SELEX strategy to function as a potential BACE1 inhibitor. To our knowledge, this is the first study to acquire a DNA aptamer that exhibited binding specificity to BACE1 and inhibited its activity.

  20. The Alzheimer's disease β-secretase enzyme, BACE1

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    Vassar Robert

    2007-11-01

    Full Text Available Abstract The pathogenesis of Alzheimer's disease is highly complex. While several pathologies characterize this disease, amyloid plaques, composed of the β-amyloid peptide are hallmark neuropathological lesions in Alzheimer's disease brain. Indeed, a wealth of evidence suggests that β-amyloid is central to the pathophysiology of AD and is likely to play an early role in this intractable neurodegenerative disorder. The BACE1 enzyme is essential for the generation of β-amyloid. BACE1 knockout mice do not produce β-amyloid and are free from Alzheimer's associated pathologies including neuronal loss and certain memory deficits. The fact that BACE1 initiates the formation of β-amyloid, and the observation that BACE1 levels are elevated in this disease provide direct and compelling reasons to develop therapies directed at BACE1 inhibition thus reducing β-amyloid and its associated toxicities. However, new data indicates that complete abolishment of BACE1 may be associated with specific behavioral and physiological alterations. Recently a number of non-APP BACE1 substrates have been identified. It is plausible that failure to process certain BACE1 substrates may underlie some of the reported abnormalities in the BACE1-deficient mice. Here we review BACE1 biology, covering aspects ranging from the initial identification and characterization of this enzyme to recent data detailing the apparent dysregulation of BACE1 in Alzheimer's disease. We pay special attention to the putative function of BACE1 during healthy conditions and discuss in detail the relationship that exists between key risk factors for AD, such as vascular disease (and downstream cellular consequences, and the pathogenic alterations in BACE1 that are observed in the diseased state.

  1. Seamless integration of dose-response screening and flow chemistry: efficient generation of structure-activity relationship data of β-secretase (BACE1) inhibitors.

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    Werner, Michael; Kuratli, Christoph; Martin, Rainer E; Hochstrasser, Remo; Wechsler, David; Enderle, Thilo; Alanine, Alexander I; Vogel, Horst

    2014-02-03

    Drug discovery is a multifaceted endeavor encompassing as its core element the generation of structure-activity relationship (SAR) data by repeated chemical synthesis and biological testing of tailored molecules. Herein, we report on the development of a flow-based biochemical assay and its seamless integration into a fully automated system comprising flow chemical synthesis, purification and in-line quantification of compound concentration. This novel synthesis-screening platform enables to obtain SAR data on b-secretase (BACE1) inhibitors at an unprecedented cycle time of only 1 h instead of several days. Full integration and automation of industrial processes have always led to productivity gains and cost reductions, and this work demonstrates how applying these concepts to SAR generation may lead to a more efficient drug discovery process. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Population PKPD modeling of BACE1 inhibitor-induced reduction in Aβ levels in vivo and correlation to in vitro potency in primary cortical neurons from mouse and guinea pig.

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    Janson, Juliette; Eketjäll, Susanna; Tunblad, Karin; Jeppsson, Fredrik; Von Berg, Stefan; Niva, Camilla; Radesäter, Ann-Cathrin; Fälting, Johanna; Visser, Sandra A G

    2014-03-01

    The aims were to quantify the in vivo time-course between the oral dose, the plasma and brain exposure and the inhibitory effect on Amyloid β (Aβ) in brain and cerebrospinal fluid, and to establish the correlation between in vitro and in vivo potency of novel β-secretase (BACE1) inhibitors. BACE1-mediated inhibition of Aβ was quantified in in vivo dose- and/or time-response studies and in vitro in SH-SY5Y cells, N2A cells, and primary cortical neurons (PCN). An indirect response model with inhibition on Aβ production rate was used to estimate unbound in vivo IC 50 in a population pharmacokinetic-pharmacodynamic modeling approach. Estimated in vivo inhibitory potencies varied between 1 and 1,000 nM. The turnover half-life of Aβ40 in brain was predicted to be 0.5 h in mouse and 1 h in guinea pig. An excellent correlation between PCN and in vivo potency was observed. Moreover, a strong correlation in potency was found between human SH-SY5Y cells and mouse PCN, being 4.5-fold larger in SH-SY5Y cells. The strong in vivo-in vitro correlation increased the confidence in using human cell lines for screening and optimization of BACE1 inhibitors. This can optimize the design and reduce the number of preclinical in vivo effect studies.

  3. The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation.

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    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

    2016-07-22

    The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation*

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    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

    2016-01-01

    The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. PMID:27302062

  5. Gadd153 and NF-κB crosstalk regulates 27-hydroxycholesterol-induced increase in BACE1 and β-amyloid production in human neuroblastoma SH-SY5Y cells.

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    Gurdeep Marwarha

    Full Text Available β-amyloid (Aβ peptide, accumulation of which is a culprit for Alzheimer's disease (AD, is derived from the initial cleavage of amyloid precursor protein by the aspartyl protease BACE1. Identification of cellular mechanisms that regulate BACE1 production is of high relevance to the search for potential disease-modifying therapies that inhibit BACE1 to reduce Aβ accumulation and AD progression. In the present study, we show that the cholesterol oxidation product 27-hydroxycholesterol (27-OHC increases BACE1 and Aβ levels in human neuroblastoma SH-SY5Y cells. This increase in BACE1 involves a crosstalk between the two transcription factors NF-κB and the endoplasmic reticulum stress marker, the growth arrest and DNA damage induced gene-153 (gadd153, also called CHOP. We specifically show that 27-OHC induces a substantial increase in NF-κB binding to the BACE1 promoter and subsequent increase in BACE1 transcription and Aβ production. The NF-κB inhibitor, sc514, significantly attenuated the 27-OHC-induced increase in NF-κB-mediated BACE1 expression and Aβ genesis. We further show that the 27-OHC-induced NF-κB activation and increased NF-κB-mediated BACE1 expression is contingent on the increased activation of gadd153. Silencing gadd153 expression with siRNA alleviated the 27-OHC-induced increase in NF-κB activation, NF-κB binding to the BACE1 promoter, and subsequent increase in BACE1 transcription and Aβ production. We also show that increased levels of BACE1 in the triple transgenic mouse model for AD is preceded by gadd153 and NF-κB activation. In summary, our study demonstrates that gadd153 and NF-κB work in concert to regulate BACE1 expression. Agents that inhibit gadd153 activation and subsequent interaction with NF-κB might be promising targets to reduce BACE1 and Aβ overproduction and may ultimately serve as disease-modifying treatments for AD.

  6. Neuronal human BACE1 knockin induces systemic diabetes in mice.

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    Plucińska, Kaja; Dekeryte, Ruta; Koss, David; Shearer, Kirsty; Mody, Nimesh; Whitfield, Phillip D; Doherty, Mary K; Mingarelli, Marco; Welch, Andy; Riedel, Gernot; Delibegovic, Mirela; Platt, Bettina

    2016-07-01

    β-Secretase 1 (BACE1) is a key enzyme in Alzheimer's disease pathogenesis that catalyses the amyloidogenic cleavage of amyloid precursor protein (APP). Recently, global Bace1 deletion was shown to protect against diet-induced obesity and diabetes, suggesting that BACE1 is a potential regulator of glucose homeostasis. Here, we investigated whether increased neuronal BACE1 is sufficient to alter systemic glucose metabolism, using a neuron-specific human BACE1 knockin mouse model (PLB4). Glucose homeostasis and adiposity were determined by glucose tolerance tests and EchoMRI, lipid species were measured by quantitative lipidomics, and biochemical and molecular alterations were assessed by western blotting, quantitative PCR and ELISAs. Glucose uptake in the brain and upper body was measured via (18)FDG-PET imaging. Physiological and molecular analyses demonstrated that centrally expressed human BACE1 induced systemic glucose intolerance in mice from 4 months of age onward, alongside a fatty liver phenotype and impaired hepatic glycogen storage. This diabetic phenotype was associated with hypothalamic pathology, i.e. deregulation of the melanocortin system, and advanced endoplasmic reticulum (ER) stress indicated by elevated central C/EBP homologous protein (CHOP) signalling and hyperphosphorylation of its regulator eukaryotic translation initiation factor 2α (eIF2α). In vivo (18)FDG-PET imaging further confirmed brain glucose hypometabolism in these mice; this corresponded with altered neuronal insulin-related signalling, enhanced protein tyrosine phosphatase 1B (PTP1B) and retinol-binding protein 4 (RBP4) levels, along with upregulation of the ribosomal protein and lipid translation machinery. Increased forebrain and plasma lipid accumulation (i.e. ceramides, triacylglycerols, phospholipids) was identified via lipidomics analysis. Our data reveal that neuronal BACE1 is a key regulator of metabolic homeostasis and provide a potential mechanism for the high

  7. BACE1 Physiological Functions May Limit Its Use as Therapeutic Target for Alzheimer's Disease.

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    Barão, Soraia; Moechars, Diederik; Lichtenthaler, Stefan F; De Strooper, Bart

    2016-03-01

    The protease β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is required for the production of the amyloid-β (Aβ) peptide, which is central to the pathogenesis of Alzheimer's disease (AD). Chronic inhibition of this protease may temper amyloid production and cure or prevent AD. However, while BACE1 inhibitors are being pushed forward as drug candidates, a remarkable gap in knowledge on the physiological functions of BACE1 and its close homolog BACE2 becomes apparent. Here we discuss the major discoveries of the past 3 years concerning BACE1 biology and to what extent these could limit the use of BACE1 inhibitors in the clinic. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Palmitoylated APP Forms Dimers, Cleaved by BACE1.

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    Raja Bhattacharyya

    Full Text Available A major rate-limiting step for Aβ generation and deposition in Alzheimer's disease brains is BACE1-mediated cleavage (β-cleavage of the amyloid precursor protein (APP. We previously reported that APP undergoes palmitoylation at two cysteine residues (Cys186 and Cys187 in the E1-ectodomain. 8-10% of total APP is palmitoylated in vitro and in vivo. Palmitoylated APP (palAPP shows greater preference for β-cleavage than total APP in detergent resistant lipid rafts. Protein palmitoylation is known to promote protein dimerization. Since dimerization of APP at its E1-ectodomain results in elevated BACE1-mediated cleavage of APP, we have now investigated whether palmitoylation of APP affects its dimerization and whether this leads to elevated β-cleavage of the protein. Here we report that over 90% of palAPP is dimerized while only ~20% of total APP forms dimers. PalAPP-dimers are predominantly cis-oriented while total APP dimerizes in both cis- and trans-orientation. PalAPP forms dimers 4.5-times more efficiently than total APP. Overexpression of the palmitoylating enzymes DHHC7 and DHHC21 that increase palAPP levels and Aβ release, also increased APP dimerization in cells. Conversely, inhibition of APP palmitoylation by pharmacological inhibitors reduced APP-dimerization in coimmunoprecipitation and FLIM/FRET assays. Finally, in vitro BACE1-activity assays demonstrate that palmitoylation-dependent dimerization of APP promotes β-cleavage of APP in lipid-rich detergent resistant cell membranes (DRMs, when compared to total APP. Most importantly, generation of sAPPβ-sAPPβ dimers is dependent on APP-palmitoylation while total sAPPβ generation is not. Since BACE1 shows preference for palAPP dimers over total APP, palAPP dimers may serve as novel targets for effective β-cleavage inhibitors of APP as opposed to BACE1 inhibitors.

  9. Reversing hypomyelination in BACE1-null mice with Akt-DD overexpression.

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    Hu, Xiangyou; Schlanger, Rita; He, Wanxia; Macklin, Wendy B; Yan, Riqiang

    2013-05-01

    β-Site amyloid precursor protein convertase enzyme 1 (BACE1), a type I transmembrane aspartyl protease required to cleave amyloid precursor protein for releasing a toxic amyloid peptide, also cleaves type I and type III neuregulin-1 (Nrg-1). BACE1 deficiency in mice causes hypomyelination during development and impairs remyelination if injured. In BACE1-null mice, the abolished cleavage of neuregulin-1 by BACE1 is speculated to cause reduced myelin sheath thickness in both the central nervous system and peripheral nervous system because reduced cleavage of Nrg-1 correlates with reduced Akt phosphorylation, a downstream signaling molecule of the Nrg-1/ErbB pathway. Here we tested specifically whether increasing Akt activity alone in oligodendrocytes would be sufficient to reverse the hypomyelination phenotype in BACE1-null mice. BACE1-null mice were bred with transgenic mice expressing constitutively active Akt (Akt-DD; mutations with D(308)T and D(473)S) in oligodendrocytes. Relative to littermate BACE1-null controls, BACE1(-/-)/Akt-DD mice exhibited enhanced expression of myelin basic protein and promoter of proteolipid protein. The elevated expression of myelin proteins correlated with a thicker myelin sheath in optic nerves; comparison of quantified g ratios with statistic significance was used to confirm this reversion. However, it appeared that myelin sheath thickness in the sciatic nerves was not increased in BACE1(-/-)/Akt-DD mice, as the g ratio was not significantly different from the control. Hence, increased Akt activity in BACE1-null myelinating cells only compensates for the loss of BACE1 activity in the central nervous system, which is consistent with the observation that overexpression of Akt-DD in Schwann cells did not induce hypermyelination. Our results suggest that signaling activity other than Akt may also contribute to proper myelination in peripheral nerves.

  10. The Alzheimer's β-secretase enzyme BACE1 is required for accurate axon guidance of olfactory sensory neurons and normal glomerulus formation in the olfactory bulb

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    Rajapaksha Tharinda W

    2011-12-01

    Full Text Available Abstract Background The β-secretase, β-site amyloid precursor protein cleaving enzyme 1 (BACE1, is a prime therapeutic target for lowering cerebral β-amyloid (Aβ levels in Alzheimer's disease (AD. Clinical development of BACE1 inhibitors is being intensely pursued. However, little is known about the physiological functions of BACE1, and the possibility exists that BACE1 inhibition may cause mechanism-based side effects. Indeed, BACE1-/- mice exhibit a complex neurological phenotype. Interestingly, BACE1 co-localizes with presynaptic neuronal markers, indicating a role in axons and/or terminals. Moreover, recent studies suggest axon guidance molecules are potential BACE1 substrates. Here, we used a genetic approach to investigate the function of BACE1 in axon guidance of olfactory sensory neurons (OSNs, a well-studied model of axon targeting in vivo. Results We bred BACE1-/- mice with gene-targeted mice in which GFP is expressed from the loci of two odorant-receptors (ORs, MOR23 and M72, and olfactory marker protein (OMP to produce offspring that were heterozygous for MOR23-GFP, M72-GFP, or OMP-GFP and were either BACE1+/+ or BACE1-/-. BACE1-/- mice had olfactory bulbs (OBs that were smaller and weighed less than OBs of BACE1+/+ mice. In wild-type mice, BACE1 was present in OSN axon terminals in OB glomeruli. In whole-mount preparations and tissue sections, many OB glomeruli from OMP-GFP; BACE1-/- mice were malformed compared to wild-type glomeruli. MOR23-GFP; BACE1-/- mice had an irregular MOR23 glomerulus that was innervated by randomly oriented, poorly fasciculated OSN axons compared to BACE1+/+ mice. Most importantly, M72-GFP; BACE1-/- mice exhibited M72 OSN axons that were mis-targeted to ectopic glomeruli, indicating impaired axon guidance in BACE1-/- mice. Conclusions Our results demonstrate that BACE1 is required for the accurate targeting of OSN axons and the proper formation of glomeruli in the OB, suggesting a role for BACE1 in

  11. Heparan sulfate regulates amyloid precursor protein processing by BACE1, the Alzheimer's β-secretase

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    Scholefield, Zoe; Yates, Edwin A.; Wayne, Gareth; Amour, Augustin; McDowell, William; Turnbull, Jeremy E.

    2003-01-01

    Cleavage of amyloid precursor protein (APP) by the Alzheimer's β-secretase (BACE1) is a key step in generating amyloid β-peptide, the main component of amyloid plaques. Here we report evidence that heparan sulfate (HS) interacts with β-site APP-cleaving enzyme (BACE) 1 and regulates its cleavage of APP. We show that HS and heparin interact directly with BACE1 and inhibit in vitro processing of peptide and APP substrates. Inhibitory activity is dependent on saccharide size and specific structural characteristics, and the mechanism of action involves blocking access of substrate to the active site. In cellular assays, HS specifically inhibits BACE1 cleavage of APP but not alternative cleavage by α-secretase. Endogenous HS immunoprecipitates with BACE1 and colocalizes with BACE1 in the Golgi complex and at the cell surface, two of its putative sites of action. Furthermore, inhibition of cellular HS synthesis results in enhanced BACE1 activity. Our findings identify HS as a natural regulator of BACE1 and suggest a novel mechanism for control of APP processing. PMID:14530380

  12. Multi-structure docking analysis of BACE1 crystal structures and non-peptidic ligands.

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    Haghighijoo, Zahra; Hemmateenejad, Bahram; Edraki, Najmeh; Miri, Ramin; Emami, Saeed

    2017-09-01

    In order to design novel non-peptidic inhibitors of BACE1, many research groups have attempted using computational studies including docking analyses. Since there are too many 3D structures for BACE1 in the protein database, the selection of suitable crystal structures is a key prerequisite for the successful application of molecular docking. We employed a multi-structure docking protocol. In which 615 ligands' structures were docked into 150 BACE1 structures. The large number of the resultant docking scores were post-processed by different data analysis methods including exploratory data analysis, regression analysis and discriminant analysis. It was found that using one crystal structure for docking did not result in high accuracy for predicting activity of the BACE1 inhibitors. Instead, using of the multi-structural docking scores, post-processed by chemometrics methods arrived to highly accurate predictive models. In this regards, the PDB accession codes of 4B70, 4DVF and 2WEZ could discriminate between active and inactive compounds, with higher accuracy. Clustering of the BACE1 structures based on principal component analysis of the crystallographic structures the revealed that the discriminant structures are in the center of the clusters. Thus, these structures can be selected as predominant crystal structures for docking studies of non-peptidic BACE1 inhibitors. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. BACE1 protein endocytosis and trafficking are differentially regulated by ubiquitination at lysine 501 and the Di-leucine motif in the carboxyl terminus.

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    Kang, Eugene L; Biscaro, Barbara; Piazza, Fabrizio; Tesco, Giuseppina

    2012-12-14

    β-Site amyloid precursor protein-cleaving enzyme (BACE1) is a membrane-tethered member of the aspartyl proteases that has been identified as β-secretase. BACE1 is targeted through the secretory pathway to the plasma membrane and then is internalized to endosomes. Sorting of membrane proteins to the endosomes and lysosomes is regulated by the interaction of signals present in their carboxyl-terminal fragment with specific trafficking molecules. The BACE1 carboxyl-terminal fragment contains a di-leucine sorting signal ((495)DDISLL(500)) and a ubiquitination site at Lys-501. Here, we report that lack of ubiquitination at Lys-501 (BACE1K501R) does not affect the rate of endocytosis but produces BACE1 stabilization and accumulation of BACE1 in early and late endosomes/lysosomes as well as at the cell membrane. In contrast, the disruption of the di-leucine motif (BACE1LLAA) greatly impairs BACE1 endocytosis and produces a delayed retrograde transport of BACE1 to the trans-Golgi network (TGN) and a delayed delivery of BACE1 to the lysosomes, thus decreasing its degradation. Moreover, the combination of the lack of ubiquitination at Lys-501 and the disruption of the di-leucine motif (BACE1LLAA/KR) produces additive effects on BACE1 stabilization and defective internalization. Finally, BACE1LLAA/KR accumulates in the TGN, while its levels are decreased in EEA1-positive compartments indicating that both ubiquitination at Lys-501 and the di-leucine motif are necessary for the trafficking of BACE1 from the TGN to early endosomes. Our studies have elucidated a differential role for the di-leucine motif and ubiquitination at Lys-501 in BACE1 endocytosis, trafficking, and degradation and suggest the involvement of multiple adaptor molecules.

  14. BACE1 Protein Endocytosis and Trafficking Are Differentially Regulated by Ubiquitination at Lysine 501 and the Di-leucine Motif in the Carboxyl Terminus*

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    Kang, Eugene L.; Biscaro, Barbara; Piazza, Fabrizio; Tesco, Giuseppina

    2012-01-01

    β-Site amyloid precursor protein-cleaving enzyme (BACE1) is a membrane-tethered member of the aspartyl proteases that has been identified as β-secretase. BACE1 is targeted through the secretory pathway to the plasma membrane and then is internalized to endosomes. Sorting of membrane proteins to the endosomes and lysosomes is regulated by the interaction of signals present in their carboxyl-terminal fragment with specific trafficking molecules. The BACE1 carboxyl-terminal fragment contains a di-leucine sorting signal (495DDISLL500) and a ubiquitination site at Lys-501. Here, we report that lack of ubiquitination at Lys-501 (BACE1K501R) does not affect the rate of endocytosis but produces BACE1 stabilization and accumulation of BACE1 in early and late endosomes/lysosomes as well as at the cell membrane. In contrast, the disruption of the di-leucine motif (BACE1LLAA) greatly impairs BACE1 endocytosis and produces a delayed retrograde transport of BACE1 to the trans-Golgi network (TGN) and a delayed delivery of BACE1 to the lysosomes, thus decreasing its degradation. Moreover, the combination of the lack of ubiquitination at Lys-501 and the disruption of the di-leucine motif (BACE1LLAA/KR) produces additive effects on BACE1 stabilization and defective internalization. Finally, BACE1LLAA/KR accumulates in the TGN, while its levels are decreased in EEA1-positive compartments indicating that both ubiquitination at Lys-501 and the di-leucine motif are necessary for the trafficking of BACE1 from the TGN to early endosomes. Our studies have elucidated a differential role for the di-leucine motif and ubiquitination at Lys-501 in BACE1 endocytosis, trafficking, and degradation and suggest the involvement of multiple adaptor molecules. PMID:23109336

  15. Oleic acid and linoleic acid from Tenebrio molitor larvae inhibit BACE1 activity in vitro: molecular docking studies.

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    Youn, Kumju; Yun, Eun-Young; Lee, Jinhyuk; Kim, Ji-Young; Hwang, Jae-Sam; Jeong, Woo-Sik; Jun, Mira

    2014-02-01

    In our ongoing research to find therapeutic compounds for Alzheimer's disease (AD) from natural resources, the inhibitory activity of the BACE1 enzyme by Tenebrio molitor larvae and its major compounds were evaluated. The T. molitor larvae extract and its fractions exhibited strong BACE1 suppression. The major components of hexane fraction possessing both high yield and strong BACE1 inhibition were determined by thin layer chromatography, gas chromatography, and nuclear magnetic resonance analysis. A remarkable composition of unsaturated long chain fatty acids, including oleic acid and linoleic acid, were identified. Oleic acid, in particular, noncompetitively attenuated BACE1 activity with a half-maximal inhibitory concentration (IC₅₀) value of 61.31 μM and Ki value of 34.3 μM. Furthermore, the fatty acids were stably interacted with BACE1 at different allosteric sites of the enzyme bound with the OH of CYS319 and the NH₃ of TYR320 for oleic acid and with the C=O group of GLN304 for linoleic acid. Here, we first revealed novel pharmacophore features of oleic acids and linoleic acid to BACE1 by in silico docking studies. The present findings would clearly suggest potential guidelines for designing novel BACE1 selective inhibitors.

  16. Quantum mechanics study of the hydroxyethylamines-BACE-1 active site interaction energies

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    Gueto-Tettay, Carlos; Drosos, Juan Carlos; Vivas-Reyes, Ricardo

    2011-06-01

    The identification of BACE-1, a key enzyme in the production of Amyloid-β (Aβ) peptides, generated by the proteolytic processing of amyloid precursor protein, was a major advance in the field of Alzheimer's disease as this pathology is characterized by the presence of extracellular senile plaques, mainly comprised of Aβ peptides. Hydroxyethylamines have demonstrated a remarkable potential, like candidate drugs, for this disease using BACE-1 as target. Density Functional Theory calculations were employed to estimate interaction energies for the complexes formed between the hydroxyethylamine derivated inhibitors and 24 residues in the BACE-1 active site. The collected data offered not only a general but a particular quantitative description that gives a deep insight of the interactions in the active site, showing at the same time how ligand structural variations affect them. Polar interactions are the major energetic contributors for complex stabilization and those ones with charged aspartate residues are highlighted, as they contribute over 90% of the total attractive interaction energy. Ligand-ARG296 residue interaction reports the most repulsive value and decreasing of the magnitude of this repulsion can be a key feature for the design of novel and more potent BACE-1 inhibitors. Also it was explained why sultam derivated BACE-1 inhibitors are better ones than lactam based. Hydrophobic interactions concentrated at S1 zone and other relevant repulsions and attractions were also evaluated. The comparison of two different theory levels (X3LYP and M062X) allowed to confirm the relevance of the detected interactions as each theory level has its own strength to depict the forces involved, as is the case of M062X which is better describing the hydrophobic interactions. Those facts were also evaluated and confirmed by comparing the quantitative trend, of selected ligand-residue interactions, with MP2 theory level as reference standard method for electrostatic plus

  17. Ubiquitin regulates GGA3-mediated degradation of BACE1.

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    Kang, Eugene L; Cameron, Andrew N; Piazza, Fabrizio; Walker, Kendall R; Tesco, Giuseppina

    2010-07-30

    BACE1 (beta-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of beta-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1-3 (Golgi-localized gamma-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and beta-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery.

  18. Ubiquitin Regulates GGA3-mediated Degradation of BACE1*

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    Kang, Eugene L.; Cameron, Andrew N.; Piazza, Fabrizio; Walker, Kendall R.; Tesco, Giuseppina

    2010-01-01

    BACE1 (β-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of β-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1–3 (Golgi-localized γ-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and β-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery. PMID:20484053

  19. Linking vascular disorders and Alzheimer’s disease: Potential involvement of BACE1

    Science.gov (United States)

    Cole, Sarah L.; Vassar, Robert

    2012-01-01

    The etiology of Alzheimer’s disease (AD) remains unknown. However, specific risk factors have been identified, and aging is the strongest AD risk factor. The majority of cardiovascular events occur in older people and a close relationship between vascular disorders and AD exists. Amyloid plaques, composed of the beta amyloid peptide (Aβ), are hallmark lesions in AD and evidence indicates that Aβ plays a central role in AD pathophysiology. The BACE1 enzyme is essential for Aβ generation, and BACE1 levels are elevated in AD brain. The cause(s) of this BACE1 elevation remains undetermined. Here we review the potential contribution of vascular disease to AD pathogenesis. We examine the putative vasoactive properties of Aβ and how the cellular changes associated with vascular disease may elevate BACE1 levels. Despite increasing evidence, the exact role(s) vascular disorders play in AD remains to be determined. However, given that vascular diseases can be addressed by lifestyle and pharmacologic interventions, the potential benefits of these therapies in delaying the clinical appearance and progression of AD may warrant investigation. PMID:18289733

  20. β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses.

    Science.gov (United States)

    Hartmann, Stephanie; Zheng, Fang; Kyncl, Michele C; Karch, Sandra; Voelkl, Kerstin; Zott, Benedikt; D'Avanzo, Carla; Lomoio, Selene; Tesco, Giuseppina; Kim, Doo Y; Alzheimer, Christian; Huth, Tobias

    2018-04-04

    The β-secretase β-site APP-cleaving enzyme 1 (BACE1) is deemed a major culprit in Alzheimer's disease, but accumulating evidence indicates that there is more to the enzyme than driving the amyloidogenic processing of the amyloid precursor protein. For example, BACE1 has emerged as an important regulator of neuronal activity through proteolytic and, most unexpectedly, also through nonproteolytic interactions with several ion channels. Here, we identify and characterize the voltage-gated K + channel 3.4 (Kv3.4) as a new and functionally relevant interaction partner of BACE1. Kv3.4 gives rise to A-type current with fast activating and inactivating kinetics and serves to repolarize the presynaptic action potential. We found that BACE1 and Kv3.4 are highly enriched and remarkably colocalized in hippocampal mossy fibers (MFs). In BACE1 -/- mice of either sex, Kv3.4 surface expression was significantly reduced in the hippocampus and, in synaptic fractions thereof, Kv3.4 was specifically diminished, whereas protein levels of other presynaptic K + channels such as K Ca 1.1 and K Ca 2.3 remained unchanged. The apparent loss of presynaptic Kv3.4 affected the strength of excitatory transmission at the MF-CA3 synapse in hippocampal slices of BACE1 -/- mice when probed with the Kv3 channel blocker BDS-I. The effect of BACE1 on Kv3.4 expression and function should be bidirectional, as predicted from a heterologous expression system, in which BACE1 cotransfection produced a concomitant upregulation of Kv3.4 surface level and current based on a physical interaction between the two proteins. Our data show that, by targeting Kv3.4 to presynaptic sites, BACE1 endows the terminal with a powerful means to regulate the strength of transmitter release. SIGNIFICANCE STATEMENT The β-secretase β-site APP-cleaving enzyme 1 (BACE1) is infamous for its crucial role in the pathogenesis of Alzheimer's disease, but its physiological functions in the intact nervous system are only gradually

  1. BACE1-Dependent Neuregulin-1 Signaling: An Implication for Schizophrenia

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    Zhengrong Zhang

    2017-09-01

    Full Text Available Schizophrenia is a chronic psychiatric disorder with a lifetime prevalence of about 1% in the general population. Recent studies have shown that Neuregulin-1 (Nrg1 is a candidate gene for schizophrenia. At least 15 alternative splicing of NRG1 isoforms all contain an extracellular epidermal growth factor (EGF-like domain, which is sufficient for Nrg1 biological activity including the formation of myelin sheaths and the regulation of synaptic plasticity. It is known that Nrg1 can be cleaved by β-secretase (BACE1 and the resulting N-terminal fragment (Nrg1-ntf binds to receptor tyrosine kinase ErbB4, which activates Nrg1/ErbB4 signaling. While changes in Nrg1 expression levels in schizophrenia still remain controversial, understanding the BACE1-cleaved Nrg1-ntf and Nrg1/ErbB4 signaling in schizophrenia neuropathogenesis is essential and important. In this review paper, we included three major parts: (1 Nrg1 structure and cleavage pattern by BACE1; (2 BACE1-dependent Nrg1 cleavage associated with schizophrenia in human studies; and (3 Animal studies of Nrg1 and BACE1 mutations with behavioral observations. Our review will provide a better understanding of Nrg1 in schizophrenia and a potential strategy for using BACE1 cleavage of Nrg1 as a unique biomarker for diagnosis, as well as a new therapeutic target, of schizophrenia.

  2. Mechanistic insights into mode of action of potent natural antagonists of BACE-1 for checking Alzheimer’s plaque pathology

    Energy Technology Data Exchange (ETDEWEB)

    Dhanjal, Jaspreet Kaur [School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067 (India); Goyal, Sukriti [Apaji Institute of Mathematics and Applied Computer Technology, Banasthali University, Tonk 304022, Rajasthan (India); Sharma, Sudhanshu [Department of Biotechnology, Delhi Technological University, New Delhi 110042 (India); Hamid, Rabia [Department of Biochemistry, University of Kashmir, Srinagar 190006 (India); Grover, Abhinav, E-mail: abhinavgr@gmail.com [School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067 (India)

    2014-01-17

    Highlights: •Accumulation of Aβ plaques is one of the major pathology associated with Alzheimer’s disease. •Inhibition of β-Secretase or BACE-1 offers a viable prospect to check the growth of these plaques. •A large virtual dataset of natural compounds was screened against BACE-1. •Top two hits were analyzed for thermodynamic and structural stability using MD simulations. •Their detailed binding mode of actions were elucidated. -- Abstract: Alzheimer’s is a neurodegenerative disorder resulting in memory loss and decline in cognitive abilities. Accumulation of extracellular beta amyloidal plaques is one of the major pathology associated with this disease. β-Secretase or BACE-1 performs the initial and rate limiting step of amyloidic pathway in which 37–43 amino acid long peptides are generated which aggregate to form plaques. Inhibition of this enzyme offers a viable prospect to check the growth of these plaques. Numerous efforts have been made in recent years for the generation of BACE-1 inhibitors but many of them failed during the preclinical or clinical trials due to drug related or drug induced toxicity. In the present work, we have used computational methods to screen a large dataset of natural compounds to search for small molecules having BACE-1 inhibitory activity with low toxicity to normal cells. Molecular dynamics simulations were performed to analyze molecular interactions between the screened compounds and the active residues of the enzyme. Herein, we report two natural compounds of inhibitory nature active against β-secretase enzyme of amyloidic pathway and are potent lead molecules against Alzheimer’s disease.

  3. Mechanistic insights into mode of action of potent natural antagonists of BACE-1 for checking Alzheimer’s plaque pathology

    International Nuclear Information System (INIS)

    Dhanjal, Jaspreet Kaur; Goyal, Sukriti; Sharma, Sudhanshu; Hamid, Rabia; Grover, Abhinav

    2014-01-01

    Highlights: •Accumulation of Aβ plaques is one of the major pathology associated with Alzheimer’s disease. •Inhibition of β-Secretase or BACE-1 offers a viable prospect to check the growth of these plaques. •A large virtual dataset of natural compounds was screened against BACE-1. •Top two hits were analyzed for thermodynamic and structural stability using MD simulations. •Their detailed binding mode of actions were elucidated. -- Abstract: Alzheimer’s is a neurodegenerative disorder resulting in memory loss and decline in cognitive abilities. Accumulation of extracellular beta amyloidal plaques is one of the major pathology associated with this disease. β-Secretase or BACE-1 performs the initial and rate limiting step of amyloidic pathway in which 37–43 amino acid long peptides are generated which aggregate to form plaques. Inhibition of this enzyme offers a viable prospect to check the growth of these plaques. Numerous efforts have been made in recent years for the generation of BACE-1 inhibitors but many of them failed during the preclinical or clinical trials due to drug related or drug induced toxicity. In the present work, we have used computational methods to screen a large dataset of natural compounds to search for small molecules having BACE-1 inhibitory activity with low toxicity to normal cells. Molecular dynamics simulations were performed to analyze molecular interactions between the screened compounds and the active residues of the enzyme. Herein, we report two natural compounds of inhibitory nature active against β-secretase enzyme of amyloidic pathway and are potent lead molecules against Alzheimer’s disease

  4. Plasma long non-coding RNA BACE1 as a novel biomarker for diagnosis of Alzheimer disease.

    Science.gov (United States)

    Feng, Liang; Liao, Yu-Ting; He, Jin-Cai; Xie, Cheng-Long; Chen, Si-Yan; Fan, Hui-Hui; Su, Zhi-Peng; Wang, Zhen

    2018-01-09

    Long non-coding RNA (LncRNA) have been reported to be involved in the pathogenesis of neurodegenerative diseases, but whether it can serve as a biomarker for Alzheimer disease (AD) is not yet known. The present study selected four specific LncRNA (17A, 51A, BACE1 and BC200) as possible AD biomarker. RT-qPCR was performed to validate the LncRNA. Receiver operating characteristic curve (ROC) and area under the ROC curve (AUC) were applied to study the potential of LncRNA as a biomarker in a population of 88 AD patients and 72 control individuals. We found that the plasma LncRNA BACE1 level of AD patients was significantly higher than that of healthy controls (p = 0.006). Plasma level of LncRNA 17A, 51A and BC200 did not show a significant difference between two groups (p = 0.098, p = 0.204 and p = 0.232, respectively). ROC curve analysis showed that LncRNA BACE1 was the best candidate of these LncRNA (95% CI: 0.553-0.781, p = 0.003). In addition, no correlation was found for expression of these LncRNA in both control and AD groups with age or MMSE scale (p > 0.05). Our present study compared the plasma level of four LncRNA between AD and non-AD patients, and found that the level of the BACE1 is increased in the plasma of AD patients and have a high specificity (88%) for AD, indicating BACE1 may be a potential candidate biomarker to predict AD.

  5. Encompassing receptor flexibility in virtual screening using ensemble docking-based hybrid QSAR: discovery of novel phytochemicals for BACE1 inhibition.

    Science.gov (United States)

    Chakraborty, Sandipan; Ramachandran, Balaji; Basu, Soumalee

    2014-10-01

    Mimicking receptor flexibility during receptor-ligand binding is a challenging task in computational drug design since it is associated with a large increase in the conformational search space. In the present study, we have devised an in silico design strategy incorporating receptor flexibility in virtual screening to identify potential lead compounds as inhibitors for flexible proteins. We have considered BACE1 (β-secretase), a key target protease from a therapeutic perspective for Alzheimer's disease, as the highly flexible receptor. The protein undergoes significant conformational transitions from open to closed form upon ligand binding, which makes it a difficult target for inhibitor design. We have designed a hybrid structure-activity model containing both ligand based descriptors and energetic descriptors obtained from molecular docking based on a dataset of structurally diverse BACE1 inhibitors. An ensemble of receptor conformations have been used in the docking study, further improving the prediction ability of the model. The designed model that shows significant prediction ability judged by several statistical parameters has been used to screen an in house developed 3-D structural library of 731 phytochemicals. 24 highly potent, novel BACE1 inhibitors with predicted activity (Ki) ≤ 50 nM have been identified. Detailed analysis reveals pharmacophoric features of these novel inhibitors required to inhibit BACE1.

  6. Molecular Docking and Dynamic Simulation of AZD3293 and Solanezumab Effects Against BACE1 to Treat Alzheimer's Disease

    Directory of Open Access Journals (Sweden)

    Mubashir Hassan

    2018-06-01

    Full Text Available The design of novel inhibitors to target BACE1 with reduced cytotoxicity effects is a promising approach to treat Alzheimer's disease (AD. Multiple clinical drugs and antibodies such as AZD3293 and Solanezumab are being tested to investigate their therapeutical potential against AD. The current study explores the binding pattern of AZD3293 and Solanezumab against their target proteins such as β-secretase (BACE1 and mid-region amyloid-beta (Aβ (PDBIDs: 2ZHV & 4XXD, respectively using molecular docking and dynamic simulation (MD approaches. The molecular docking results show that AZD3293 binds within the active region of BACE1 by forming hydrogen bonds against Asp32 and Lys107 with distances 2.95 and 2.68 Å, respectively. However, the heavy chain of Solanezumab interacts with Lys16 and Asp23 of amyloid beta having bond length 2.82, 2.78, and 3.00 Å, respectively. The dynamic cross correlations and normal mode analyses show that BACE1 depicted good residual correlated motions and fluctuations, as compared to Solanezumab. Using MD, the Root Mean Square Deviation and Fluctuation (RMSD/F graphs show that AZD3293 residual fluctuations and RMSD value (0.2 nm was much better compared to Solanezumab (0.7 nm. Moreover, the radius of gyration (Rg results also depicts the significance of AZD3293 docked complex compared to Solanezumab through residual compactness. Our comparative results show that AZD3293 is a better therapeutic agent for treating AD than Solanezumab.

  7. Increased CSF-BACE1 activity associated with decreased hippocampus volume in Alzheimer's disease.

    LENUS (Irish Health Repository)

    Ewers, Michael

    2012-02-01

    The enzyme beta-secretase (BACE1) is essentially involved in the production of cerebral amyloidogenic pathology in Alzheimer\\'s disease (AD). The measurement of BACE1 activity in cerebrospinal fluid (CSF) has been reported, which may render CSF measurement of BACE1 a potential biomarker candidate of AD. In order to investigate whether BACE1 protein activity is correlated with regional brain atrophy in AD, we investigated the association between CSF levels of BACE1 and MRI-assessed hippocampus volume in patients with AD (n = 30). An increase in CSF-BACE1 activity was associated with decreased left and right hippocampus volume corrected for global head volume in the AD patients. Boot-strapped regression analysis showed that increased CSF levels of BACE1 activity were associated with increased CSF concentration of total tau but not amyloid-beta1-42 in AD. White matter hyperintensities did not influence the results. BACE1 activity and protein levels were significantly increased in AD compared to 19 elderly healthy controls. Thus, the CSF biomarker candidate of BACE1 activity was associated with hippocampus atrophy in AD in a robust manner and may reflect neurotoxic amyloid-beta-related processes.

  8. Astrocytic expression of the Alzheimer's disease beta-secretase (BACE1) is stimulus-dependent

    DEFF Research Database (Denmark)

    Hartlage-Rübsamen, Maike; Zeitschel, Ulrike; Apelt, Jenny

    2003-01-01

    The beta-site APP-cleaving enzyme (BACE1) is a prerequisite for the generation of beta-amyloid peptides, which give rise to cerebrovascular and parenchymal beta-amyloid deposits in the brain of Alzheimer's disease patients. BACE1 is neuronally expressed in the brains of humans and experimental...... paradigms studied. In contrast, BACE1 expression by reactive astrocytes was evident in chronic but not in acute models of gliosis. Additionally, we observed BACE1-immunoreactive astrocytes in proximity to beta-amyloid plaques in the brains of aged Tg2576 mice and Alzheimer's disease patients....

  9. Glimepiride attenuates Aβ production via suppressing BACE1 activity in cortical neurons.

    Science.gov (United States)

    Liu, Feiyang; Wang, Yijin; Yan, Ming; Zhang, Luyong; Pang, Tao; Liao, Hong

    2013-12-17

    Numerous lines of evidence suggest a strong link between diabetes mellitus and Alzheimer's disease (AD). Impaired insulin signaling and insulin resistance occur not only in diabetes but also in the brain of AD. Recent evidence has indicated that peroxisome proliferator-activated receptor γ (PPARγ) agonists thiazolidinediones (TZDs) can decrease β-amyloid peptide (Aβ) deposition, which is the core component of senile plaques in AD, but the underlying mechanisms still remain unclear. In this study, we investigated whether glimepiride with PPARγ-stimulating activity, an oral anti-diabetic drug, has similar effects on Aβ production in primary cortical neurons. We demonstrated that glimepiride decreased extracellular Aβ40 and Aβ42 levels. The effect of glimepiride on reduction of Aβ40 generation was mediated by downregulation of β-site APP-cleaving enzyme 1 (BACE1) mRNA and protein expression, and by suppression of BACE1 activity. In addition, we found that high glucose condition enhanced Aβ40 production and glimepiride significantly decreased high glucose-induced Aβ40 production. Finally, a specific PPARγ antagonist GW9662 reversed glimepiride inhibitory effect on Aβ40 generation, suggesting a PPARγ-dependent mechanism may be involved. Our data indicated that glimepiride may serve as a promising drug for the treatment of AD associated with diabetes. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  10. Differential regulation of BACE1 expression by oxidative and nitrosative signals

    Directory of Open Access Journals (Sweden)

    Xu Huaxi

    2011-03-01

    Full Text Available Abstract Background It is well established that both cerebral hypoperfusion/stroke and type 2 diabetes are risk factors for Alzheimer's disease (AD. Recently, the molecular link between ischemia/hypoxia and amyloid precursor protein (APP processing has begun to be established. However, the role of the key common denominator, namely nitric oxide (NO, in AD is largely unknown. In this study, we investigated redox regulation of BACE1, the rate-limiting enzyme responsible for the β-cleavage of APP to Aβ peptides. Results Herein, we studied events such as S-nitrosylation, a covalent modification of cysteine residues by NO, and H2O2-mediated oxidation. We found that NO and H2O2 differentially modulate BACE1 expression and enzymatic activity: NO at low concentrations (2O2 (1-10 μM induces BACE1 expression via transcriptional activation, resulting in increased enzymatic activity. The differential effects of NO and H2O2 on BACE1 expression and activity are also reflected in their opposing effects on Aβ generation in cultured neurons in a dose-dependent manner. Furthermore, we found that BACE1 is highly S-nitrosylated in normal aging brains while S-nitrosylation is markedly reduced in AD brains. Conclusion This study demonstrates for the first time that BACE1 is highly modified by NO via multiple mechanisms: low and high levels of NO suppress BACE1 via transcriptional and post translational regulation, in contrast with the upregulation of BACE1 by H2O2-mediated oxidation. These novel NO-mediated regulatory mechanisms likely protect BACE1 from being further oxidized by excessive oxidative stress, as from H2O2 and peroxynitrite which are known to upregulate BACE1 and activate the enzyme, resulting in excessive cleavage of APP and Aβ generation; they likely represent the crucial house-keeping mechanism for BACE1 expression/activation under physiological conditions.

  11. Depletion of GGA1 and GGA3 mediates post-injury elevation of BACE1

    Science.gov (United States)

    Walker, Kendall R.; Kang, Eugene L.; Whalen, Michael J.; Shen, Yong; Tesco, Giuseppina

    2012-01-01

    Traumatic brain injury (TBI) is one of the most robust environmental risk factors for Alzheimer’s disease (AD). Compelling evidence is accumulating that a single event of TBI is associated with increased levels of Aβ. However, the underlying molecular mechanisms remain unknown. We report here that the BACE1 interacting protein, GGA3, is depleted while BACE1 levels increase in the acute phase post-injury (48hrs) in a mouse model of TBI. We further demonstrated the role of GGA3 in the regulation of BACE1 in vivo by showing that BACE1 levels are increased in the brain of GGA3 null mice. We next found that head trauma potentiates BACE1 elevation in GGA3 null mice in the acute phase post-TBI and discovered that GGA1, a GGA3 homologue, is a novel caspase-3 substrate depleted at 48 hrs post-TBI. Moreover, GGA1 silencing potentiates BACE1 elevation induced by GGA3 deletion in neurons in vitro indicating that GGA1 and GGA3 synergistically regulate BACE1. Accordingly, we found that levels of both GGA1 and GGA3 are depleted while BACE1 levels are increased in a series of post-mortem AD brains. Finally, we show that GGA3 haploinsufficiency results in sustained elevation of BACE1 and Aβ levels while GGA1 levels are restored in the subacute phase (7 days) post-injury. In conclusion, our data indicate that depletion of GGA1 and GGA3 engender a rapid and robust elevation of BACE1 in the acute phase post-injury. However, the efficient disposal of the acutely accumulated BACE1 solely depends on GGA3 levels in the sub-acute phase of injury. PMID:22836275

  12. Depletion of GGA1 and GGA3 mediates postinjury elevation of BACE1.

    Science.gov (United States)

    Walker, Kendall R; Kang, Eugene L; Whalen, Michael J; Shen, Yong; Tesco, Giuseppina

    2012-07-25

    Traumatic brain injury (TBI) is one of the most robust environmental risk factors for Alzheimer's disease (AD). Compelling evidence is accumulating that a single event of TBI is associated with increased levels of Aβ. However, the underlying molecular mechanisms remain unknown. We report here that the BACE1 interacting protein, GGA3, is depleted while BACE1 levels increase in the acute phase after injury (48 h) in a mouse model of TBI. We further demonstrated the role of GGA3 in the regulation of BACE1 in vivo by showing that BACE1 levels are increased in the brain of GGA3-null mice. We next found that head trauma potentiates BACE1 elevation in GGA3-null mice in the acute phase after TBI, and discovered that GGA1, a GGA3 homolog, is a novel caspase-3 substrate depleted at 48 h after TBI. Moreover, GGA1 silencing potentiates BACE1 elevation induced by GGA3 deletion in neurons in vitro, indicating that GGA1 and GGA3 synergistically regulate BACE1. Accordingly, we found that levels of both GGA1 and GGA3 are depleted while BACE1 levels are increased in a series of postmortem AD brains. Finally, we show that GGA3 haploinsufficiency results in sustained elevation of BACE1 and Aβ levels while GGA1 levels are restored in the subacute phase (7 d) after injury. In conclusion, our data indicate that depletion of GGA1 and GGA3 engender a rapid and robust elevation of BACE1 in the acute phase after injury. However, the efficient disposal of the acutely accumulated BACE1 solely depends on GGA3 levels in the subacute phase of injury.

  13. Fluorescence Microspectroscopy for Testing the Dimerization Hypothesis of BACE1 Protein in Cultured HEK293 Cells

    Science.gov (United States)

    Gardeen, Spencer; Johnson, Joseph L.; Heikal, Ahmed A.

    2016-06-01

    Alzheimer's Disease (AD) is a neurodegenerative disorder that results from the formation of beta-amyloid plaques in the brain that trigger the known symptoms of memory loss in AD patients. The beta-amyloid plaques are formed by the proteolytic cleavage of the amyloid precursor protein (APP) by the proteases BACE1 and gamma-secretase. These enzyme-facilitated cleavages lead to the production of beta-amyloid fragments that aggregate to form plaques, which ultimately lead to neuronal cell death. Recent detergent protein extraction studies suggest that BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. In this contribution, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using complementary fluorescence spectroscopy and microscopy methods. Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal, and differential interference contrast to monitor the localization and distribution of intracellular BACE1. Complementary fluorescence lifetime and anisotropy measurements enabled us to examine the conformational and environmental changes of BACE1 as a function of substrate binding. Using fluorescence correlation spectroscopy, we also quantified the diffusion coefficient of BACE1-EGFP on the plasma membrane as a means to test the dimerization hypothesis as a fucntion of substrate-analog inhibitition. Our results represent an important first towards examining the substrate-mediated dimerization hypothesis of BACE1 in live cells.

  14. Dose-dependent inhibition of BACE-1 by the monoterpenoid 2,3,4,4-tetramethyl-5-methylenecyclopent-2-enone in cellular and mouse models of Alzheimer's disease.

    Science.gov (United States)

    Videira, Rita; Castanheira, Pedro; Grãos, Mário; Resende, Rosa; Salgueiro, Lígia; Faro, Carlos; Cavaleiro, Carlos

    2014-06-27

    BACE-1 is an aspartic protease involved in the conversion of amyloid precursor protein (APP) to amyloid-β (Aβ) in vivo, which is one of the key steps in the development and progression of Alzheimer's disease. In a previous screening procedure for inhibitors of BACE-1 activity, the oil of Lavandula luisieri was identified as the most potent among several essential oils. The inhibitory effect of this essential oil on Aβ production was also demonstrated in a cellular assay. The composition of the volatile oil and the isolation of the compound responsible for the inhibitory activity were also reported. The present work focused on the characterization of the inhibition of BACE-1 by this active compound, a monoterpene necrodane ketone, 2,3,4,4-tetramethyl-5-methylenecyclopent-2-enone (1), with assessment of its Ki value and the type of inhibition. The dose-related effects of the compound were also evaluated using two different cell lines, with determinations of the respective EC50 values. The entire oil and the 2,3,4,4-tetramethyl-5-methylenecyclopent-2-enone (1) were tested on a triple transgenic mouse model of Alzheimer's disease. The overall results showed that compound 1 displayed a dose-dependent inhibition of BACE-1 in cellular and mouse models of Alzheimer's disease and is therefore capable of passing through cellular membranes and the blood-brain barrier.

  15. A PM7 dynamic residue-ligand interactions energy landscape of the BACE1 inhibitory pathway by hydroxyethylamine compounds. Part I: The flap closure process.

    Science.gov (United States)

    Gueto-Tettay, Carlos; Martinez-Consuegra, Alejandro; Zuchniarz, Joshua; Gueto-Tettay, Luis Roberto; Drosos-Ramírez, Juan Carlos

    2017-09-01

    BACE1 is an enzyme of scientific interest because it participates in the progression of Alzheimer's disease. Hydroxyethylamines (HEAs) are a family of compounds which exhibit inhibitory activity toward BACE1 at a nanomolar level, favorable pharmacokinetic properties and oral bioavailability. The first step in the inhibition of BACE1 by HEAs consists of their entrance into the protease active site and the resultant conformational change in the protein, from Apo to closed form. These two conformations differ in the position of an antiparallel loop (called the flap) which covers the entrance to the catalytic site. For BACE1, closure of this flap is vital to its catalytic activity and to inhibition of the enzyme due to the new interactions thereby formed with the ligand. In the present study a dynamic energy landscape of residue-ligand interaction energies (ReLIE) measured for 112 amino acids in the BACE1 active site and its immediate vicinity during the closure of the flap induced by 8 HEAs of different inhibitory power is presented. A total of 6.272 million ReLIE calculations, based on the PM7 semiempirical method, provided a deep and quantitative view of the first step in the inhibition of the aspartyl protease. The information suggests that residues Asp93, Asp289, Thr292, Thr293, Asn294 and Arg296 are anchor points for the ligand, accounting for approximately 45% of the total protein-ligand interaction. Additionally, flap closure improved the BACE1-HEA interaction by around 25%. Furthermore, the inhibitory activity of HEAs could be related to the capacity of these ligands to form said anchor point interactions and maintain them over time: the lack of some of these anchor interactions delayed flap closure or impeded it completely, or even caused the flap to reopen. The methodology employed here could be used as a tool to evaluate future structural modifications which lead to improvements in the favorability and stability of BACE1-HEA ReLIEs, aiding in the design of

  16. BACE1 Deficiency Causes Abnormal Neuronal Clustering in the Dentate Gyrus

    Directory of Open Access Journals (Sweden)

    Hailong Hou

    2017-07-01

    Full Text Available BACE1 is validated as Alzheimer's β-secretase and a therapeutic target for Alzheimer's disease. In examining BACE1-null mice, we discovered that BACE1 deficiency develops abnormal clusters of immature neurons, forming doublecortin-positive neuroblasts, in the developing dentate gyrus, mainly in the subpial zone (SPZ. Such clusters were rarely observed in wild-type SPZ and not reported in other mouse models. To understand their origins and fates, we examined how neuroblasts in BACE1-null SPZ mature and migrate during early postnatal development. We show that such neuroblasts are destined to form Prox1-positive granule cells in the dentate granule cell layer, and mainly mature to form excitatory neurons, but not inhibitory neurons. Mechanistically, higher levels of reelin potentially contribute to abnormal neurogenesis and timely migration in BACE1-null SPZ. Altogether, we demonstrate that BACE1 is a critical regulator in forming the dentate granule cell layer through timely maturation and migration of SPZ neuroblasts.

  17. GGA1 regulates signal-dependent sorting of BACE1 to recycling endosomes, which moderates Aβ production

    Science.gov (United States)

    Toh, Wei Hong; Chia, Pei Zhi Cheryl; Hossain, Mohammed Iqbal; Gleeson, Paul A.

    2018-01-01

    The diversion of the membrane-bound β-site amyloid precursor protein–(APP) cleaving enzyme (BACE1) from the endolysosomal pathway to recycling endosomes represents an important transport step in the regulation of amyloid beta (Aβ) production. However, the mechanisms that regulate endosome sorting of BACE1 are poorly understood. Here we assessed the transport of BACE1 from early to recycling endosomes and have identified essential roles for the sorting nexin 4 (SNX4)-mediated, signal-independent pathway and for a novel signal-mediated pathway. The signal-mediated pathway is regulated by the phosphorylation of the DXXLL-motif sequence DISLL in the cytoplasmic tail of BACE1. The phosphomimetic S498D BACE1 mutant was trafficked to recycling endosomes at a faster rate compared with wild-type BACE1 or the nonphosphorylatable S498A mutant. The rapid transit of BACE1 S498D from early endosomes was coupled with reduced levels of amyloid precursor protein processing and Aβ production, compared with the S498A mutant. We show that the adaptor, GGA1, and retromer are essential to mediate rapid trafficking of phosphorylated BACE1 to recycling endosomes. In addition, the BACE1 DISLL motif is phosphorylated and regulates endosomal trafficking, in primary neurons. Therefore, post-translational phosphorylation of DISLL enhances the exit of BACE1 from early endosomes, a pathway mediated by GGA1 and retromer, which is important in regulating Aβ production. PMID:29142073

  18. β-Secretase BACE1 regulates hippocampal and reconstituted M-currents in a β-subunit-like fashion.

    Science.gov (United States)

    Hessler, Sabine; Zheng, Fang; Hartmann, Stephanie; Rittger, Andrea; Lehnert, Sandra; Völkel, Meike; Nissen, Matthias; Edelmann, Elke; Saftig, Paul; Schwake, Michael; Huth, Tobias; Alzheimer, Christian

    2015-02-25

    The β-secretase BACE1 is widely known for its pivotal role in the amyloidogenic pathway leading to Alzheimer's disease, but how its action on transmembrane proteins other than the amyloid precursor protein affects the nervous system is only beginning to be understood. We report here that BACE1 regulates neuronal excitability through an unorthodox, nonenzymatic interaction with members of the KCNQ (Kv7) family that give rise to the M-current, a noninactivating potassium current with slow kinetics. In hippocampal neurons from BACE1(-/-) mice, loss of M-current enhanced neuronal excitability. We relate the diminished M-current to the previously reported epileptic phenotype of BACE1-deficient mice. In HEK293T cells, BACE1 amplified reconstituted M-currents, altered their voltage dependence, accelerated activation, and slowed deactivation. Biochemical evidence strongly suggested that BACE1 physically associates with channel proteins in a β-subunit-like fashion. Our results establish BACE1 as a physiologically essential constituent of regular M-current function and elucidate a striking new feature of how BACE1 impacts on neuronal activity in the intact and diseased brain. Copyright © 2015 the authors 0270-6474/15/353298-14$15.00/0.

  19. VPS35 regulates developing mouse hippocampal neuronal morphogenesis by promoting retrograde trafficking of BACE1

    Directory of Open Access Journals (Sweden)

    Chun-Lei Wang

    2012-10-01

    VPS35, a major component of the retromer, plays an important role in the selective endosome-to-Golgi retrieval of membrane proteins. Dysfunction of retromer is a risk factor for neurodegenerative disorders, but its function in developing mouse brain remains poorly understood. Here we provide evidence for VPS35 promoting dendritic growth and maturation, and axonal protein transport in developing mouse hippocampal neurons. Embryonic hippocampal CA1 neurons suppressing Vps35 expression by in utero electroporation of its micro RNAs displayed shortened apical dendrites, reduced dendritic spines, and swollen commissural axons in the neonatal stage, those deficits reflecting a defective protein transport/trafficking in developing mouse neurons. Further mechanistic studies showed that Vps35 depletion in neurons resulted in an impaired retrograde trafficking of BACE1 (β1-secretase and altered BACE1 distribution. Suppression of BACE1 expression in CA1 neurons partially rescued both dendritic and axonal deficits induced by Vps35-deficiency. These results thus demonstrate that BACE1 acts as a critical cargo of retromer in vitro and in vivo, and suggest that VPS35 plays an essential role in regulating apical dendritic maturation and in preventing axonal spheroid formation in developing hippocampal neurons.

  20. Triazolyl tryptoline derivatives as β-secretase inhibitors

    OpenAIRE

    Jiaranaikulwanitch, Jutamas; Boonyarat, Chantana; Fokin, Valery V.; Vajragupta, Opa

    2010-01-01

    Tryptoline, a core structure of ochrolifuanine E, which is a hit compound from virtual screening of the Thai herbal database against BACE1 was used as a scaffold for the design of BACE1 inhibitors. The tryptoline was linked with different side chains by 1,2,3-triazole ring readily synthesized by catalytic azide-alkyne cycloaddition reactions. Twenty two triazolyl tryptoline derivatives were synthesized and screened for the inhibitory action against BACE1. JJCA-140 was the most potent inhibito...

  1. Anti-BACE1 and Antimicrobial Activities of Steroidal Compounds Isolated from Marine Urechis unicinctus

    OpenAIRE

    Yong-Zhe Zhu; Jing-Wen Liu; Xue Wang; In-Hong Jeong; Young-Joon Ahn; Chuan-Jie Zhang

    2018-01-01

    The human β-site amyloid cleaving enzyme (BACE1) has been considered as an effective drug target for treatment of Alzheimer’s disease (AD). In this study, Urechis unicinctus (U. unicinctus), which is a Far East specialty food known as innkeeper worm, ethanol extract was studied by bioassay-directed fractionation and isolation to examine its potential β-site amyloid cleaving enzyme inhibitory and antimicrobial activity. The following compounds were characterized: hecogenin, cholest-4-en-3-one,...

  2. Anti-BACE1 and Antimicrobial Activities of Steroidal Compounds Isolated from Marine Urechis unicinctus

    Directory of Open Access Journals (Sweden)

    Yong-Zhe Zhu

    2018-03-01

    Full Text Available The human β-site amyloid cleaving enzyme (BACE1 has been considered as an effective drug target for treatment of Alzheimer’s disease (AD. In this study, Urechis unicinctus (U. unicinctus, which is a Far East specialty food known as innkeeper worm, ethanol extract was studied by bioassay-directed fractionation and isolation to examine its potential β-site amyloid cleaving enzyme inhibitory and antimicrobial activity. The following compounds were characterized: hecogenin, cholest-4-en-3-one, cholesta-4,6-dien-3-ol, and hurgadacin. These compounds were identified by their mass spectrometry, 1H, and 13C NMR spectral data, comparing those data with NIST/EPA/NIH Mass spectral database (NIST11 and published values. Hecogenin and cholest-4-en-3-one showed significant inhibitory activity against BACE1 with EC50 values of 116.3 and 390.6 µM, respectively. Cholesta-4,6-dien-3-ol and hurgadacin showed broad spectrum antimicrobial activity, particularly strongly against Escherichia coli (E. coli, Salmonella enterica (S. enterica, Pasteurella multocida (P. multocida, and Physalospora piricola (P. piricola, with minimal inhibitory concentration (MIC ranging from 0.46 to 0.94 mg/mL. This is the first report regarding those four known compounds that were isolated from U. unicinctus and their anti-BACE1 and antimicrobial activity, highlighting the fact that known natural compounds may be a critical source of new medicine leads. These findings provide scientific evidence for potential application of those bioactive compounds for the development of AD drugs and antimicrobial agents.

  3. Anti-BACE1 and Antimicrobial Activities of Steroidal Compounds Isolated from Marine Urechis unicinctus.

    Science.gov (United States)

    Zhu, Yong-Zhe; Liu, Jing-Wen; Wang, Xue; Jeong, In-Hong; Ahn, Young-Joon; Zhang, Chuan-Jie

    2018-03-14

    The human β-site amyloid cleaving enzyme (BACE1) has been considered as an effective drug target for treatment of Alzheimer's disease (AD). In this study, Urechis unicinctus (U. unicinctus) , which is a Far East specialty food known as innkeeper worm, ethanol extract was studied by bioassay-directed fractionation and isolation to examine its potential β-site amyloid cleaving enzyme inhibitory and antimicrobial activity. The following compounds were characterized: hecogenin, cholest-4- en -3-one, cholesta-4,6- dien -3-ol, and hurgadacin. These compounds were identified by their mass spectrometry, ¹H, and 13 C NMR spectral data, comparing those data with NIST/EPA/NIH Mass spectral database (NIST11) and published values. Hecogenin and cholest-4- en -3-one showed significant inhibitory activity against BACE1 with EC 50 values of 116.3 and 390.6 µM, respectively. Cholesta-4,6- dien -3-ol and hurgadacin showed broad spectrum antimicrobial activity, particularly strongly against Escherichia coli (E. coli) , Salmonella enterica (S. enterica) , Pasteurella multocida (P. multocida) , and Physalospora piricola (P. piricola) , with minimal inhibitory concentration (MIC) ranging from 0.46 to 0.94 mg/mL. This is the first report regarding those four known compounds that were isolated from U. unicinctus and their anti-BACE1 and antimicrobial activity, highlighting the fact that known natural compounds may be a critical source of new medicine leads. These findings provide scientific evidence for potential application of those bioactive compounds for the development of AD drugs and antimicrobial agents.

  4. Mild oxidative stress induces redistribution of BACE1 in non-apoptotic conditions and promotes the amyloidogenic processing of Alzheimer's disease amyloid precursor protein.

    Directory of Open Access Journals (Sweden)

    Jiang-Li Tan

    Full Text Available BACE1 is responsible for β-secretase cleavage of the amyloid precursor protein (APP, which represents the first step in the production of amyloid β (Aβ peptides. Previous reports, by us and others, have indicated that the levels of BACE1 protein and activity are increased in the brain cortex of patients with Alzheimer's disease (AD. The association between oxidative stress (OS and AD has prompted investigations that support the potentiation of BACE1 expression and enzymatic activity by OS. Here, we have established conditions to analyse the effects of mild, non-lethal OS on BACE1 in primary neuronal cultures, independently from apoptotic mechanisms that were shown to impair BACE1 turnover. Six-hour treatment of mouse primary cortical cells with 10-40 µM hydrogen peroxide did not significantly compromise cell viability but it did produce mild oxidative stress (mOS, as shown by the increased levels of reactive radical species and activation of p38 stress kinase. The endogenous levels of BACE1 mRNA and protein were not significantly altered in these conditions, whereas a toxic H2O2 concentration (100 µM caused an increase in BACE1 protein levels. Notably, mOS conditions resulted in increased levels of the BACE1 C-terminal cleavage product of APP, β-CTF. Subcellular fractionation techniques showed that mOS caused a major rearrangement of BACE1 localization from light to denser fractions, resulting in an increased distribution of BACE1 in fractions containing APP and markers for trans-Golgi network and early endosomes. Collectively, these data demonstrate that mOS does not modify BACE1 expression but alters BACE1 subcellular compartmentalization to favour the amyloidogenic processing of APP, and thus offer new insight in the early molecular events of AD pathogenesis.

  5. Rationalizing fragment based drug discovery for BACE1: insights from FB-QSAR, FB-QSSR, multi objective (MO-QSPR) and MIF studies.

    Science.gov (United States)

    Manoharan, Prabu; Vijayan, R S K; Ghoshal, Nanda

    2010-10-01

    The ability to identify fragments that interact with a biological target is a key step in FBDD. To date, the concept of fragment based drug design (FBDD) is increasingly driven by bio-physical methods. To expand the boundaries of QSAR paradigm, and to rationalize FBDD using In silico approach, we propose a fragment based QSAR methodology referred here in as FB-QSAR. The FB-QSAR methodology was validated on a dataset consisting of 52 Hydroxy ethylamine (HEA) inhibitors, disclosed by GlaxoSmithKline Pharmaceuticals as potential anti-Alzheimer agents. To address the issue of target selectivity, a major confounding factor in the development of selective BACE1 inhibitors, FB-QSSR models were developed using the reported off target activity values. A heat map constructed, based on the activity and selectivity profile of the individual R-group fragments, and was in turn used to identify superior R-group fragments. Further, simultaneous optimization of multiple properties, an issue encountered in real-world drug discovery scenario, and often overlooked in QSAR approaches, was addressed using a Multi Objective (MO-QSPR) method that balances properties, based on the defined objectives. MO-QSPR was implemented using Derringer and Suich desirability algorithm to identify the optimal level of independent variables (X) that could confer a trade-off between selectivity and activity. The results obtained from FB-QSAR were further substantiated using MIF (Molecular Interaction Fields) studies. To exemplify the potentials of FB-QSAR and MO-QSPR in a pragmatic fashion, the insights gleaned from the MO-QSPR study was reverse engineered using Inverse-QSAR in a combinatorial fashion to enumerate some prospective novel, potent and selective BACE1 inhibitors.

  6. Rationalizing fragment based drug discovery for BACE1: insights from FB-QSAR, FB-QSSR, multi objective (MO-QSPR) and MIF studies

    Science.gov (United States)

    Manoharan, Prabu; Vijayan, R. S. K.; Ghoshal, Nanda

    2010-10-01

    The ability to identify fragments that interact with a biological target is a key step in FBDD. To date, the concept of fragment based drug design (FBDD) is increasingly driven by bio-physical methods. To expand the boundaries of QSAR paradigm, and to rationalize FBDD using In silico approach, we propose a fragment based QSAR methodology referred here in as FB-QSAR. The FB-QSAR methodology was validated on a dataset consisting of 52 Hydroxy ethylamine (HEA) inhibitors, disclosed by GlaxoSmithKline Pharmaceuticals as potential anti-Alzheimer agents. To address the issue of target selectivity, a major confounding factor in the development of selective BACE1 inhibitors, FB-QSSR models were developed using the reported off target activity values. A heat map constructed, based on the activity and selectivity profile of the individual R-group fragments, and was in turn used to identify superior R-group fragments. Further, simultaneous optimization of multiple properties, an issue encountered in real-world drug discovery scenario, and often overlooked in QSAR approaches, was addressed using a Multi Objective (MO-QSPR) method that balances properties, based on the defined objectives. MO-QSPR was implemented using Derringer and Suich desirability algorithm to identify the optimal level of independent variables ( X) that could confer a trade-off between selectivity and activity. The results obtained from FB-QSAR were further substantiated using MIF (Molecular Interaction Fields) studies. To exemplify the potentials of FB-QSAR and MO-QSPR in a pragmatic fashion, the insights gleaned from the MO-QSPR study was reverse engineered using Inverse-QSAR in a combinatorial fashion to enumerate some prospective novel, potent and selective BACE1 inhibitors.

  7. BACE1 Is Necessary for Experience-Dependent Homeostatic Synaptic Plasticity in Visual Cortex

    Directory of Open Access Journals (Sweden)

    Emily Petrus

    2014-01-01

    Full Text Available Alzheimer’s disease (AD is the most common form of age-related dementia, which is thought to result from overproduction and/or reduced clearance of amyloid-beta (Aβ peptides. Studies over the past few decades suggest that Aβ is produced in an activity-dependent manner and has physiological relevance to normal brain functions. Similarly, physiological functions for β- and γ-secretases, the two key enzymes that produce Aβ by sequentially processing the amyloid precursor protein (APP, have been discovered over recent years. In particular, activity-dependent production of Aβ has been suggested to play a role in homeostatic regulation of excitatory synaptic function. There is accumulating evidence that activity-dependent immediate early gene Arc is an activity “sensor,” which acts upstream of Aβ production and triggers AMPA receptor endocytosis to homeostatically downregulate the strength of excitatory synaptic transmission. We previously reported that Arc is critical for sensory experience-dependent homeostatic reduction of excitatory synaptic transmission in the superficial layers of visual cortex. Here we demonstrate that mice lacking the major neuronal β-secretase, BACE1, exhibit a similar phenotype: stronger basal excitatory synaptic transmission and failure to adapt to changes in visual experience. Our results indicate that BACE1 plays an essential role in sensory experience-dependent homeostatic synaptic plasticity in the neocortex.

  8. Effects of curcuminoids identified in rhizomes of Curcuma longa on BACE-1 inhibitory and behavioral activity and lifespan of Alzheimer’s disease Drosophila models

    Science.gov (United States)

    2014-01-01

    Background Alzheimer’s disease (AD) is the most common type of presenile and senile dementia. The human β-amyloid precursor cleavage enzyme (BACE-1) is a key enzyme responsible for amyloid plaque production, which implicates the progress and symptoms of AD. Here we assessed the anti-BACE-1 and behavioral activities of curcuminoids from rhizomes of Curcuma longa (Zingiberaceae), diarylalkyls curcumin (CCN), demethoxycurcumin (DMCCN), and bisdemethoxycurcumin (BDMCCN) against AD Drosophila melanogaster models. Methods Neuro-protective ability of the curcuminoids was assessed using Drosophila melanogaster model system overexpressing BACE-1 and its substrate APP in compound eyes and entire neurons. Feeding and climbing activity, lifespan, and morphostructural changes in fly eyes also were evaluated. Results BDMCCN has the strongest inhibitory activity toward BACE-1 with 17 μM IC50, which was 20 and 13 times lower than those of CCN and DMCCN respectively. Overexpression of APP/BACE-1 resulted in the progressive and measurable defects in morphology of eyes and locomotion. Remarkably, supplementing diet with either 1 mM BDMCCN or 1 mM CCN rescued APP/BACE1-expressing flies and kept them from developing both morphological and behavioral defects. Our results suggest that structural characteristics, such as degrees of saturation, types of carbon skeleton and functional group, and hydrophobicity appear to play a role in determining inhibitory potency of curcuminoids on BACE-1. Conclusion Further studies will warrant possible applications of curcuminoids as therapeutic BACE-1 blockers. PMID:24597901

  9. BACE1 elevation engendered by GGA3 deletion increases β-amyloid pathology in association with APP elevation and decreased CHL1 processing in 5XFAD mice.

    Science.gov (United States)

    Kim, WonHee; Ma, Liang; Lomoio, Selene; Willen, Rachel; Lombardo, Sylvia; Dong, Jinghui; Haydon, Philip G; Tesco, Giuseppina

    2018-02-02

    β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the rate-limiting enzyme in the production of amyloid beta (Aβ), the toxic peptide that accumulates in the brains of Alzheimer's disease (AD) patients. Our previous studies have shown that the clathrin adaptor Golgi-localized γ-ear-containing ARF binding protein 3 (GGA3) plays a key role in the trafficking of BACE1 to lysosomes, where it is normally degraded. GGA3 depletion results in BACE1 stabilization both in vitro and in vivo. Moreover, levels of GGA3 are reduced and inversely related to BACE1 levels in post-mortem brains of AD patients. In order to assess the effect of GGA3 deletion on AD-like phenotypes, we crossed GGA3 -/- mice with 5XFAD mice. BACE1-mediated processing of APP and the cell adhesion molecule L1 like protein (CHL1) was measured as well as levels of Aβ42 and amyloid burden. In 5XFAD mice, we found that hippocampal and cortical levels of GGA3 decreased while BACE1 levels increased with age, similar to what is observed in human AD brains. GGA3 deletion prevented age-dependent elevation of BACE1 in GGA3KO;5XFAD mice. We also found that GGA3 deletion resulted in increased hippocampal levels of Aβ42 and amyloid burden in 5XFAD mice at 12 months of age. While levels of BACE1 did not change with age and gender in GGAKO;5XFAD mice, amyloid precursor protein (APP) levels increased with age and were higher in female mice. Moreover, elevation of APP was associated with a decreased BACE1-mediated processing of CHL1 not only in 12 months old 5XFAD mice but also in human brains from subjects affected by Down syndrome, most likely due to substrate competition. This study demonstrates that GGA3 depletion is a leading candidate mechanism underlying elevation of BACE1 in AD. Furthermore, our findings suggest that BACE1 inhibition could exacerbate mechanism-based side effects in conditions associated with APP elevation (e.g. Down syndrome) owing to impairment of BACE1-mediated processing of CHL1

  10. Discovery and X-ray crystallographic analysis of a spiropiperidine iminohydantoin inhibitor of beta-secretase.

    Science.gov (United States)

    Barrow, James C; Stauffer, Shaun R; Rittle, Kenneth E; Ngo, Phung L; Yang, ZhiQiang; Selnick, Harold G; Graham, Samuel L; Munshi, Sanjeev; McGaughey, Georgia B; Holloway, M Katharine; Simon, Adam J; Price, Eric A; Sankaranarayanan, Sethu; Colussi, Dennis; Tugusheva, Katherine; Lai, Ming-Tain; Espeseth, Amy S; Xu, Min; Huang, Qian; Wolfe, Abigail; Pietrak, Beth; Zuck, Paul; Levorse, Dorothy A; Hazuda, Daria; Vacca, Joseph P

    2008-10-23

    A high-throughput screen at 100 microM inhibitor concentration for the BACE-1 enzyme revealed a novel spiropiperidine iminohydantoin aspartyl protease inhibitor template. An X-ray cocrystal structure with BACE-1 revealed a novel mode of binding whereby the inhibitor interacts with the catalytic aspartates via bridging water molecules. Using the crystal structure as a guide, potent compounds with good brain penetration were designed.

  11. G Protein-Coupled Receptors (GPCRs in Alzheimer’s Disease: A Focus on BACE1 Related GPCRs

    Directory of Open Access Journals (Sweden)

    Juan eZhao

    2016-03-01

    Full Text Available The G protein coupled receptors (GPCRs have been considered as one of the largest families of validated drug targets, which involve in almost overall physiological functions and pathological processes. Meanwhile, Alzheimer’s disease (AD, the most common type of dementia, affects thinking, learning, memory and behavior of elderly people, that has become the hotspot nowadays for its increasing risks and incurability. The above fields have been intensively studied, and the link between the two has been demonstrated, whereas the way how GPCRs perturb AD progress are yet to be further explored given their complexities. In this review, we summarized recent progress regarding the GPCRs interacted with β-site APP cleaving enzyme 1 (BACE1, a key secretase in AD pathogenesis. Then we discussed the current findings on the regulatory roles of GPCRs on BACE1, and the possibility for pharmaceutical treatment of AD patients by the allosteric modulators and biased ligands of GPCRs. We hope this review can provide new insights into the understanding of mechanistic link between GPCRs and BACE1, and highlight the potential of GPCRs as therapeutic target for AD.

  12. Identification of myo-inositol hexakisphosphate (IP6) as a β-secretase 1 (BACE1) inhibitory molecule in rice grain extract and digest.

    Science.gov (United States)

    Abe, Takako K; Taniguchi, Masayuki

    2014-01-01

    Alzheimer's disease (AD) is widely considered to be caused by amyloid-β peptide (Aβ) accumulation in the brain. Aβ is excised from amyloid-β precursor protein through sequential cleavage by β-secretase 1 (BACE1) and γ-secretase. Thus, BACE1 inhibition could prevent Aβ accumulation. Here, we identified myo-inositol hexakisphosphate (IP6) as a BACE1 inhibitory molecule in rice grain extract and digest. The rice digest and IP6 significantly inhibited Aβ production in neuroblastoma cells without cytotoxicity. These results suggested that rice components, including IP6, may be promising starting materials for the development of potent and safe drugs and/or food to prevent AD.

  13. Increased CSF-BACE 1 Activity Is Associated with ApoE-[Epsilon]4 Genotype in Subjects with Mild Cognitive Impairment and Alzheimer's Disease

    Science.gov (United States)

    Ewers, Michael; Zhong, Zhenyu; Burger, Katharina; Wallin, Anders; Blennow, Kaj; Teipel, Stefan J.; Shen, Yong; Hampel, Harald

    2008-01-01

    The Apolipoprotein (ApoE) [epsilon]4 allele is a major genetic risk factor of Alzheimer's disease, and may affect the production of amyloid beta (A[beta][subscript 1-42]). Recently, we have shown that [beta]-secretase (BACE 1) activity can be reliably detected within the brain and human CSF. Here, we have examined an association between the ApoE…

  14. β-secretase inhibitor; a promising novel therapeutic drug in AD

    Directory of Open Access Journals (Sweden)

    Kelly Willemijn Menting

    2014-07-01

    Full Text Available Alzheimer’s disease (AD and vascular dementia are responsible for up to 90% of dementia cases. According to the World Health Organization (WHO, a staggering number of 35.6 million people are currently diagnosed with dementia. Blocking disease progression or preventing AD altogether is desirable for both social and economic reasons and recently focus has shifted to a new and promising drug: the β-secretase inhibitor. Much of AD research has investigated the amyloid cascade hypothesis, which postulates that AD is caused by changes in amyloid beta (Aβ stability and aggregation. Blocking Aβ production by inhibiting the first protease required for its generation, β-secretase/BACE1, may be the next step in blocking AD progression. In April 2012, promising phase I data on inhibitor MK-8931 was presented. This drug reduced Aβ CSF levels up to 92% and was well tolerated by patients. In March 2013 data was added from a one week trial in 32 mild to moderate AD patients, showing CSF Aβ levels decreased up to 84%. However, BACE1 inhibitors require further research. First, greatly reducing Aβ levels through BACE1 inhibition may have harmful side effects. Second, BACE1 inhibitors have yet to pass clinical trial phase II/III and no data on possible side effects on AD patients are available. And third, there remains doubt about the clinical efficacy of BACE1 inhibitors. In moderate AD patients, Aβ plaques have already been formed. BACE1 inhibitors prevent production of new Aβ plaques, but hypothetically do not influence already existing Aβ peptides. Therefore, BACE1 inhibitors are potentially better at preventing AD instead of having therapeutic use.

  15. Role of synthetic route on the transport properties of BaCe1-xYxO3 proton conductor

    International Nuclear Information System (INIS)

    Chiodelli, Gaetano; Malavasi, Lorenzo; Tealdi, Cristina; Barison, Simona; Battagliarin, Marino; Doubova, Lioudmila; Fabrizio, Monica; Mortalo, Cecilia; Gerbasi, Rosalba

    2009-01-01

    In this paper the role of the preparation route on the transport properties of the BaCe 1-x Y x O 3-δ solid solution with x = 0, 0.1, 0.15, and 0.2 has been studied. In particular, the samples were synthesized by means of the solid-state reaction and by a modified Pechini method. The effect of grain size on the sintering behaviour of the two samples batches was investigated by means of impedance spectroscopy and electron microscopy. It was found that a good sintering of the pellets can be achieved at 1250 deg. C for the samples prepared through the Pechini method. The ceramic route, even at higher temperatures, does not lead to good density values. The extensive conductivity measurements as a function of doping and gas environment (pure oxygen and argon + 10% H 2 + water) we carried out showed that the optimal Y-doping is around 15% and that a significant proton conductivity can be achieved for T lower than 500 deg. C. Above this temperature, the role of oxygen defects starts becoming relevant. Finally, the presence of a slope change in all the Arrhenius plots, irrespective to the gas environment, at about 450-500 deg. C was observed and qualitatively interpreted as due to a change in the nature of the main charge carriers involved

  16. BACE1 inhibition by microdose lithium formulation NP03 rescues memory loss and early stage amyloid neuropathology.

    Science.gov (United States)

    Wilson, E N; Do Carmo, S; Iulita, M F; Hall, H; Ducatenzeiler, A; Marks, A R; Allard, S; Jia, D T; Windheim, J; Cuello, A C

    2017-08-01

    Lithium is first-line therapy for bipolar affective disorder and has recently been shown to have protective effects in populations at risk for Alzheimer's disease (AD). However, the mechanism underlying this protection is poorly understood and consequently limits its possible therapeutic application in AD. Moreover, conventional lithium formulations have a narrow therapeutic window and are associated with a severe side effect profile. Here we evaluated a novel microdose formulation of lithium, coded NP03, in a well-characterized rat model of progressive AD-like amyloid pathology. This formulation allows microdose lithium delivery to the brain in the absence of negative side effects. We found that NP03 rescued key initiating components of AD pathology, including inactivating GSK-3β, reducing BACE1 expression and activity, and reducing amyloid levels. Notably, NP03 rescued memory loss, impaired CRTC1 promoter binding of synaptic plasticity genes and hippocampal neurogenesis. These results raise the possibility that NP03 be of therapeutic value in the early or preclinical stages of AD.

  17. The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA Proteins Alter Amyloid-β Precursor Protein (APP Processing through Interaction of Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1.

    Directory of Open Access Journals (Sweden)

    Bjoern von Einem

    Full Text Available Proteolytic processing of amyloid-β precursor protein (APP by beta-site APP cleaving enzyme 1 (BACE1 is the initial step in the production of amyloid beta (Aβ, which accumulates in senile plaques in Alzheimer's disease (AD. Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aβ generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα, sAPPβ, and Aβ upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.

  18. Cleavage of ST6Gal I by Radiation-Induced BACE1 Inhibits Golgi-Anchored ST6Gal I-Mediated Sialylation of Integrin β1 and Migration in Colon Cancer Cells

    International Nuclear Information System (INIS)

    Lee, Minyoung; Park, Jung-Jin; Ko, Young-Gyu; Lee, Yun-Sil

    2012-01-01

    Previously, we found that β-galactoside α2,6-sialyltransferase (ST6Gal I), an enzyme that adds sialic acids to N-linked oligosaccharides of glycoproteins and is frequently overexpressed in cancer cells, is up-regulated by ionizing radiation (IR) and cleaved to a form possessing catalytic activity comparable to that of the Golgi-localized enzyme. Moreover, this soluble form is secreted into the culture media. Induction of ST6Gal I significantly increased the migration of colon cancer cells via sialylation of integrin β1. Here, we further investigated the mechanisms underlying ST6Gal I cleavage, solubilization and release from cells, and addressed its functions, focusing primarily on cancer cell migration. We performed immunoblotting and lectin affinity assay to analyze the expression of ST6 Gal I and level of sialylated integrin β1. After ionizing radiation, migration of cells was measured by in vitro migration assay. α2, 6 sialylation level of cell surface was analyzed by flow cytometry. Cell culture media were concentrated and then analyzed for soluble ST6Gal I levels using an α2, 6 sialyltransferase sandwich ELISA. We found that ST6Gal I was cleaved by BACE1 (β-site amyloid precursor protein-cleaving enzyme), which was specifically overexpressed in response to IR. The soluble form of ST6Gal I, which also has sialyltransferase enzymatic activity, was cleaved from the Golgi membrane and then released into the culture media. Both non-cleaved and cleaved forms of ST6Gal I significantly increased colon cancer cell migration in a sialylation-dependent manner. The pro-migratory effect of the non-cleaved form of ST6Gal I was dependent on integrin β1 sialylation, whereas that of the cleaved form of ST6Gal I was not, suggesting that other intracellular sialylated molecules apart from cell surface molecules such as integrin β1 might be involved in mediating the pro-migratory effects of the soluble form of ST6Gal I. Moreover, production of soluble form ST6Gal I by

  19. Acetylation site specificities of lysine deacetylase inhibitors in human cells

    DEFF Research Database (Denmark)

    Schölz, Christian; Weinert, Brian Tate; Wagner, Sebastian A

    2015-01-01

    Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acety......1-α, providing a possible mechanistic explanation of its adverse, pro-inflammatory effects. Our results offer a systems view of KDACI specificities, providing a framework for studying function of acetylation and deacetylases....

  20. Fetzima (levomilnacipran), a drug for major depressive disorder as a dual inhibitor for human serotonin transporters and beta-site amyloid precursor protein cleaving enzyme-1.

    Science.gov (United States)

    Rizvi, Syed Mohd Danish; Shaikh, Sibhghatulla; Khan, Mahiuddin; Biswas, Deboshree; Hameed, Nida; Shakil, Shazi

    2014-01-01

    Pharmacological management of Major Depressive Disorder includes the use of serotonin reuptake inhibitors which targets serotonin transporters (SERT) to increase the synaptic concentrations of serotonin. Beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1) is responsible for amyloid β plaque formation. Hence it is an interesting target for Alzheimer's disease (AD) therapy. This study describes molecular interactions of a new Food and Drug Administration approved antidepressant drug named 'Fetzima' with BACE-1 and SERT. Fetzima is chemically known as levomilnacipran. The study has explored a possible link between the treatment of Depression and AD. 'Autodock 4.2' was used for docking study. The free energy of binding (ΔG) values for 'levomilnacipran-SERT' interaction and 'levomilnacipran-BACE1' interaction were found to be -7.47 and -8.25 kcal/mol, respectively. Levomilnacipran was found to interact with S438, known to be the most important amino acid residue of serotonin binding site of SERT during 'levomilnacipran-SERT' interaction. In the case of 'levomilnacipran-BACE1' interaction, levomilnacipran interacted with two very crucial aspartic acid residues of BACE-1, namely, D32 and D228. These residues are accountable for the cleavage of amyloid precursor protein and the subsequent formation of amyloid β plaques in AD brain. Hence, Fetzima (levomilnacipran) might act as a potent dual inhibitor of SERT and BACE-1 and expected to form the basis of a future dual therapy against depression and AD. It is an established fact that development of AD is associated with Major Depressive Disorder. Therefore, the design of new BACE-1 inhibitors based on antidepressant drug scaffolds would be particularly beneficial.

  1. Thermodynamic and kinetic characterization of hydroxyethylamine β-secretase-1 inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Mondal, Kalyani; Regnstrom, Karin; Morishige, Winse; Barbour, Robin; Probst, Gary; Xu, Ying-Zi; Artis, Dean R.; Yao, Nanhua; Beroza, Paul; Bova, Michael P., E-mail: mpbova2001@yahoo.com

    2013-11-15

    Highlights: •Kinetic and thermodynamic characterization of 10 hydroxyethylamine BACE-1 inhibitors. •Equilibrium binding of inhibitors was enthalpy driven for BACE-1. •Negative entropy of binding was observed towards BACE-1, but not Cathepsin-D. •Structural analysis demonstrates ligand binding induces a major conformational change. •Structural analysis and SPR analysis corroborate induced fit and negative entropy of binding. -- Abstract: Alzheimer’s disease (AD) is a devastating neurodegenerative disease affecting millions of people. β-Secretase-1 (BACE-1), an enzyme involved in the processing of the amyloid precursor protein (APP) to form Aβ, is a well validated target for AD. Herein, the authors characterize 10 randomly selected hydroxyethylamine (HEA) BACE-1 inhibitors in terms of their association and dissociation rate constants and thermodynamics of binding using surface plasmon resonance (SPR). Rate constants of association (k{sub a}) measured at 25 °C ranged from a low of 2.42 × 10{sup 4} M{sup −1} s{sup −1} to the highest value of 8.3 × 10{sup 5} M{sup −1} s{sup −1}. Rate constants of dissociation (k{sub d}) ranged from 1.09 × 10{sup −4} s{sup −1} (corresponding to a residence time of close to three hours), to the fastest of 0.028 s{sup −1}. Three compounds were selected for further thermodynamic analysis where it was shown that equilibrium binding was enthalpy driven while unfavorable entropy of binding was observed. Structural analysis revealed that upon ligand binding, the BACE-1flap folds down over the bound ligand causing an induced fit. The maximal difference between alpha carbon positions in the open and closed conformations of the flap was over 5 Å. Thus the negative entropy of binding determined using SPR analysis was consistent with an induced fit observed by structural analysis.

  2. Membrane-Associated Effects of Glucocorticoid on BACE1 Upregulation and Aβ Generation: Involvement of Lipid Raft-Mediated CREB Activation.

    Science.gov (United States)

    Choi, Gee Euhn; Lee, Sei-Jung; Lee, Hyun Jik; Ko, So Hee; Chae, Chang Woo; Han, Ho Jae

    2017-08-30

    Glucocorticoid has been widely accepted to induce Alzheimer's disease, but the nongenomic effect of glucocorticoid on amyloid β (Aβ) generation has yet to be studied. Here, we investigated the effect of the nongenomic pathway induced by glucocorticoid on amyloid precursor protein processing enzymes as well as Aβ production using male ICR mice and human neuroblastoma SK-N-MC cells. Mice groups exposed to restraint stress or intracerebroventricular injection of Aβ showed impaired cognition, decreased intracellular glucocorticoid receptor (GR) level, but elevated level of membrane GR (mGR). In this respect, we identified the mGR-dependent pathway evoked by glucocorticoid using impermeable cortisol conjugated to BSA (cortisol-BSA) on SK-N-MC cells. Cortisol-BSA augmented the expression of β-site amyloid precursor protein cleaving enzyme 1 (BACE1), the level of C-terminal fragment β of amyloid precursor protein (C99) and Aβ production, which were maintained even after blocking intracellular GR. We also found that cortisol-BSA enhanced the interaction between mGR and Gαs, which colocalized in the lipid raft. The subsequently activated CREB by cortisol-BSA bound to the CRE site of the BACE1 promoter increasing its expression, which was downregulated by inhibiting CBP. Consistently, blocking CBP attenuated cognitive impairment and Aβ production induced by corticosterone treatment or intracerebroventricular injection of Aβ more efficiently than inhibiting intracellular GR in mice. In conclusion, glucocorticoid couples mGR with Gαs and triggers cAMP-PKA-CREB axis dependent on the lipid raft to stimulate BACE1 upregulation and Aβ generation. SIGNIFICANCE STATEMENT Patients with Alzheimer's disease (AD) have been growing sharply and stress is considered as the major environment factor of AD. Glucocorticoid is the primarily responsive factor to stress and is widely known to induce AD. However, most AD patients usually have impaired genomic pathway of glucocorticoid

  3. Iminopyrimidinones: A novel pharmacophore for the development of orally active renin inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    McKittrick, Brian A.; Caldwell, John P.; Bara, Thomas; Boykow, George; Chintala, Madhu; Clader, John; Czarniecki, Michael; Courneya, Brandy; Duffy, Ruth; Fleming, Linda; Giessert, Rachel; Greenlee, William J.; Heap, Charles; Hong, Liwu; Huang, Ying; Iserloh, Ulrich; Josien, Hubert; Khan, Tanweer; Korfmacher, Walter; Liang, Xian; Mazzola, Robert; Mitra, Soumya; Moore, Kristina; Orth, Peter; Rajagopalan, Murali; Roy, Sudipta; Sakwa, Samuel; Strickland, Corey; Vaccaro, Henry; Voigt, Johannes; Wang, Hongwu; Wong, Jesse; Zhang, Rumin; Zych, Andrew (Merck); (Albany MR)

    2015-04-01

    The development of renin inhibitors with favorable oral pharmacokinetic profiles has been a longstanding challenge for the pharmaceutical industry. As part of our work to identify inhibitors of BACE1, we have previously developed iminopyrimidinones as a novel pharmacophore for aspartyl protease inhibition. In this letter we describe how we modified substitution around this pharmacophore to develop a potent, selective and orally active renin inhibitor.

  4. Differential contributions of ApoE4 and female sex to BACE1 activity and expression mediate Aβ deposition and learning and memory in mouse models of Alzheimer’s disease

    Directory of Open Access Journals (Sweden)

    Xu eHou

    2015-10-01

    Full Text Available Alzheimer’s disease (AD, the most common form of dementia, disproportionately affects women in both prevalence and severity. This increased vulnerability to AD in women is strongly associated with age-related ovarian hormone loss and apolipoprotein E 4 allele (ApoE4, the most important genetic risk factor for sporadic AD. Up to date, the mechanism involved in the interaction between ApoE4 and sex/gender in AD is still unclear. This study evaluated the sex-dependent ApoE4 effects on learning and memory, Aβ deposition and potential mechanisms, using mice bearing both sporadic (ApoE4 and familial (APPSwe, PS1M146V, tauP301L; 3xTg AD risk factors and compared with sex- and age-matched 3xTg or nonTg mice. Compared to nonTg mice, transgenic mice of both sexes showed spatial learning and memory deficits in the radial arm water maze and novel arm discrimination tests at 20 months of age. However, at 10 months, only ApoE4/3xTg mice showed significant learning and memory impairment. Moreover, molecular studies of hippocampal tissue revealed significantly higher protein levels of Aβ species, β-site APP cleavage enzyme (BACE1 and Sp1, a transcription factor of BACE1, in female ApoE4/3xTg when compared with female nonTg, female 3xTg and male ApoE4/3xTg mice. Significantly increased BACE1 enzymatic activities were observed in both male and female mice carrying ApoE4; however, only the females showed significant higher BACE1 expressions. Together, these data suggest that ApoE4 allele is associated with increased BACE1 enzymatic activity, while female sex plays an important role in increasing BACE1 expression. The combination of both provides a molecular basis for high Aβ pathology and the resultant hippocampus-dependent learning and memory deficits in female ApoE4 carriers.

  5. Prion protein is decreased in Alzheimer's brain and inversely correlates with BACE1 activity, amyloid-β levels and Braak stage.

    Directory of Open Access Journals (Sweden)

    Isobel J Whitehouse

    Full Text Available The cellular prion protein (PrP(C has been implicated in the development of Alzheimer's disease (AD. PrP(C decreases amyloid-β (Aβ production, which is involved in AD pathogenesis, by inhibiting β-secretase (BACE1 activity. Contactin 5 (CNTN5 has also been implicated in the development of AD by a genome-wide association study. Here we measured PrP(C and CNTN5 in frontal cortex samples from 24 sporadic AD and 24 age-matched control brains and correlated the expression of these proteins with markers of AD. PrP(C was decreased in sporadic AD compared to controls (by 49%, p = 0.014 but there was no difference in CNTN5 between sporadic AD and controls (p = 0.217. PrP(C significantly inversely correlated with BACE1 activity (rs = -0.358, p = 0.006, Aβ load (rs = -0.456, p = 0.001, soluble Aβ (rs = -0.283, p = 0.026 and insoluble Aβ (rs = -0.353, p = 0.007 and PrP(C also significantly inversely correlated with the stage of disease, as indicated by Braak tangle stage (rs = -0.377, p = 0.007. CNTN5 did not correlate with Aβ load (rs = 0.040, p = 0.393, soluble Aβ (rs = 0.113, p = 0.223 or insoluble Aβ (rs = 0.169, p = 0.125. PrP(C was also measured in frontal cortex samples from 9 Down's syndrome (DS and 8 age-matched control brains. In contrast to sporadic AD, there was no difference in PrP(C in the DS brains compared to controls (p = 0.625. These data are consistent with a role for PrP(C in regulating Aβ production and indicate that brain PrP(C level may be important in influencing the onset and progression of sporadic AD.

  6. Cholesterol-dependent energy transfer between fluorescent proteins-insights into protein proximity of APP and BACE1 in different membranes in Niemann-Pick type C disease cells.

    Science.gov (United States)

    von Einem, Bjoern; Weber, Petra; Wagner, Michael; Malnar, Martina; Kosicek, Marko; Hecimovic, Silva; Arnim, Christine A F von; Schneckenburger, Herbert

    2012-11-26

    Förster resonance energy transfer (FRET) -based techniques have recently been applied to study the interactions between β-site APP-cleaving enzyme-GFP (BACE1-GFP) and amyloid precursor protein-mRFP (APP-mRFP) in U373 glioblastoma cells. In this context, the role of APP-BACE1 proximity in Alzheimer's disease (AD) pathogenesis has been discussed. FRET was found to depend on intracellular cholesterol levels and associated alterations in membrane stiffness. Here, NPC1 null cells (CHO-NPC1-/-), exhibiting increased cholesterol levels and disturbed cholesterol transport similar to that observed in Niemann-Pick type C disease (NPC), were used to analyze the influence of altered cholesterol levels on APP-BACE1 proximity. Fluorescence lifetime measurements of whole CHO-wild type (WT) and CHO-NPC1-/- cells (EPI-illumination microscopy), as well as their plasma membranes (total internal reflection fluorescence microscopy, TIRFM), were performed. Additionally, generalized polarization (GP) measurements of CHO-WT and CHO-NPC1-/- cells incubated with the fluorescence marker laurdan were performed to determine membrane stiffness of plasma- and intracellular-membranes. CHO-NPC1-/- cells showed higher membrane stiffness at intracellular- but not plasma-membranes, equivalent to cholesterol accumulation in late endosomes/lysosomes. Along with higher membrane stiffness, the FRET efficiency between BACE1-GFP and APP-mRFP was reduced at intracellular membranes, but not within the plasma membrane of CHO-NPC1-/-. Our data show that FRET combined with TIRF is a powerful technique to determine protein proximity and membrane fluidity in cellular models of neurodegenerative diseases.

  7. Kinetics and molecular docking studies of loganin, morroniside and 7-O-galloyl-D-sedoheptulose derived from Corni fructus as cholinesterase and β-secretase 1 inhibitors.

    Science.gov (United States)

    Bhakta, Himanshu Kumar; Park, Chan Hum; Yokozawa, Takako; Min, Byung-Sun; Jung, Hyun Ah; Choi, Jae Sue

    2016-06-01

    We evaluated the major active components isolated from Corni Fructus: loganin, morroniside, and 7-O-galloyl-D-sedoheptulose as inhibitors of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) for use in Alzheimer's disease treatment. These compounds exhibited predominant cholinesterase (ChEs) inhibitory effects with IC50 values of 0.33, 3.95, and 10.50 ± 1.16 µM, respectively, for AChE, and 33.02, 37.78, and 87.94 ± 4.66 µM, respectively, for BChE. Kinetics studies revealed that loganin and 7-O-galloyl-D-sedoheptulose inhibited AChE with characteristics typical of mixed inhibitors, while morroniside was found to be a noncompetitive inhibitor against AChE and also exerted mixed BChE inhibitory activities. For BACE1, loganin showed noncompetitive type inhibitory effects, while morroniside and 7-O-galloyl-D-sedoheptulose were found to be mixed inhibitors. Furthermore, these compounds exhibited dose-dependent inhibitory activity with ONOO(-)-mediated protein tyrosine nitration. Molecular docking simulation of these compounds demonstrated negative binding energies for ChEs, and BACE1, indicating high affinity and tighter binding capacity for the active site of the enzyme. Loganin was the most potent inhibitor against both ChEs and BACE1. The data suggest that these compounds together can act as a triple inhibitor of AChE, BChE, and BACE1, providing a preventive and therapeutic strategy for Alzheimer's disease treatment.

  8. Inhibitor specificity of recombinant and endogenous caspase-9.

    Science.gov (United States)

    Ryan, Ciara A; Stennicke, Henning R; Nava, Victor E; Burch, Jennifer B; Hardwick, J Marie; Salvesen, Guy S

    2002-01-01

    Apoptosis triggered through the intrinsic pathway by radiation and anti-neoplastic drugs is initiated by the activation of caspase-9. To elucidate control mechanisms in this pathway we used a range of synthetic and natural reagents. The inhibitory potency of acetyl-Asp-Glu-Val-Asp-aldehyde ('Ac-DEVD-CHO'), benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone ('Z-VAD-FMK') and the endogenous caspase inhibitor X-chromosome-linked inhibitor of apoptosis protein ('XIAP') against recombinant caspase-9 were predictive of the efficacy of these compounds in a cell-free system. However, the viral proteins CrmA and p35, although potent inhibitors of recombinant caspase-9, had almost no ability to block caspase-9 in this system. These findings were also mirrored in cell expression studies. We hypothesize that the viral inhibitors CrmA and p35 are excluded from reacting productively with the natural form of active caspase-9 in vivo, making the potency of inhibitors highly context-dependent. This is supported by survival data from a mouse model of apoptosis driven by Sindbis virus expressing either p35 or a catalytic mutant of caspase-9. These results consolidate previous findings that CrmA is a potent inhibitor of caspase-9 in vitro, yet fails to block caspase-9-mediated cell death. PMID:12067274

  9. Cortical Astrocytes Acutely Exposed to the Monomethylarsonous Acid (MMAIII) Show Increased Pro-inflammatory Cytokines Gene Expression that is Consistent with APP and BACE-1: Over-expression.

    Science.gov (United States)

    Escudero-Lourdes, C; Uresti-Rivera, E E; Oliva-González, C; Torres-Ramos, M A; Aguirre-Bañuelos, P; Gandolfi, A J

    2016-10-01

    Long-term exposure to inorganic arsenic (iAs) through drinking water has been associated with cognitive impairment in children and adults; however, the related pathogenic mechanisms have not been completely described. Increased or chronic inflammation in the brain is linked to impaired cognition and neurodegeneration; iAs induces strong inflammatory responses in several cells, but this effect has been poorly evaluated in central nervous system (CNS) cells. Because astrocytes are the most abundant cells in the CNS and play a critical role in brain homeostasis, including regulation of the inflammatory response, any functional impairment in them can be deleterious for the brain. We propose that iAs could induce cognitive impairment through inflammatory response activation in astrocytes. In the present work, rat cortical astrocytes were acutely exposed in vitro to the monomethylated metabolite of iAs (MMA III ), which accumulates in glial cells without compromising cell viability. MMA III LD 50 in astrocytes was 10.52 μM, however, exposure to sub-toxic MMA III concentrations (50-1000 nM) significantly increased IL-1β, IL-6, TNF-α, COX-2, and MIF-1 gene expression. These effects were consistent with amyloid precursor protein (APP) and β-secretase (BACE-1) increased gene expression, mainly for those MMA III concentrations that also induced TNF-α over-expression. Other effects of MMA III on cortical astrocytes included increased proliferative and metabolic activity. All tested MMA III concentrations led to an inhibition of intracellular lactate dehydrogenase (LDH) activity. Results suggest that MMA III induces important metabolic and functional changes in astrocytes that may affect brain homeostasis and that inflammation may play a major role in cognitive impairment-related pathogenicity in As-exposed populations.

  10. Long-term electromagnetic pulse exposure induces Abeta deposition and cognitive dysfunction through oxidative stress and overexpression of APP and BACE1.

    Science.gov (United States)

    Jiang, Da-Peng; Li, Jin-Hui; Zhang, Jie; Xu, Sheng-Long; Kuang, Fang; Lang, Hai-Yang; Wang, Ya-Feng; An, Guang-Zhou; Li, Jing; Guo, Guo-Zhen

    2016-07-01

    A progressively expanded literature has been devoted in the past years to the noxious or beneficial effects of electromagnetic field (EMF) to Alzheimer׳s disease (AD). This study concerns the relationship between electromagnetic pulse (EMP) exposure and the occurrence of AD in rats and the underlying mechanisms, focusing on the role of oxidative stress (OS). 55 healthy male Sprague Dawley (SD) rats were used and received continuous exposure for 8 months. Morris water maze (MWM) test was conducted to test the ability of cognitive and memory. The level of OS was detected by superoxide dismutase (SOD) activity and glutathione (GSH) content. We found that long-term EMP exposure induced cognitive damage in rats. The content of β-amyloid (Aβ) protein in hippocampus was increased after long-term EMP exposure. OS of hippocampal neuron was detected. Western blotting and immunohistochemistry (IHC) assay showed that the content of Aβ protein and its oligomers in EMP-exposed rats were higher than that of sham-exposed rats. The content of Beta Site App Cleaving Enzyme (BACE1) and microtubule-associated protein 1 light chain 3-II (LC3-II) in EMP-exposed rats hippocampus were also higher than that of sham-exposed rats. SOD activity and GSH content in EMP-exposed rats were lower than sham-exposed rats (p<0.05). Several mechanisms were proposed based on EMP exposure-induced OS, including increased amyloid precursor protein (APP) aberrant cleavage. Although further study is needed, the present results suggest that long-term EMP exposure is harmful to cognitive ability in rats and could induce AD-like pathological manifestation. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Substrate and inhibitor specificity of kynurenine monooxygenase from Cytophaga hutchinsonii.

    Science.gov (United States)

    Phillips, Robert S; Anderson, Andrew D; Gentry, Harvey G; Güner, Osman F; Bowen, J Phillip

    2017-04-15

    Kynurenine monooxygenase (KMO) is a potential drug target for treatment of neurodegenerative disorders such as Huntington's and Alzheimer's diseases. We have evaluated substituted kynurenines as substrates or inhibitors of KMO from Cytophaga hutchinsonii. Kynurenines substituted with a halogen at the 5-position are excellent substrates, with values of k cat and k cat /K m comparable to or higher than kynurenine. However, kynurenines substituted in the 3-position are competitive inhibitors, with K I values lower than the K m for kynurenine. Bromination also enhances inhibition, and 3,5-dibromokynurenine is a potent competitive inhibitor with a K I value of 1.5μM. A pharmacophore model of KMO was developed, and predicted that 3,4-dichlorohippuric acid would be an inhibitor. The K I for this compound was found to be 34μM, thus validating the pharmacophore model. We are using these results and our model to design more potent inhibitors of KMO. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Structure-Based Design of an Iminoheterocyclic β-Site Amyloid Precursor Protein Cleaving Enzyme (BACE) Inhibitor that Lowers Central Aβ in Nonhuman Primates

    Energy Technology Data Exchange (ETDEWEB)

    Mandal, Mihirbaran; Wu, Yusheng; Misiaszek, Jeffrey; Li, Guoqing; Buevich, Alexei; Caldwell, John P.; Liu, Xiaoxiang; Mazzola, Robert D.; Orth, Peter; Strickland, Corey; Voigt, Johannes; Wang, Hongwu; Zhu, Zhaoning; Chen, Xia; Grzelak, Michael; Hyde, Lynn A.; Kuvelkar, Reshma; Leach, Prescott T.; Terracina, Giuseppe; Zhang, Lili; Zhang, Qi; Michener, Maria S.; Smith, Brad; Cox, Kathleen; Grotz, Diane; Favreau, Leonard; Mitra, Kaushik; Kazakevich, Irina; McKittrick, Brian A.; Greenlee, William; Kennedy, Matthew E.; Parker, Eric M.; Cumming, Jared N.; Stamford, Andrew W. (Merck)

    2016-04-14

    We describe successful efforts to optimize the in vivo profile and address off-target liabilities of a series of BACE1 inhibitors represented by 6 that embodies the recently validated fused pyrrolidine iminopyrimidinone scaffold. Employing structure-based design, truncation of the cyanophenyl group of 6 that binds in the S3 pocket of BACE1 followed by modification of the thienyl group in S1 was pursued. Optimization of the pyrimidine substituent that binds in the S2'–S2'' pocket of BACE1 remediated time-dependent CYP3A4 inhibition of earlier analogues in this series and imparted high BACE1 affinity. These efforts resulted in the discovery of difluorophenyl analogue 9 (MBi-4), which robustly lowered CSF and cortex Aβ40 in both rats and cynomolgus monkeys following a single oral dose. Compound 9 represents a unique molecular shape among BACE inhibitors reported to potently lower central Aβ in nonrodent preclinical species.

  13. Nitrite and nitroso compounds can serve as specific catalase inhibitors.

    Science.gov (United States)

    Titov, Vladimir Yu; Osipov, Anatoly N

    2017-03-01

    We present evidence that nitrite and nitrosothiols, nitrosoamines and non-heme dinitrosyl iron complexes can reversibly inhibit catalase with equal effectiveness. Catalase activity was evaluated by the permanganatometric and calorimetric assays. This inhibition is not the result of chemical transformations of these compounds to a single inhibitor, as well as it is not the result of NO release from these substances (as NO traps have no effect on the extent of inhibition). It was found that chloride and bromide in concentration above 80 mM and thiocyanate in concentration above 20 μM enhance catalase inhibition by nitrite and the nitroso compounds more than 100 times. The inhibition degree in this case is comparable with that induced by azide. We propose that the direct catalase inhibitor is a positively charged NO-group. This group acquires a positive charge in the active center of enzyme by interaction of nitrite or nitroso compounds with some enzyme groups. Halides and thiocyanate protect the NO + group from hydration and thus increase its inhibition effect. It is probable that a comparatively low chloride concentration in many cells is the main factor to protect catalase from inhibition by nitrite and nitroso compounds.

  14. An integrated in silico approach to design specific inhibitors targeting human poly(a-specific ribonuclease.

    Directory of Open Access Journals (Sweden)

    Dimitrios Vlachakis

    Full Text Available Poly(A-specific ribonuclease (PARN is an exoribonuclease/deadenylase that degrades 3'-end poly(A tails in almost all eukaryotic organisms. Much of the biochemical and structural information on PARN comes from the human enzyme. However, the existence of PARN all along the eukaryotic evolutionary ladder requires further and thorough investigation. Although the complete structure of the full-length human PARN, as well as several aspects of the catalytic mechanism still remain elusive, many previous studies indicate that PARN can be used as potent and promising anti-cancer target. In the present study, we attempt to complement the existing structural information on PARN with in-depth bioinformatics analyses, in order to get a hologram of the molecular evolution of PARNs active site. In an effort to draw an outline, which allows specific drug design targeting PARN, an unequivocally specific platform was designed for the development of selective modulators focusing on the unique structural and catalytic features of the enzyme. Extensive phylogenetic analysis based on all the publicly available genomes indicated a broad distribution for PARN across eukaryotic species and revealed structurally important amino acids which could be assigned as potentially strong contributors to the regulation of the catalytic mechanism of PARN. Based on the above, we propose a comprehensive in silico model for the PARN's catalytic mechanism and moreover, we developed a 3D pharmacophore model, which was subsequently used for the introduction of DNP-poly(A amphipathic substrate analog as a potential inhibitor of PARN. Indeed, biochemical analysis revealed that DNP-poly(A inhibits PARN competitively. Our approach provides an efficient integrated platform for the rational design of pharmacophore models as well as novel modulators of PARN with therapeutic potential.

  15. Inhibitors

    Science.gov (United States)

    ... JM, and the Hemophilia Inhibitor Research Study Investigators. Validation of Nijmegen-Bethesda assay modifications to allow inhibitor ... webinars on blood disorders Language: English (US) Español (Spanish) File Formats Help: How do I view different ...

  16. Inhibitin: a specific inhibitor of sodium/sodium exchange in erythrocytes.

    OpenAIRE

    Morgan, K; Brown, R C; Spurlock, G; Southgate, K; Mir, M A

    1986-01-01

    An inhibitor of ouabain-insensitive sodium/sodium exchange in erythrocytes has been isolated from leukemic promyelocytes. To explore the specific effects of this inhibitor, named inhibitin, sodium transport experiments were carried out in human erythrocytes. Inhibitin reduced ouabain-insensitive bidirectional sodium transport. It did not change net sodium fluxes, had no significant effect on rubidium influx, and did not inhibit sodium-potassium-ATPase activity. The inhibitory effect of inhibi...

  17. Rostafuroxin: an ouabain-inhibitor counteracting specific forms of hypertension.

    Science.gov (United States)

    Ferrari, Patrizia

    2010-12-01

    An innovative approach to the therapy of essential hypertension (EH) and the related complications has been pursued by our group with the aim of defining specific genetic-molecular mechanisms underlying the disease in sub-sets of patients. This approach is anticipated to have a major effect on the clinical practice, diagnostics and development of new drugs able to selectively target such mechanisms. The final achievement is the definition of biomarkers for identifying patients who more likely should benefit for a given therapy both in terms of efficacy and reduction of the adverse reactions. Among many, two mechanisms have been defined and addressed:Both alterations lead to hypertension, organ hypertrophy, negative vascular remodeling and increased cardiovascular risk by affecting the renal Na(+) handling, through the up-regulation of the Na(+)-K(+) pump and the activation of the Src-dependent signal transduction pathway. A novel antihypertensive agent, rostafuroxin (PST2238), has been selected and developed for its ability to correct the renal Na(+)-K(+) pump abnormalities sustained by the mutant adducin and EO-dependent mechanisms. It is endowed with high potency and efficacy in reducing blood pressure (BP) and preventing organ hypertrophy in animal models representative of both adducin and EO mechanisms. At molecular level, in the kidney, rostafuroxin normalizes the enhanced activity of the Na(+)-K(+) pump induced by mutant adducin and antagonizes the EO triggering of the Src-EGFr-dependent signaling pathway leading to renal Na(+)-K(+) pump and ERK phosphorylation and activation. In the vasculature, it normalizes the increased myogenic tone caused by ouabain. A very high safety ratio and the absence of interaction with other mechanisms involved in BP regulation, together with evidence of high tolerability and efficacy in hypertensive patients indicate rostafuroxin as the first example of a new class of antihypertensive agents designed to antagonize adducin and

  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. A specific pharmacophore model of sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors.

    Science.gov (United States)

    Tang, Chunlei; Zhu, Xiaoyun; Huang, Dandan; Zan, Xin; Yang, Baowei; Li, Ying; Du, Xiaoyong; Qian, Hai; Huang, Wenlong

    2012-06-01

    Sodium-dependent glucose co-transporter 2 (SGLT2) plays a pivotal role in maintaining glucose equilibrium in the human body, emerging as one of the most promising targets for the treatment of diabetes mellitus type 2. Pharmacophore models of SGLT2 inhibitors have been generated with a training set of 25 SGLT2 inhibitors using Discovery Studio V2.1. The best hypothesis (Hypo1(SGLT2)) contains one hydrogen bond donor, five excluded volumes, one ring aromatic and three hydrophobic features, and has a correlation coefficient of 0.955, cost difference of 68.76, RMSD of 0.85. This model was validated by test set, Fischer randomization test and decoy set methods. The specificity of Hypo1(SGLT2) was evaluated. The pharmacophore features of Hypo1(SGLT2) were different from the best pharmacophore model (Hypo1(SGLT1)) of SGLT1 inhibitors we developed. Moreover, Hypo1(SGLT2) could effectively distinguish selective inhibitors of SGLT2 from those of SGLT1. These results indicate that a highly predictive and specific pharmacophore model of SGLT2 inhibitors has been successfully obtained. Then Hypo1(SGLT2) was used as a 3D query to screen databases including NCI and Maybridge for identifying new inhibitors of SGLT2. The hit compounds were subsequently subjected to filtering by Lipinski's rule of five. And several compounds selected from the top ranked hits have been suggested for further experimental assay studies.

  20. Potent and Selective Peptidyl Boronic Acid Inhibitors of the Serine Protease Prostate-Specific Antigen

    Science.gov (United States)

    LeBeau, Aaron M.; Singh, Pratap; Isaacs, John T.; Denmeade, Samuel R.

    2012-01-01

    SUMMARY Prostate cancer cells produce high (microgram to milligram/milliliter) levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the extracellular fluid surrounding prostate cancers but is found at 1,000- to 10,000-fold lower concentrations in the circulation, where it is inactivated due to binding to abundant serum protease inhibitors. The exclusive presence of high levels of active PSA within prostate cancer sites makes PSA an attractive candidate for targeted imaging and therapeutics. A synthetic approach based on a peptide substrate identified first peptide aldehyde and then boronic acid inhibitors of PSA. The best of these had the sequence Cbz-Ser-Ser-Lys-Leu-(boro)Leu, with a Ki for PSA of 65 nM. The inhibitor had a 60-fold higher Ki for chymotrypsin. A validated model of PSA’s catalytic site confirmed the critical interactions between the inhibitor and residues within the PSA enzyme. PMID:18635003

  1. Identification of STAT1 and STAT3 specific inhibitors using comparative virtual screening and docking validation.

    Directory of Open Access Journals (Sweden)

    Malgorzata Szelag

    Full Text Available Signal transducers and activators of transcription (STATs facilitate action of cytokines, growth factors and pathogens. STAT activation is mediated by a highly conserved SH2 domain, which interacts with phosphotyrosine motifs for specific STAT-receptor contacts and STAT dimerization. The active dimers induce gene transcription in the nucleus by binding to a specific DNA-response element in the promoter of target genes. Abnormal activation of STAT signaling pathways is implicated in many human diseases, like cancer, inflammation and auto-immunity. Searches for STAT-targeting compounds, exploring the phosphotyrosine (pTyr-SH2 interaction site, yielded many small molecules for STAT3 but sparsely for other STATs. However, many of these inhibitors seem not STAT3-specific, thereby questioning the present modeling and selection strategies of SH2 domain-based STAT inhibitors. We generated new 3D structure models for all human (hSTATs and developed a comparative in silico docking strategy to obtain further insight into STAT-SH2 cross-binding specificity of a selection of previously identified STAT3 inhibitors. Indeed, by primarily targeting the highly conserved pTyr-SH2 binding pocket the majority of these compounds exhibited similar binding affinity and tendency scores for all STATs. By comparative screening of a natural product library we provided initial proof for the possibility to identify STAT1 as well as STAT3-specific inhibitors, introducing the 'STAT-comparative binding affinity value' and 'ligand binding pose variation' as selection criteria. In silico screening of a multi-million clean leads (CL compound library for binding of all STATs, likewise identified potential specific inhibitors for STAT1 and STAT3 after docking validation. Based on comparative virtual screening and docking validation, we developed a novel STAT inhibitor screening tool that allows identification of specific STAT1 and STAT3 inhibitory compounds. This could increase our

  2. PEGylated DX-1000: Pharmacokinetics and Antineoplastic Activity of a Specific Plasmin Inhibitor

    Directory of Open Access Journals (Sweden)

    Laetitia Devy

    2007-11-01

    Full Text Available Novel inhibitors of the urokinase-mediated plasminogen (plg activation system are potentially of great clinical benefit as anticancer treatments. Using phage display, we identified DX-1000 a tissue factor pathway inhibitor-derived Kunitz domain protein which is a specific high-affinity inhibitor of plasmin (pin (Ki = 99 pM. When tested in vitro, DX-1000 blocks plasminmediated pro-matrix metal loproteinase-9 (proMMP-9 activation on cells and dose-dependently inhibits tube formation, while not significantly affecting hemostasis and coagulation. However, this low-molecular weight protein inhibitor (~ 7 kDa exhibits rapid plasma clearance in mice and rabbits, limiting its potential clinical use in chronic diseases. After site-specific PEGylation, DX-1000 retains its activity and exhibits a decreased plasma clearance. This PEGylated derivative is effective in vitro, as well as potent in inhibiting tumor growth of green fluorescent protein (GFP-labeled MDA-MB-231 cells. 4PEG-DX-1000 treatment causes a significant reduction of urokinase-type plasminogen activator (uPA and plasminogen expressions, a reduction of tumor proliferation, and vascularization. 4PEG-DX-1000 treatment significantly decreases the level of active mitogenactivated protein kinase (MAPK in the primary tumors and reduces metastasis incidence. Together, our results demonstrate the potential value of plasmin inhibitors as therapeutic agents for blocking breast cancer growth and metastasis.

  3. Selective serotonin reuptake inhibitor antidepressant use in first trimester pregnancy and risk of specific congenital anomalies

    DEFF Research Database (Denmark)

    Wemakor, Anthony; Casson, Karen; Garne, Ester

    2015-01-01

    Evidence of an association between early pregnancy exposure to selective serotonin reuptake inhibitors (SSRI) and congenital heart defects (CHD) has contributed to recommendations to weigh benefits and risks carefully. The objective of this study was to determine the specificity of association be...

  4. Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa

    DEFF Research Database (Denmark)

    Larsen, Katrine S; Østergaard, Henrik; Bjelke, Jais R

    2007-01-01

    The remarkably high specificity of the coagulation proteases towards macromolecular substrates is provided by numerous interactions involving the catalytic groove and remote exosites. For FVIIa [activated FVII (Factor VII)], the principal initiator of coagulation via the extrinsic pathway, several...... for FVIIa by marked changes in primary substrate specificity and decreased rates of antithrombin III inhibition. Interestingly, these changes do not necessarily coincide with an altered ability to activate Factor X, demonstrating that inhibitor and macromolecular substrate selectivity may be engineered...

  5. Propiconazole is a specific and accessible brassinosteroid (BR) biosynthesis inhibitor for Arabidopsis and maize.

    Science.gov (United States)

    Hartwig, Thomas; Corvalan, Claudia; Best, Norman B; Budka, Joshua S; Zhu, Jia-Ying; Choe, Sunghwa; Schulz, Burkhard

    2012-01-01

    Brassinosteroids (BRs) are steroidal hormones that play pivotal roles during plant development. In addition to the characterization of BR deficient mutants, specific BR biosynthesis inhibitors played an essential role in the elucidation of BR function in plants. However, high costs and limited availability of common BR biosynthetic inhibitors constrain their key advantage as a species-independent tool to investigate BR function. We studied propiconazole (Pcz) as an alternative to the BR inhibitor brassinazole (Brz). Arabidopsis seedlings treated with Pcz phenocopied BR biosynthetic mutants. The steady state mRNA levels of BR, but not gibberellic acid (GA), regulated genes increased proportional to the concentrations of Pcz. Moreover, root inhibition and Pcz-induced expression of BR biosynthetic genes were rescued by 24epi-brassinolide, but not by GA(3) co-applications. Maize seedlings treated with Pcz showed impaired mesocotyl, coleoptile, and true leaf elongation. Interestingly, the genetic background strongly impacted the tissue specific sensitivity towards Pcz. Based on these findings we conclude that Pcz is a potent and specific inhibitor of BR biosynthesis and an alternative to Brz. The reduced cost and increased availability of Pcz, compared to Brz, opens new possibilities to study BR function in larger crop species.

  6. Pre-equilibrium competitive library screening for tuning inhibitor association rate and specificity toward serine proteases.

    Science.gov (United States)

    Cohen, Itay; Naftaly, Si; Ben-Zeev, Efrat; Hockla, Alexandra; Radisky, Evette S; Papo, Niv

    2018-04-16

    High structural and sequence similarity within protein families can pose significant challenges to the development of selective inhibitors, especially toward proteolytic enzymes. Such enzymes usually belong to large families of closely similar proteases and may also hydrolyze, with different rates, protein- or peptide-based inhibitors. To address this challenge, we employed a combinatorial yeast surface display library approach complemented with a novel pre-equilibrium, competitive screening strategy for facile assessment of the effects of multiple mutations on inhibitor association rates and binding specificity. As a proof of principle for this combined approach, we utilized this strategy to alter inhibitor/protease association rates and to tailor the selectivity of the amyloid β-protein precursor Kunitz protease inhibitor domain (APPI) for inhibition of the oncogenic protease mesotrypsin, in the presence of three competing serine proteases, anionic trypsin, cationic trypsin and kallikrein-6. We generated a variant, designated APPI P13W/M17G/I18F/F34V , with up to 30-fold greater specificity relative to the parental APPI M17G/I18F/F34V protein, and 6500- to 230 000-fold improved specificity relative to the wild-type APPI protein in the presence of the other proteases tested. A series of molecular docking simulations suggested a mechanism of interaction that supported the biochemical results. These simulations predicted that the selectivity and specificity are affected by the interaction of the mutated APPI residues with nonconserved enzyme residues located in or near the binding site. Our strategy will facilitate a better understanding of the binding landscape of multispecific proteins and will pave the way for design of new drugs and diagnostic tools targeting proteases and other proteins. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  7. First demonstration of cerebrospinal fluid and plasma A beta lowering with oral administration of a beta-site amyloid precursor protein-cleaving enzyme 1 inhibitor in nonhuman primates.

    Science.gov (United States)

    Sankaranarayanan, Sethu; Holahan, Marie A; Colussi, Dennis; Crouthamel, Ming-Chih; Devanarayan, Viswanath; Ellis, Joan; Espeseth, Amy; Gates, Adam T; Graham, Samuel L; Gregro, Allison R; Hazuda, Daria; Hochman, Jerome H; Holloway, Katharine; Jin, Lixia; Kahana, Jason; Lai, Ming-tain; Lineberger, Janet; McGaughey, Georgia; Moore, Keith P; Nantermet, Philippe; Pietrak, Beth; Price, Eric A; Rajapakse, Hemaka; Stauffer, Shaun; Steinbeiser, Melissa A; Seabrook, Guy; Selnick, Harold G; Shi, Xiao-Ping; Stanton, Matthew G; Swestock, John; Tugusheva, Katherine; Tyler, Keala X; Vacca, Joseph P; Wong, Jacky; Wu, Guoxin; Xu, Min; Cook, Jacquelynn J; Simon, Adam J

    2009-01-01

    beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic A beta 42 peptides in Alzheimer's disease. Although previous mouse studies have shown brain A beta lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of A beta in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC(50) approximately 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain A beta levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma A beta levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPP beta, A beta 40, A beta 42, and plasma A beta 40 levels. CSF A beta 42 lowering showed an EC(50) of approximately 20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF A beta lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

  8. Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor

    OpenAIRE

    Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min

    2015-01-01

    Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits P. falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report 3 crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all 3 structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of...

  9. Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours

    Energy Technology Data Exchange (ETDEWEB)

    Lasko, Loren M.; Jakob, Clarissa G.; Edalji, Rohinton P.; Qiu, Wei; Montgomery, Debra; Digiammarino, Enrico L.; Hansen, T. Matt; Risi, Roberto M.; Frey, Robin; Manaves, Vlasios; Shaw, Bailin; Algire, Mikkel; Hessler, Paul; Lam, Lloyd T.; Uziel, Tamar; Faivre, Emily; Ferguson, Debra; Buchanan, Fritz G.; Martin, Ruth L.; Torrent, Maricel; Chiang, Gary G.; Karukurichi, Kannan; Langston, J. William; Weinert, Brian T.; Choudhary, Chunaram; de Vries, Peter; Van Drie, John H.; McElligott, David; Kesicki, Ed; Marmorstein, Ronen; Sun, Chaohong; Cole, Philip A.; Rosenberg, Saul H.; Michaelides, Michael R.; Lai, Albert; Bromberg, Kenneth D. (AbbVie); (UCopenhagen); (Petra Pharma); (UPENN); (JHU); (Van Drie); (Faraday)

    2017-09-27

    The dynamic and reversible acetylation of proteins, catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is a major epigenetic regulatory mechanism of gene transcription1 and is associated with multiple diseases. Histone deacetylase inhibitors are currently approved to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind2. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have also been implicated in human pathological conditions (including cancer3). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products4, bi-substrate analogues5 and the widely used small molecule C6466,7, lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like catalytic inhibitor of p300 and CBP. We present a high resolution (1.95 Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 competes with acetyl coenzyme A (acetyl-CoA). A-485 selectively inhibited proliferation in lineage-specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft model. These results demonstrate the feasibility of using small molecule inhibitors to selectively target the catalytic activity of histone acetyltransferases, which may provide effective treatments for transcriptional activator-driven malignancies and diseases.

  10. Specific CLK inhibitors from a novel chemotype for regulation of alternative splicing

    DEFF Research Database (Denmark)

    Fedorov, Oleg; Huber, Kilian; Eisenreich, Andreas

    2011-01-01

    There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile KH-CB19......, a potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Cocrystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix aC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19...... effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue...

  11. Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours

    DEFF Research Database (Denmark)

    Lasko, Loren M; Jakob, Clarissa G; Edalji, Rohinton P

    2017-01-01

    -specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft...... to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have...... also been implicated in human pathological conditions (including cancer). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products, bi-substrate analogues and the widely used small molecule C646, lack potency or selectivity. Here, we describe A-485, a potent...

  12. Inhibitors of serotonin reuptake and specific imipramine binding in human blood plasma

    International Nuclear Information System (INIS)

    Brusov, O.S.; Fomenko, A.M.; Katasonov, A.B.; Lidemann, R.R.

    1985-01-01

    This paper describes a method of extraction of endogenous inhibitors of specific IMI binding and of 5-HT reuptake, from human blood plasma and the heterogeneity of these compounds is demonstrated. Specific binding was determined as the difference between binding of 3 H-IMI in the absence and in the presence of 50 microM IMI. Under these conditions, specific binding amounted to 70-80% of total binding of 3 H-IMI. It is shown that extract obtained from human blood contains a material which inhibits dose-dependently both 5-HT reuptake and specific binding of 3 H-IMI. Gel-chromatography of extracts of human blood plasma on Biogel P-2 is also shown

  13. Optimizing fluorescently tethered Hsp90 inhibitor dose for maximal specific uptake by breast tumors

    Science.gov (United States)

    Crouch, Brian T.; Duer, Joy; Wang, Roujia; Gallagher, Jennifer; Hall, Allison; Soo, Mary Scott; Hughes, Philip; Haystead, Timothy A. J.; Ramanujam, Nirmala

    2018-03-01

    Despite improvements in surgical resection, 20-40% of patients undergoing breast conserving surgery require at least one additional re-excision. Leveraging the unique surface expression of heat shock protein 90 (Hsp90), a chaperone protein involved in several key hallmarks of cancer, in breast cancer provides an exciting opportunity to identify residual disease during surgery. We developed a completely non-destructive strategy using HS-27, a fluorescently-tethered Hsp90 inhibitor, to assay surface Hsp90 expression on intact tissue specimens using a fluorescence microendoscope with a field of view of 750 μm and subcellular resolution of 4 μm. HS-27 consists of an FDA approved Hsp90 inhibitor tethered to fluorescein isothiocyanate (EX 488nm, EM 525nm). Here, we optimized ex vivo HS-27 administration in pre-clinical breast cancer models and validated our approach on 21 patients undergoing standard of care ultrasound guided core needle biopsy. HS-27 administration time was fixed at 1- minute to minimize imaging impact on clinical workflow. HS-27 and HS-217 (non-specific control) doses were modulated from 1 μM up to 100 μM to identify the dose maximizing the ratio of specific uptake (HS-27 fluorescence) to non-specific uptake (HS-217 fluorescence). The specificity ratio was maximized at 100 μM and was significantly greater than all other doses (pcancer makes this technology attractive for assessing tumor margins, as one agent can be used for all subtypes.

  14. Anti-tumor effects of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase α

    International Nuclear Information System (INIS)

    Maeda, Naoki; Kokai, Yasuo; Ohtani, Seiji; Sahara, Hiroeki; Kuriyama, Isoko; Kamisuki, Shinji; Takahashi, Shunya; Sakaguchi, Kengo; Sugawara, Fumio; Yoshida, Hiromi; Sato, Noriyuki; Mizushina, Yoshiyuki

    2007-01-01

    In the screening of selective inhibitors of eukaryotic DNA polymerases (pols), dehydroaltenusin was found to be an inhibitor of pol α from a fungus (Alternaria tennuis). We succeeded in chemically synthesizing dehydroaltenusin, and the compound inhibited only mammalian pol α with IC 50 value of 0.5 μM, and did not influence the activities of other replicative pols such as pols δ and ε, but also showed no effect on pol α activity from another vertebrate, fish, or from a plant species. Dehydroaltenusin also had no influence on the other pols and DNA metabolic enzymes tested. The compound also inhibited the proliferation of human cancer cells with LD 50 values of 38.0-44.4 μM. In an in vivo anti-tumor assay on nude mice bearing solid tumors of HeLa cells, dehydroaltenusin was shown to be a promising suppressor of solid tumors. Histopathological examination revealed that increased tumor necrosis and decreased mitotic index were apparently detected by the compound in vivo. Therefore, dehydroaltenusin could be of interest as not only a mammalian pol α-specific inhibitor, but also as a candidate drug for anti-cancer treatment

  15. Isolation and characterization of a specific endogenous Na+, K+-ATPase inhibitor from bovine adrenal

    International Nuclear Information System (INIS)

    Tamura, M.; Lam, T.T.; Inagami, T.

    1988-01-01

    In order to identify a specific endogenous Na + ,K + -ATPase inhibitor which could possibly be related to salt-dependent hypertension, the authors looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na + ,K + -ATPase; (ii) competitive displacing activity against [ 3 H]ouabain binding to the enzyme; (iii) inhibitory activity for 86 Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C 18 open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na + ,K + -ATPase. These results strongly suggest that this water-soluble nonpeptidic Na + ,K + -ATPase inhibitor may be a specific endogenous regulator for the ATPase

  16. 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.

  17. Wheat Subtilisin/Chymotrypsin Inhibitor (WSCI) as a scaffold for novel serine protease inhibitors with a given specificity.

    Science.gov (United States)

    Tedeschi, Francesca; Di Maro, Antimo; Facchiano, Angelo; Costantini, Susan; Chambery, Angela; Bruni, Natalia; Capuzzi, Valeria; Ficca, Anna Grazia; Poerio, Elia

    2012-10-30

    WSCI (Wheat Subtilisin/Chymotrypsin Inhibitor) is a small protein belonging to the Potato inhibitor I family exhibiting a high content of essential amino acid. In addition to bacterial subtilisins and mammalian chymotrypsins, WSCI inhibits chymotrypsin-like activities isolated from digestive traits of a number of insect larvae. In vivo, as suggested for many plant proteinase inhibitors, WSCI seems to play a role of natural defence against attacks of pests and pathogens. The functional region of WSCI, containing the inhibitor reactive site (Met48-Glu49), corresponds to an extended flexible loop (Val42-Asp53) whose architecture is somehow stabilized by a number of secondary interactions established with a small β-sheet located underneath. The aim of this study was to employ a WSCI molecule as a stable scaffold to obtain recombinant inhibitors with new acquired anti-proteinase activity or, alternatively, inactive WSCI variants. A gene sequence coding for the native WSCI, along with genes coding for muteins with different specficities, could be exploited to obtain transformed non-food use plants with improved insect resistance. On the other hand, the genetic transformation of cereal plants over-expressing inactive WSCI muteins could represent a possible strategy to improve the nutritional quality of cereal-based foods, without risk of interference with human or animal digestive enzymes. Here, we described the characterization of four muteins containing single/multiple amino acid substitutions at the WSCI reactive site and/or at its proximity. Modalities of interaction of these muteins with proteinases (subtilisin, trypsin and chymotrypsin) were investigated by time course hydrolysis and molecular simulations studies.

  18. Docking Studies and Biological Evaluation of a Potential β-Secretase Inhibitor of 3-Hydroxyhericenone F from Hericium erinaceus

    Directory of Open Access Journals (Sweden)

    Chen Diling

    2017-05-01

    Full Text Available Alzheimer’s disease (AD is the most common neurodegenerative disorder, affecting approximately more than 5% of the population worldwide over the age 65, annually. The incidence of AD is expected to be higher in the next 10 years. AD patients experience poor prognosis and as a consequence new drugs and therapeutic strategies are required in order to improve the clinical responses and outcomes of AD. The purpose of the present study was to screen a certain number of potential compounds from herbal sources and investigate their corresponding mode of action. In the present study, the learning and memory effects of ethanol:water (8:2 extracts from Hericium erinaceus were evaluated on a dementia rat model. The model was established by intraperitoneal injection of 100 mg/kg/d D-galactose in rats. The results indicated that the extracts can significantly ameliorate the learning and memory abilities. Specific active ingredients were screened in vivo assays and the results were combined with molecular docking studies. Potential receptor–ligand interactions on the BACE1-inhibitor namely, 3-Hydroxyhericenone F (3HF were investigated. The isolation of a limited amount of 3HF from the fruit body of H. erinaceus by chemical separation was conducted, and the mode of action of this compound was verified in NaN3-induced PC12 cells. The cell-based assays demonstrated that 3HF can significantly down-regulate the expression of BACE1 (p < 0.01, while additional AD intracellular markers namely, p-Tau and Aβ1-42 were further down-regulated (p < 0.05. The data further indicate that 3HF can ameliorate certain mitochondrial dysfunction conditions by the reversal of the decreasing level of mitochondrial respiratory chain complexes, the calcium ion levels ([Ca2+], the inhibiton in the production of ROS, the increase in the mitochondrial membrane potential and ATP levels, and the regulation of the expression levels of the genes encoding for the p21, COX I, COX II, PARP1

  19. Docking Studies and Biological Evaluation of a Potential β-Secretase Inhibitor of 3-Hydroxyhericenone F from Hericium erinaceus.

    Science.gov (United States)

    Diling, Chen; Tianqiao, Yong; Jian, Yang; Chaoqun, Zheng; Ou, Shuai; Yizhen, Xie

    2017-01-01

    Alzheimer's disease (AD) is the most common neurodegenerative disorder, affecting approximately more than 5% of the population worldwide over the age 65, annually. The incidence of AD is expected to be higher in the next 10 years. AD patients experience poor prognosis and as a consequence new drugs and therapeutic strategies are required in order to improve the clinical responses and outcomes of AD. The purpose of the present study was to screen a certain number of potential compounds from herbal sources and investigate their corresponding mode of action. In the present study, the learning and memory effects of ethanol:water (8:2) extracts from Hericium erinaceus were evaluated on a dementia rat model. The model was established by intraperitoneal injection of 100 mg/kg/d D-galactose in rats. The results indicated that the extracts can significantly ameliorate the learning and memory abilities. Specific active ingredients were screened in vivo assays and the results were combined with molecular docking studies. Potential receptor-ligand interactions on the BACE1-inhibitor namely, 3-Hydroxyhericenone F (3HF) were investigated. The isolation of a limited amount of 3HF from the fruit body of H. erinaceus by chemical separation was conducted, and the mode of action of this compound was verified in NaN 3 -induced PC12 cells. The cell-based assays demonstrated that 3HF can significantly down-regulate the expression of BACE1 ( p < 0.01), while additional AD intracellular markers namely, p-Tau and Aβ 1-42 were further down-regulated ( p < 0.05). The data further indicate that 3HF can ameliorate certain mitochondrial dysfunction conditions by the reversal of the decreasing level of mitochondrial respiratory chain complexes, the calcium ion levels ([Ca 2+ ]), the inhibiton in the production of ROS, the increase in the mitochondrial membrane potential and ATP levels, and the regulation of the expression levels of the genes encoding for the p21, COX I, COX II, PARP1, and NF

  20. CD4-specific designed ankyrin repeat proteins are novel potent HIV entry inhibitors with unique characteristics.

    Directory of Open Access Journals (Sweden)

    Andreas Schweizer

    2008-07-01

    Full Text Available Here, we describe the generation of a novel type of HIV entry inhibitor using the recently developed Designed Ankyrin Repeat Protein (DARPin technology. DARPin proteins specific for human CD4 were selected from a DARPin DNA library using ribosome display. Selected pool members interacted specifically with CD4 and competed with gp120 for binding to CD4. DARPin proteins derived in the initial selection series inhibited HIV in a dose-dependent manner, but showed a relatively high variability in their capacity to block replication of patient isolates on primary CD4 T cells. In consequence, a second series of CD4-specific DARPins with improved affinity for CD4 was generated. These 2nd series DARPins potently inhibit infection of genetically divergent (subtype B and C HIV isolates in the low nanomolar range, independent of coreceptor usage. Importantly, the actions of the CD4 binding DARPins were highly specific: no effect on cell viability or activation, CD4 memory cell function, or interference with CD4-independent virus entry was observed. These novel CD4 targeting molecules described here combine the unique characteristics of DARPins-high physical stability, specificity and low production costs-with the capacity to potently block HIV entry, rendering them promising candidates for microbicide development.

  1. Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor.

    Science.gov (United States)

    Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min

    2015-06-18

    Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits Plasmodium falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report three crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all three structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of ATP. Three residues holding the methyltetrahydropyran moiety of cladosporin are critical for the specificity of cladosporin against LysRS over other class II tRNA synthetase families. The species-exclusive inhibition of PfLysRS is linked to a structural divergence beyond the active site that mounts a lysine-specific stabilizing response to binding cladosporin. These analyses reveal that inherent divergence of tRNA synthetase structural assembly may allow for highly specific inhibition even through the otherwise universal substrate binding pocket and highlight the potential for structure-driven drug development. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Discovery of the 3-Imino-1,2,4-thiadiazinane 1,1-Dioxide Derivative Verubecestat (MK-8931)–A β-Site Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor for the Treatment of Alzheimer’s Disease

    Energy Technology Data Exchange (ETDEWEB)

    Scott, Jack D.; Li, Sarah W.; Brunskill, Andrew P.J.; Chen, Xia; Cox, Kathleen; Cumming, Jared N.; Forman, Mark; Gilbert, Eric J.; Hodgson, Robert A.; Hyde, Lynn A.; Jiang, Qin; Iserloh, Ulrich; Kazakevich, Irina; Kuvelkar, Reshma; Mei, Hong; Meredith, John; Misiaszek, Jeffrey; Orth, Peter; Rossiter, Lana M.; Slater, Meagan; Stone, Julie; Strickland, Corey O.; Voigt, Johannes H.; Wang, Ganfeng; Wang, Hongwu; Wu, Yusheng; Greenlee, William J.; Parker, Eric M.; Kennedy, Matthew E.; Stamford, Andrew W. (Merck)

    2016-12-08

    Verubecestat 3 (MK-8931), a diaryl amide-substituted 3-imino-1,2,4-thiadiazinane 1,1-dioxide derivative, is a high-affinity β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor currently undergoing Phase 3 clinical evaluation for the treatment of mild to moderate and prodromal Alzheimer’s disease. Although not selective over the closely related aspartyl protease BACE2, verubecestat has high selectivity for BACE1 over other key aspartyl proteases, notably cathepsin D, and profoundly lowers CSF and brain Aβ levels in rats and nonhuman primates and CSF Aβ levels in humans. In this annotation, we describe the discovery of 3, including design, validation, and selected SAR around the novel iminothiadiazinane dioxide core as well as aspects of its preclinical and Phase 1 clinical characterization.

  3. Covalent cross-linking of insulin-like growth factor-1 to a specific inhibitor from human serum

    International Nuclear Information System (INIS)

    Ooi, G.T.; Herington, A.C.

    1986-01-01

    Previous studies have shown that a specific inhibitor of insulin-like growth factor (IGF) action in vitro can be isolated from normal human serum and subsequently partially purified on an IGF-affinity column. The ability of the inhibitor to bind the IGFs has now been confirmed directly using covalent cross-linking techniques. When 125 I-IGF-1 was cross-linked to inhibitor using disuccinimidyl suberate, five specifically labelled bands were seen on SDS-PAGE and autoradiography. Two bands (MW 21.5 K and 25.5 K) were intensely labelled, while the remaining three (MW 37 K, 34 K and 18 K) appeared as minor bands only. Inhibitor bioactivity, following further analysis by hydrophobic interaction chromatography or Con A-Sepharose affinity chromatography, was always associated with the presence of the 21.5 K and/or 25.5 K bands

  4. Purification of charybdotoxine, a specific inhibitor of the high-conductance Ca2+-activated K+ channel

    International Nuclear Information System (INIS)

    Smith, C.; Phillips, M.; Miller, C.

    1986-01-01

    Charybdotoxim is a high-affinity specific inhibitor of the high-conductance Ca 2+ -activated K + channel found in the plasma membranes of many vertebrate cell types. Using Ca 2+ -activated K + channels reconstituted into planar lipid bilayer membranes as an assay, the authors have purified the toxin from the venom of the scorpion Leiurus quinquestriatus by a two-step procedure involving chromatofocusing on SP-Sephadex, followed by reversed-phase high-performance liquid chromatography. Charybdotoxin is shown to be a highly basic protein with a mass of 10 kDa. Under the standard assay conditions, the purified toxin inhibits the Ca 2+ -activated K + channel with an apparent dissociation constant of 3.5 nM. The protein is unusually stable, with inhibitory potency being insensitive to boiling or exposure to organic solvents. The toxin's activity is sensitive to chymotrypsin treatment and to acylation of lysine groups. The protein may be radioiodinated without loss of activity

  5. Diphenyleneiodonium, an inhibitor of NOXes and DUOXes, is also an iodide-specific transporter

    Directory of Open Access Journals (Sweden)

    C. Massart

    2014-01-01

    Full Text Available NADPH oxidases (NOXes and dual oxidases (DUOXes generate O2.− and H2O2. Diphenyleneiodonium (DPI inhibits the activity of these enzymes and is often used as a specific inhibitor. It is shown here that DPI, at concentrations similar to those which inhibit the generation of O2 derivatives, activated the efflux of radioiodide but not of its analog 99mTcO4− nor of the K+ cation mimic 86Rb+ in thyroid cells, in the PCCl3 rat thyroid cell line and in COS cell lines expressing the iodide transporter NIS. Effects obtained with DPI, especially in thyroid cells, should therefore be interpreted with caution.

  6. Task-specific ionic liquid as a new green inhibitor of mild steel corrosion

    Energy Technology Data Exchange (ETDEWEB)

    Kowsari, E., E-mail: kowsarie@aut.ac.ir [Department of Chemistry, Amirkabir University of Technology, No. 424, Hafez Avenue, 1591634311 Tehran (Iran, Islamic Republic of); Payami, M. [Department of Chemistry, Amirkabir University of Technology, No. 424, Hafez Avenue, 1591634311 Tehran (Iran, Islamic Republic of); Amini, R.; Ramezanzadeh, B. [Department of Surface Coating and Corrosion, Institute for Color Science and Technology (ICST), PO 16765-654 Tehran (Iran, Islamic Republic of); Javanbakht, M. [Department of Chemistry, Amirkabir University of Technology, No. 424, Hafez Avenue, 1591634311 Tehran (Iran, Islamic Republic of)

    2014-01-15

    The corrosion inhibition effects of an imidazolium-based task specific ionic liquid (TSIL) were investigated on a low carbon steel in 1 M HCl solution. Samples were exposed to 1 M HCl solution without and with different concentrations of TSIL. Weight loss measurements, potentiodynamic polarization test, electrochemical impedance spectroscopy (EIS), scanning electron microscope (SEM), atomic force microscope (AFM) and contact angle measurements were utilized to investigate the inhibition effects of TSIL. The results obtained from the polarization studies revealed that both the anodic and cathodic branches slopes affected in the presence of TSIL. It was shown that TSIL behaved as a mixed type inhibitor with a dominant effect on the anodic reaction rate depression. It was shown that the increase in polarization resistance and the decrease in corrosion current density were more pronounced using 100 mg/L of TSIL after 2 h immersion time. It was also shown that the adsorption of TSIL followed a Langmuir adsorption isotherm.

  7. Identification of brain-specific angiogenesis inhibitor 2 as an interaction partner of glutaminase interacting protein

    International Nuclear Information System (INIS)

    Zencir, Sevil; Ovee, Mohiuddin; Dobson, Melanie J.; Banerjee, Monimoy; Topcu, Zeki; Mohanty, Smita

    2011-01-01

    Highlights: → Brain-specific angiogenesis inhibitor 2 (BAI2) is a new partner protein for GIP. → BAI2 interaction with GIP was revealed by yeast two-hybrid assay. → Binding of BAI2 to GIP was characterized by NMR, CD and fluorescence. → BAI2 and GIP binding was mediated through the C-terminus of BAI2. -- Abstract: The vast majority of physiological processes in living cells are mediated by protein-protein interactions often specified by particular protein sequence motifs. PDZ domains, composed of 80-100 amino acid residues, are an important class of interaction motif. Among the PDZ-containing proteins, glutaminase interacting protein (GIP), also known as Tax Interacting Protein TIP-1, is unique in being composed almost exclusively of a single PDZ domain. GIP has important roles in cellular signaling, protein scaffolding and modulation of tumor growth and interacts with a number of physiological partner proteins, including Glutaminase L, β-Catenin, FAS, HTLV-1 Tax, HPV16 E6, Rhotekin and Kir 2.3. To identify the network of proteins that interact with GIP, a human fetal brain cDNA library was screened using a yeast two-hybrid assay with GIP as bait. We identified brain-specific angiogenesis inhibitor 2 (BAI2), a member of the adhesion-G protein-coupled receptors (GPCRs), as a new partner of GIP. BAI2 is expressed primarily in neurons, further expanding GIP cellular functions. The interaction between GIP and the carboxy-terminus of BAI2 was characterized using fluorescence, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy assays. These biophysical analyses support the interaction identified in the yeast two-hybrid assay. This is the first study reporting BAI2 as an interaction partner of GIP.

  8. Isozyme-specific fluorescent inhibitor of glutathione s-transferase omega 1.

    Science.gov (United States)

    Son, Junghyun; Lee, Jae-Jung; Lee, Jun-Seok; Schüller, Andreas; Chang, Young-Tae

    2010-05-21

    Recently, the glutathione S-transferase omega 1 (GSTO1) is suspected to be involved in certain cancers and neurodegenerative diseases. However, profound investigation on the pathological roles of GSTO1 has been hampered by the lack of specific methods to determine or modulate its activity in biological systems containing other isoforms with similar catalytic function. Here, we report a fluorescent compound that is able to inhibit and monitor the activity of GSTO1. We screened 43 fluorescent chemicals and found a compound (6) that binds specifically to the active site of GSTO1. We observed that compound 6 inhibits GSTO1 by covalent modification but spares other isoforms in HEK293 cells and demonstrated that compound 6 could report the activity of GSTO1 in NIH/3T3 or HEK293 cells by measuring the fluorescence intensity of the labeled amount of GSTO1 in SDS-PAGE. Compound 6 is a useful tool to study GSTO1, applicable as a specific inhibitor and an activity reporter.

  9. Optimal Classes of Chemotherapeutic Agents Sensitized by Specific Small-Molecule Inhibitors of Akt In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Yan Shi

    2005-11-01

    Full Text Available Akt is a serine/threonine kinase that transduces survival signals from survival/growth factors. Deregulation and signal imbalance in cancer cells make them prone to apoptosis. Upregulation or activation of Akt to aid the survival of cancer cells is a common theme in human malignancies. We have developed small-molecule Akt inhibitors that are potent and specific. These Akt inhibitors can inhibit Akt activity and block phosphorylation by Akt on multiple downstream targets in cells. Synergy in apoptosis induction was observed when Akt inhibitors were combined with doxorubicin or camptothecin. Akt inhibitor-induced enhancement of topoisomerase inhibitor cytotoxicity was also evident in long-term cell survival assay. Synergy with paclitaxel in apoptosis induction was evident in cells pretreated with paclitaxel, and enhancement of tumor delay by paclitaxel was demonstrated through cotreatment with Akt inhibitor Compound A (A-443654. Combination with other classes of chemotherapeutic agents did not yield any enhancement of cytotoxicity. These findings provide important guidance in selecting appropriate classes of chemotherapeutic agents for combination with Akt inhibitors in cancer treatment.

  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. Effects of specific inhibitors on anammox and denitrification in marine sediments.

    Science.gov (United States)

    Jensen, Marlene Mark; Thamdrup, Bo; Dalsgaard, Tage

    2007-05-01

    The effects of three metabolic inhibitors (acetylene, methanol, and allylthiourea [ATU]) on the pathways of N2 production were investigated by using short anoxic incubations of marine sediment with a 15N isotope technique. Acetylene inhibited ammonium oxidation through the anammox pathway as the oxidation rate decreased exponentially with increasing acetylene concentration; the rate decay constant was 0.10+/-0.02 microM-1, and there was 95% inhibition at approximately 30 microM. Nitrous oxide reduction, the final step of denitrification, was not sensitive to acetylene concentrations below 10 microM. However, nitrous oxide reduction was inhibited by higher concentrations, and the sensitivity was approximately one-half the sensitivity of anammox (decay constant, 0.049+/-0.004 microM-1; 95% inhibition at approximately 70 microM). Methanol specifically inhibited anammox with a decay constant of 0.79+/-0.12 mM-1, and thus 3 to 4 mM methanol was required for nearly complete inhibition. This level of methanol stimulated denitrification by approximately 50%. ATU did not have marked effects on the rates of anammox and denitrification. The profile of inhibitor effects on anammox agreed with the results of studies of the process in wastewater bioreactors, which confirmed the similarity between the anammox bacteria in bioreactors and natural environments. Acetylene and methanol can be used to separate anammox and denitrification, but the effects of these compounds on nitrification limits their use in studies of these processes in systems where nitrification is an important source of nitrate. The observed differential effects of acetylene and methanol on anammox and denitrification support our current understanding of the two main pathways of N2 production in marine sediments and the use of 15N isotope methods for their quantification.

  12. Investigating PKA-RII specificity using analogs of the PKA:AKAP peptide inhibitor STAD-2.

    Science.gov (United States)

    Bendzunas, N George; Dörfler, Sabrina; Autenrieth, Karolin; Bertinetti, Daniela; Machal, Erik M F; Kennedy, Eileen J; Herberg, Friedrich W

    2018-03-15

    Generation of the second messenger molecule cAMP mediates a variety of cellular responses which are essential for critical cellular processes. In response to elevated cAMP levels, cAMP dependent protein kinase (PKA) phosphorylates serine and threonine residues on a wide variety of target substrates. In order to enhance the precision and directionality of these signaling events, PKA is localized to discrete locations within the cell by A-kinase anchoring proteins (AKAPs). The interaction between PKA and AKAPs is mediated via an amphipathic α-helix derived from AKAPs which binds to a stable hydrophobic groove formed in the dimerization/docking (D/D) domain of PKA-R in an isoform-specific fashion. Although numerous AKAP disruptors have previously been identified that can inhibit either RI- or RII-selective AKAPs, no AKAP disruptors have been identified that have isoform specificity for RIα versus RIβ or RIIα versus RIIβ. As a strategy to identify isoform-specific AKAP inhibitors, a library of chemically stapled protein-protein interaction (PPI) disruptors was developed based on the RII-selective AKAP disruptor, STAD-2. An alanine was substituted at each position in the sequence, and from this library it was possible to delineate the importance of longer aliphatic residues in the formation of a region which complements the hydrophobic cleft formed by the D/D domain. Interestingly, lysine residues that were added to both terminal ends of the peptide sequence to facilitate water solubility appear to contribute to isoform specificity for RIIα over RIIβ while having only weak interaction with RI. This work supports current hypotheses on the mechanisms of AKAP binding and highlights the significance of particular residue positions that aid in distinguishing between the RII isoforms and may provide insight into future design of isoform-selective AKAP disruptors. Copyright © 2018 Elsevier Ltd. All rights reserved.

  13. Effect of specific enzyme inhibitors on replication, total genome DNA repair and on gene-specific DNA repair after UV irradiation in CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Jones, J.C.; Stevsner, Tinna; Bohr, Vilhelm A. (National Cancer Institute, NIH, Bethesda, MD (USA). Division of Cancer Treatment, Laboratory of Molecular Pharmacology); Mattern, M.R. (Smith Kline Beecham Pharmaceuticals, King of Prussia, PA (USA). Department of Biomolecular Discovery)

    1991-09-01

    The effects were studied of some specific enzyme inhibitors on DNA repair and replication after UV damage in Chinese hamster ovary cells. The DNA repair was studied at the level of the average, overall genome and also in the active dihydrofolate reductase gene. Replication was measured in the overall genome. The inhibitors were tested of DNA poly-merase {alpha} and {delta} (aphidicolin), of poly(ADPr) polymerase (3-aminobenzamide), of ribonucleotide reductase (hydroxyurea), of topo-isomerase I (camptothecin), and of topoisomerase II (merbarone, VP-16). In addition, the effects were tested of the potential topoisomerase I activator, {beta}-lapachone. All of these compounds inhibited genome replication and all topoisomerase inhibitors affected the overall genome repair; {beta}-lapachone stimulated it. None of these compounds had any effect on the gene-specific repair. (author). 36 refs.; 3 figs.; 2 tabs.

  14. Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

    Science.gov (United States)

    Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

    2015-01-01

    ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can

  15. A specific and potent inhibitor of glucosylceramide synthase for substrate inhibition therapy of Gaucher disease.

    Science.gov (United States)

    McEachern, Kerry Anne; Fung, John; Komarnitsky, Svetlana; Siegel, Craig S; Chuang, Wei-Lien; Hutto, Elizabeth; Shayman, James A; Grabowski, Gregory A; Aerts, Johannes M F G; Cheng, Seng H; Copeland, Diane P; Marshall, John

    2007-07-01

    An approach to treating Gaucher disease is substrate inhibition therapy which seeks to abate the aberrant lysosomal accumulation of glucosylceramide. We have identified a novel inhibitor of glucosylceramide synthase (Genz-112638) and assessed its activity in a murine model of Gaucher disease (D409V/null). Biochemical characterization of Genz-112638 showed good potency (IC(50) approximately 24nM) and specificity against the target enzyme. Mice that received drug prior to significant accumulation of substrate (10 weeks of age) showed reduced levels of glucosylceramide and number of Gaucher cells in the spleen, lung and liver when compared to age-matched control animals. Treatment of older mice that already displayed significant amounts of tissue glucosylceramide (7 months old) resulted in arrest of further accumulation of the substrate and appearance of additional Gaucher cells in affected organs. These data indicate that substrate inhibition therapy with Genz-112638 represents a viable alternate approach to enzyme therapy to treat the visceral pathology in Gaucher disease.

  16. Evaluation of the specificity of antigen assays for plasminogen activator inhibitor 1 : Comparison of two new commercial kits

    NARCIS (Netherlands)

    Huisman, L.G.M.; Meijer, P.; Griensven, J. van; Kluft, C.

    1992-01-01

    t-PA depleted citrated plasma was used to prepare standards of different molecular forms of plasminogen activator inhibitor 1 (PAI-1). These standards were used to evaluate the specificity of two new PAI-1 antigen assays: the TintElize PAI-1 antigen assay (cat. no. 210221) and the Innotest PAI-1.

  17. Two-dimensional NMR studies of squash family inhibitors. Sequence-specific proton assignments and secondary structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III

    Energy Technology Data Exchange (ETDEWEB)

    Krisnamoorthi, R.; Yuxi Gong; Chanlan Sun Lin (Kansas State Univ., Manhattan (United States)); VanderVelde, D. (Univ. of Kansas, Lawrence (United States))

    1992-01-28

    The solution structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI-III*) was investigated by two-dimensional proton nuclear magnetic resonance (2D NMR) spectroscopy. CMTI-III*, prepared by reacting CMTI-III with trypsin which cleaved the Arg5-Ile6 peptide bond, had the two fragments held together by a disulfide linkage. Sequence-specific {sup 1}H NMR resonance assignments were made for all the 29 amino acid residues of the protein. The secondary structure of CMTI-III*, as deduced from NOESY cross peaks and identification of slowly exchanging hydrogens, contains two turns, a 3{sub 10}-helix, and a triple-stranded {beta}-sheet. Sequential proton assignments were also made for the virgin inhibitor, CMTI-III, at pH 4.71, 30C. Comparison of backbone hydrogen chemical shifts of CMTI-III and CMTI-III* revealed significant changes for residues located far away from the reactive-site region as well as for those located near it, indicating tertiary structural changes that are transmitted through most of the 29 residues of the inhibitor protein. These chemical shift changes were relatively small compared to changes that occurred upon hydrolysis of the reactive-site peptide bond between Arg 5 and Ile6 in CMTI-III.

  18. Two-dimensional NMR studies of squash family inhibitors. Sequence-specific proton assignments and secondary structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III.

    Science.gov (United States)

    Krishnamoorthi, R; Gong, Y X; Lin, C L; VanderVelde, D

    1992-01-28

    The solution structure of reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI-III*) was investigated by two-dimensional proton nuclear magnetic resonance (2D NMR) spectroscopy. CMTI-III*, prepared by reacting CMTI-III with trypsin which cleaved the Arg5-Ile6 peptide bond, had the two fragments held together by a disulfide linkage. Sequence-specific 1H NMR resonance assignments were made for all the 29 amino acid residues of the protein. The secondary structure of CMTI-III*, as deduced from NOESY cross peaks and identification of slowly exchanging hydrogens, contains two turns (residues 8-12 and 24-27), a 3(10)-helix (residues 13-16), and a triple-stranded beta-sheet (residues 8-10, 29-27, and 21-25). This secondary structure is similar to that of CMTI-I [Holak, T. A., Gondol, D., Otlewski, J., & Wilusz, T. (1989) J. Mol. Biol. 210, 635-648], which has a Glu instead of a Lys at position 9. Sequential proton assignments were also made for the virgin inhibitor, CMTI-III, at pH 4.71, 30 degrees C. Comparison of backbone hydrogen chemical shifts of CMTI-III and CMTI-III* revealed significant changes for residues located far away from the reactive-site region as well as for those located near it, indicating tertiary structural changes that are transmitted through most of the 29 residues of the inhibitor protein. Many of these residues are functionally important in that they make contact with atoms of the enzyme in the trypsin-inhibitor complex, as revealed by X-ray crystallography [Bode, W., Greyling, H. J., Huber, R., Otlewski, J., & Wilusz, T. (1989) FEBS Lett. 242, 285-292].(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Oligopeptidase B from Serratia proteamaculans. III. Inhibition analysis. Specific interactions with metalloproteinase inhibitors.

    Science.gov (United States)

    Mikhailova, A G; Khairullin, R F; Kolomijtseva, G Ya; Rumsh, L D

    2012-03-01

    Inhibition of the novel oligopeptidase B from Serratia proteamaculans (PSP) by basic pancreatic trypsin inhibitor, Zn2+ ions, and o- and m-phenanthroline was investigated. A pronounced effect of calcium ions on the interaction of PSP with inhibitors was demonstrated. Inversion voltamperometry and atomic absorption spectrometry revealed no zinc ions in the PSP molecule. Hydrophobic nature of the enzyme inhibition by o- and m-phenanthroline was established.

  20. Intracellular lysyl oxidase: Effect of a specific inhibitor on nuclear mass in proliferating cells

    Energy Technology Data Exchange (ETDEWEB)

    Saad, Fawzy A. [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Torres, Marie [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Wang, Hao [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States); Graham, Lila, E-mail: lilagraham@cs.com [Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedics, Children' s Hospital Boston, 300 Longwood Avenue EN926, Boston, MA 02115 (United States); Harvard Medical School, Boston, MA 02115 (United States)

    2010-06-11

    LOX, the principal enzyme involved in crosslinking of collagen, was the first of several lysyl oxidase isotypes to be characterized. Its active form was believed to be exclusively extracellular. Active LOX was later reported to be present in cell nuclei; its function there is unknown. LOX expression opposes the effect of mutationally activated Ras, which is present in about 30% of human cancers. The mechanism of LOX in countering the action of Ras is also unknown. In the present work, assessment of nuclear protein for possible effects of lysyl oxidase activity led to the discovery that proliferating cells dramatically increase their nuclear protein content when exposed to BAPN ({beta}-aminopropionitrile), a highly specific lysyl oxidase inhibitor that reportedly blocks LOX inhibition of Ras-induced oocyte maturation. In three cell types (PC12 cells, A7r5 smooth muscle cells, and NIH 3T3 fibroblasts), BAPN caused a 1.8-, 1.7-, and 2.1-fold increase in total nuclear protein per cell, respectively, affecting all major components in both nuclear matrix and chromatin fractions. Since nuclear size is correlated with proliferative status, enzyme activity restricting nuclear growth may be involved in the lysyl oxidase tumor suppressive effect. Evidence is also presented for the presence of apparent lysyl oxidase isotype(s) containing a highly conserved LOX active site sequence in the nuclei of PC12 cells, which do not manufacture extracellular lysyl oxidase substrates. Results reported here support the hypothesis that nuclear lysyl oxidase regulates nuclear growth, and thereby modulates cell proliferation.

  1. Changing the inhibitory specificity and function of Cucurbita maxima trypsin inhibitor-V by site-directed mutagenesis.

    Science.gov (United States)

    Wen, L; Lee, I; Chen, G; Huang, J K; Gong, Y; Krishnamoorthi, R

    1995-02-27

    Cucurbita maxima trypsin inhibitor-V (CMTI-V) is also a specific inhibitor of human blood coagulation factor beta-factor XIIa. A recombinant version of CMTI-V has allowed probing of roles of individual amino acid residues including the reactive site residue, lysine (P1), by site-directed mutagenesis. The K44R showed at least a 5-fold increase in inhibitory activity toward human beta-factor XIIa, while there was no change toward bovine trypsin. This result demonstrates that beta-factor-XIIa prefers an arginine residue over lysine residue, while trypsin is non-specific to lysine or arginine in its binding pocket. On the other hand, the specificity of CMTI-V could be changed from trypsin to chymotrypsin inhibition by mutation of the P1 residue to either leucine or methionine (K44L or K44M).

  2. Identification of tyrosinase specific inhibitors from Xanthium strumarium fruit extract using ultrafiltration-high performance liquid chromatography.

    Science.gov (United States)

    Wang, Zhiqiang; Hwang, Seung Hwan; Huang, Bo; Lim, Soon Sung

    2015-10-01

    In this study, a strategy based on ultrafiltration-high performance liquid chromatography coupled with diode array detection (UF-HPLC-DAD) was proposed for screening tyrosinase specific inhibitors in Xanthii fructus. The false negatives were distinguished by optimizing the UF-HPLC-DAD parameters to reduce the background noise; the false positives were distinguished by introducing a blocked tyrosinase in the control group for comparison. To obtain the best blocker, the competitive experiments were performed using various known ligands. Using this strategy, three competitive inhibitors (protocatechuic acid; 3,5-di-O-caffeoylquinic acid; and 1,5-di-O-caffeoylquinic acid) and one mixed-type inhibitor (chlorogenic acid) were identified. These results were verified using tyrosinase inhibition assay, kinetic analysis, and structural simulation of the complex. Our experimental results suggest that the proposed strategy could be useful for high-throughput identification of tyrosinase specific inhibitors in natural products. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. The Structural Basis of Cryptosporidium-Specific IMP Dehydrogenase Inhibitor Selectivity

    Energy Technology Data Exchange (ETDEWEB)

    MacPherson, Iain S.; Kirubakaran, Sivapriya; Gorla, Suresh Kumar; Riera, Thomas V.; D’Aquino, J. Alejandro; Zhang, Minjia; Cuny, Gregory D.; Hedstrom, Lizbeth (BWH); (Brandeis)

    2010-03-29

    Cryptosporidium parvum is a potential biowarfare agent, an important AIDS pathogen, and a major cause of diarrhea and malnutrition. No vaccines or effective drug treatment exist to combat Cryptosporidium infection. This parasite relies on inosine 5{prime}-monophosphate dehydrogenase (IMPDH) to obtain guanine nucleotides, and inhibition of this enzyme blocks parasite proliferation. Here, we report the first crystal structures of CpIMPDH. These structures reveal the structural basis of inhibitor selectivity and suggest a strategy for further optimization. Using this information, we have synthesized low-nanomolar inhibitors that display 10{sup 3} selectivity for the parasite enzyme over human IMPDH2.

  4. Chemical probing of the human sirtuin 5 active site reveals its substrate acyl specificity and peptide-based inhibitors.

    Science.gov (United States)

    Roessler, Claudia; Nowak, Theresa; Pannek, Martin; Gertz, Melanie; Nguyen, Giang T T; Scharfe, Michael; Born, Ilona; Sippl, Wolfgang; Steegborn, Clemens; Schutkowski, Mike

    2014-09-26

    Sirtuins are NAD(+)-dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin 5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide-based inhibitors that interact with the NAD(+) binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X-ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Critical Data-Based Re-Evaluation of Minocycline as a Putative Specific Microglia Inhibitor

    NARCIS (Netherlands)

    Moller, Thomas; Bard, Frederique; Bhattacharya, Anindya; Biber, Knut; Campbell, Brian; Dale, Elena; Eder, Claudia; Gan, Li; Garden, Gwenn A.; Hughes, Zoe A.; Pearse, Damien D.; Staal, Roland G. W.; Sayed, Faten A.; Wes, Paul D.; Boddeke, Hendrikus W. G. M.

    2016-01-01

    Minocycline, a second generation broad-spectrum antibiotic, has been frequently postulated to be a "microglia inhibitor." A considerable number of publications have used minocycline as a tool and concluded, after achieving a pharmacological effect, that the effect must be due to "inhibition" of

  6. Molecular dynamics of conformation-specific dopamine transporter-inhibitor complexes.

    Science.gov (United States)

    Jean, Bernandie; Surratt, Christopher K; Madura, Jeffry D

    2017-09-01

    The recreational psychostimulant cocaine inhibits dopamine reuptake from the synapse, resulting in excessive stimulation of postsynaptic dopamine receptors in brain areas associated with reward and addiction. Cocaine binds to and stabilizes the outward- (extracellular-) facing conformation of the dopamine transporter (DAT) protein, while the low abuse potential DAT inhibitor benztropine prefers the inward- (cytoplasmic-) facing conformation. A correlation has been previously postulated between psychostimulant abuse potential and preference for the outward-facing DAT conformation. The 3β-aryltropane cocaine analogs LX10 and LX11, however, differ only in stereochemistry and share a preference for the outward-facing DAT, yet are reported to vary widely in abuse potential in an animal model. In search of the molecular basis for DAT conformation preference, complexes of cocaine, benztropine, LX10 or LX11 bound to each DAT conformation were subjected to 100ns of all-atom molecular dynamics simulation. Results were consistent with previous findings from cysteine accessibility assays used to assess an inhibitor's DAT conformation preference. The respective 2β- and 2α-substituted phenyltropanes of LX10 and LX11 interacted with hydrophobic regions of the DAT S1 binding site that were inaccessible to cocaine. Solvent accessibility measurements also revealed subtle differences in inhibitor positioning within a given DAT conformation. This work serves to advance our understanding of the conformational selectivity of DAT inhibitors and suggests that MD may be useful in antipsychostimulant therapeutic design. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Fasxiator, a novel factor XIa inhibitor from snake venom, and its site-specific mutagenesis to improve potency and selectivity.

    Science.gov (United States)

    Chen, W; Carvalho, L P D; Chan, M Y; Kini, R M; Kang, T S

    2015-02-01

    Bleeding remains a major limitation of standard anticoagulant drugs that target the extrinsic and common coagulation pathways. Recently, intrinsic coagulation factors are increasingly being investigated as alternative targets for developing anticoagulant drugs with lower bleeding risk. Goals were to (i) identify novel anticoagulants selectively targeting intrinsic coagulation pathway and (ii) characterize and further improve the properties of the identified anticoagulants. We have isolated and sequenced a specific factor XIa (FXIa) inhibitor, henceforth named Fasxiator, from the venom of the banded krait snake, Bungarus fasciatus. It is a Kunitz-type protease inhibitor that prolonged activated partial thromboplastin time without significant effects on prothrombin time. Fasxiator was recombinantly expressed (rFasxiator), purified, and characterized to be a slow-type inhibitor of FXIa that exerts its anticoagulant activities (doubled activated partial thromboplastin time at ~ 3 μmol L(-1) ) by selectively inhibiting human FXIa in in vitro assays. A series of mutants were subsequently generated to improve the potency and selectivity of recombinant rFasxiator. rFasxiatorN17R,L19E showed the best balance between potency (IC50 ~ 1 nmol L(-1) ) and selectivity (> 100 times). rFasxiatorN17R,L19E is a competitive slow-type inhibitor of FXIa (Ki  = 0.86 nmol L(-1) ), possesses anticoagulant activity that is ~ 10 times stronger in human plasma than in murine plasma, and prolonged the occlusion time of mice carotid artery in FeCl3 -induced thrombosis models. We have isolated an exogenous FXIa specific inhibitor, engineered it to improve its potency by ~ 1000 times and demonstrated its in vitro and in vivo efficacy. These proof-of-principle data supported the further development of Fasxiator as a novel anticoagulant candidate. © 2014 International Society on Thrombosis and Haemostasis.

  8. 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.

  9. 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.

  10. [Specific inhibitors of cyclooxygenase-2 (COX-2): current knowledge and perspectives].

    Science.gov (United States)

    Rioda, W T; Nervetti, A

    2001-01-01

    The Authors summarize the current knowledge on a new class of nonsteroidal anti-inflammatory drugs (NSAIDs), the coxib (celecoxib and rofecoxib), in the treatment of rheumatic diseases. Celecoxib and rofecoxib are selective cyclooxygenase-2 (COX-2) inhibitors which possess the same anti-inflammatory and analgesic activities, but a better gastric tolerability compared to the non-selective COX-1 and COX-2 inhibitors. The Authors also report other possible therapeutic effects of these NSADIs as evidenced by the more recent data of the literature. Celecoxib seems to reduce the incidence of new polyps in patients with familial adenomatous polyposis. It has been suggested the use of celecoxib as a protective drug against the development of colorectal cancer. Other (neoplastic) or pre-neoplastic conditions, such as bladder dysplasia, Barret esophagus, attinic keratosis and Alzheimer's disease seem to have benefit from this class of drugs.

  11. Breakdown of the FLT3-ITD/STAT5 axis and synergistic apoptosis induction by the histone deacetylase inhibitor panobinostat and FLT3-specific inhibitors.

    Science.gov (United States)

    Pietschmann, Kristin; Bolck, Hella Anna; Buchwald, Marc; Spielberg, Steffi; Polzer, Harald; Spiekermann, Karsten; Bug, Gesine; Heinzel, Thorsten; Böhmer, Frank-Dietmar; Krämer, Oliver H

    2012-11-01

    Activating mutations of the class III receptor tyrosine kinase FLT3 are the most frequent molecular aberration in acute myeloid leukemia (AML). Mutant FLT3 accelerates proliferation, suppresses apoptosis, and correlates with poor prognosis. Therefore, it is a promising therapeutic target. Here, we show that RNA interference against FLT3 with an internal tandem duplication (FLT3-ITD) potentiates the efficacy of the histone deacetylase inhibitor (HDACi) panobinostat (LBH589) against AML cells expressing FLT3-ITD. Similar to RNA interference, tyrosine kinase inhibitors (TKI; AC220/cpd.102/PKC412) in combination with LBH589 exhibit superior activity against AML cells. Median dose-effect analyses of drug-induced apoptosis rates of AML cells (MV4-11 and MOLM-13) revealed combination index (CI) values indicating strong synergism. AC220, the most potent and FLT3-specific TKI, shows highest synergism with LBH589 in the low nanomolar range. A 4-hour exposure to LBH589 + AC220 already generates more than 50% apoptosis after 24 hours. Different cell lines lacking FLT3-ITD as well as normal peripheral blood mononuclear cells are not significantly affected by LBH589 + TKI, showing the specificity of this treatment regimen. Immunoblot analyses show that LBH589 + TKI induce apoptosis via degradation of FLT3-ITD and its prosurvival target STAT5. Previously, we showed the LBH589-induced proteasomal degradation of FLT3-ITD. Here, we show that activated caspase-3 also contributes to the degradation of FLT3-ITD and that STAT5 is a direct target of this protease. Our data strongly emphasize HDACi/TKI drug combinations as promising modality for the treatment of FLT3-ITD-positive AMLs. ©2012 AACR.

  12. Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology.

    Science.gov (United States)

    Soutter, Holly H; Centrella, Paolo; Clark, Matthew A; Cuozzo, John W; Dumelin, Christoph E; Guie, Marie-Aude; Habeshian, Sevan; Keefe, Anthony D; Kennedy, Kaitlyn M; Sigel, Eric A; Troast, Dawn M; Zhang, Ying; Ferguson, Andrew D; Davies, Gareth; Stead, Eleanor R; Breed, Jason; Madhavapeddi, Prashanti; Read, Jon A

    2016-12-06

    Millions of individuals are infected with and die from tuberculosis (TB) each year, and multidrug-resistant (MDR) strains of TB are increasingly prevalent. As such, there is an urgent need to identify novel drugs to treat TB infections. Current frontline therapies include the drug isoniazid, which inhibits the essential NADH-dependent enoyl-acyl-carrier protein (ACP) reductase, InhA. To inhibit InhA, isoniazid must be activated by the catalase-peroxidase KatG. Isoniazid resistance is linked primarily to mutations in the katG gene. Discovery of InhA inhibitors that do not require KatG activation is crucial to combat MDR TB. Multiple discovery efforts have been made against InhA in recent years. Until recently, despite achieving high potency against the enzyme, these efforts have been thwarted by lack of cellular activity. We describe here the use of DNA-encoded X-Chem (DEX) screening, combined with selection of appropriate physical properties, to identify multiple classes of InhA inhibitors with cell-based activity. The utilization of DEX screening allowed the interrogation of very large compound libraries (10 11 unique small molecules) against multiple forms of the InhA enzyme in a multiplexed format. Comparison of the enriched library members across various screening conditions allowed the identification of cofactor-specific inhibitors of InhA that do not require activation by KatG, many of which had bactericidal activity in cell-based assays.

  13. Cyclic peptide inhibitors of lysine-specific demethylase 1 with improved potency identified by alanine scanning mutagenesis.

    Science.gov (United States)

    Kumarasinghe, Isuru R; Woster, Patrick M

    2018-03-25

    Lysine-specific demethylase 1 (LSD1) is a chromatin-remodeling enzyme that plays an important role in cancer. Over-expression of LSD1 decreases methylation at histone 3 lysine 4, and aberrantly silences tumor suppressor genes. Inhibitors of LSD1 have been designed as chemical probes and potential antitumor agents. We recently reported the cyclic peptide 9, which potently and reversibly inhibits LSD1 (IC 50 2.1 μM; K i 385 nM). Systematic alanine mutagenesis of 9 revealed residues that are critical for LSD1 inhibition, and these mutated peptides were evaluated as LSD1 inhibitors. Alanine substitution at positions 2, 3, 4, 6 and 11-17 preserved inhibition, while substitution of alanine at positions 8 and 9 resulted in complete loss of activity. Cyclic mutant peptides 11 and 16 produced the greatest LSD1 inhibition, and 11, 16, 27 and 28 increased global H3K4me2 in K562 cells. In addition, 16, 27 and 28 promoted significant increases in H3K4me2 levels at the promoter sites of the genes IGFBP2 and FEZ1. Data from these LSD1 inhibitors will aid in the design of peptidomimetics with improved stability and pharmacokinetics. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  14. Isolation and characterization of a specific endogenous Na/sup +/, K/sup +/-ATPase inhibitor from bovine adrenal

    Energy Technology Data Exchange (ETDEWEB)

    Tamura, M.; Lam, T.T.; Inagami, T.

    1988-06-14

    In order to identify a specific endogenous Na/sup +/,K/sup +/-ATPase inhibitor which could possibly be related to salt-dependent hypertension, the authors looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na/sup +/,K/sup +/-ATPase; (ii) competitive displacing activity against (/sup 3/H)ouabain binding to the enzyme; (iii) inhibitory activity for /sup 86/Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C/sub 18/ open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na/sup +/,K/sup +/-ATPase. These results strongly suggest that this water-soluble nonpeptidic Na/sup +/,K/sup +/-ATPase inhibitor may be a specific endogenous regulator for the ATPase.

  15. In silico simulations of STAT1 and STAT3 inhibitors predict SH2 domain cross-binding specificity.

    Science.gov (United States)

    Szelag, Malgorzata; Sikorski, Krzysztof; Czerwoniec, Anna; Szatkowska, Katarzyna; Wesoly, Joanna; Bluyssen, Hans A R

    2013-11-15

    Signal transducers and activators of transcription (STATs) comprise a family of transcription factors that are structurally related and which participate in signaling pathways activated by cytokines, growth factors and pathogens. Activation of STAT proteins is mediated by the highly conserved Src homology 2 (SH2) domain, which interacts with phosphotyrosine motifs for specific contacts between STATs and receptors and for STAT dimerization. By generating new models for human (h)STAT1, hSTAT2 and hSTAT3 we applied comparative in silico docking to determine SH2-binding specificity of the STAT3 inhibitor stattic, and of fludarabine (STAT1 inhibitor). Thus, we provide evidence that by primarily targeting the highly conserved phosphotyrosine (pY+0) SH2 binding pocket stattic is not a specific hSTAT3 inhibitor, but is equally effective towards hSTAT1 and hSTAT2. This was confirmed in Human Micro-vascular Endothelial Cells (HMECs) in vitro, in which stattic inhibited interferon-α-induced phosphorylation of all three STATs. Likewise, fludarabine inhibits both hSTAT1 and hSTAT3 phosphorylation, but not hSTAT2, by competing with the highly conserved pY+0 and pY-X binding sites, which are less well-preserved in hSTAT2. Moreover we observed that in HMECs in vitro fludarabine inhibits cytokine and lipopolysaccharide-induced phosphorylation of hSTAT1 and hSTAT3 but does not affect hSTAT2. Finally, multiple sequence alignment of STAT-SH2 domain sequences confirmed high conservation between hSTAT1 and hSTAT3, but not hSTAT2, with respect to stattic and fludarabine binding sites. Together our data offer a molecular basis that explains STAT cross-binding specificity of stattic and fludarabine, thereby questioning the present selection strategies of SH2 domain-based competitive small inhibitors. © 2013 Elsevier B.V. All rights reserved.

  16. Discovery of Specific Inhibitors for Intestinal E. coli  β-Glucuronidase through In Silico Virtual Screening

    Directory of Open Access Journals (Sweden)

    Ta-Chun Cheng

    2015-01-01

    Full Text Available Glucuronidation is a major metabolism process of detoxification for carcinogens, 4-(methylnitrosamino-1-(3-pyridy-1-butanone (NNK and 1,2-dimethylhydrazine (DMH, of reactive oxygen species (ROS. However, intestinal E. coli   β-glucuronidase (eβG has been considered pivotal to colorectal carcinogenesis. Specific inhibition of eβG may prevent reactivating the glucuronide-carcinogen and protect the intestine from ROS-mediated carcinogenesis. In order to develop specific eβG inhibitors, we found that 59 candidate compounds obtained from the initial virtual screening had high inhibition specificity against eβG but not human βG. In particular, we found that compounds 7145 and 4041 with naphthalenylidene-benzenesulfonamide (NYBS are highly effective and selective to inhibit eβG activity. Compound 4041  (IC50=2.8 μM shows a higher inhibiting ability than compound 7145  (IC50=31.6 μM against eβG. Furthermore, the molecular docking analysis indicates that compound 4041 has two hydrophobic contacts to residues L361 and I363 in the bacterial loop, but 7145 has one contact to L361. Only compound 4041 can bind to key residue (E413 at active site of eβG via hydrogen-bonding interactions. These novel NYBS-based eβG specific inhibitors may provide as novel candidate compounds, which specifically inhibit eβG to reduce eβG-based carcinogenesis and intestinal injury.

  17. Novel β-amyloid aggregation inhibitors possessing a turn mimic.

    Science.gov (United States)

    Hamada, Yoshio; Miyamoto, Naoko; Kiso, Yoshiaki

    2015-04-01

    Amyloid β peptide, the main component of senile plaques found in the brain of Alzheimer disease (AD) patients, is a molecular target for AD therapeutic intervention. A number of potential AD therapeutics have been reported, including inhibitors of β-secretase, γ-secretase, and Aβ aggregation, and anti-amyloid agents, such as neprilysin, insulin degrading enzyme (IDE), and Aβ antibodies. Recently, we reported potent small-sized β-secretase (BACE1) inhibitors, which could serve as anti-AD drugs. However AD is a progressive disorder, where dementia symptoms gradually worsen over several decades, and therefore may require many years to get cured. One possible way to achieve a greater therapeutic effect is through simultaneous administration of multiple drugs, similar to those used in Highly Active Anti-Retroviral Therapy (HAART) used to treat AIDS. In order to overcome AD, we took a drug discovery approach to evaluate, novel β-amyloid aggregation inhibitors. Previously, we reported that a tong-type compound possessing a turn mimic as the inhibitor of HIV-1 protease dimerization. Oligomerized amyloid β peptides contain a turn structure within the molecule. Here, we designed and synthesized novel β-amyloid aggregation inhibitors with a turn-mimic template, based on the turn conformer of the oligomerized amyloid β peptides. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Use of a specific MMP inhibitor (Galardin) for preservation of hybrid layer

    Science.gov (United States)

    Breschi, Lorenzo; Martin, Patrizia; Mazzoni, Annalisa; Nato, Fernando; Carrilho, Marcela; Tjäderhane, Leo; Visintini, Erika; Cadenaro, Milena; Tay, Franklin R; De Stefano Dorigo, Elettra; Pashley, David H

    2013-01-01

    Objective Dentinal MMPs have been claimed to contribute to the auto-degradation of collagen fibrils within incompletely resin-infiltrated hybrid layers and their inhibition may, therefore, slow the degradation of hybrid layer. This study aimed to determine the contribution of a synthetic MMPs inhibitor (Galardin) to the proteolytic activity of dentinal MMPs and to the morphological and mechanical features of hybrid layers after aging. Methods Dentin powder obtained from human molars was treated with Galardin or chlorhexidine digluconate and zymographically analyzed. Microtensile bond strength was also evaluated in extracted human teeth. Exposed dentin was etched with 35% phosphoric acid and specimens were assigned to (1) pre-treatment with Galardin as additional primer for 30s; (2) no pre-treatment. A two-step etch-and-rinse adhesive (Adper Scotchbond 1XT, 3M ESPE) was then applied in accordance with manufacturer's instructions and resin composite build-ups were created. Specimens were immediately tested for their microtensile bond strength or stored in artificial saliva for 12 months prior to being tested. Data were evaluated by two-way ANOVA and Tukey's tests (〈=0.05). Additional specimens were prepared for interfacial nanoleakage analysis under light microscopy and TEM, quantified by two independent observers and statistically analyzed (|2 test, 〈=0.05). Results The inhibitory effect of Galardin on dentinal MMPs was confirmed by zymographic analysis, as complete inhibition of both MMP-2 and -9 was observed. The use of Galardin had no effect on immediate bond strength, while it significantly decreased bond degradation after 1 year (padhesive after artificial aging. PMID:20299089

  19. HAEdb: a novel interactive, locus-specific mutation database for the C1 inhibitor gene.

    Science.gov (United States)

    Kalmár, Lajos; Hegedüs, Tamás; Farkas, Henriette; Nagy, Melinda; Tordai, Attila

    2005-01-01

    Hereditary angioneurotic edema (HAE) is an autosomal dominant disorder characterized by episodic local subcutaneous and submucosal edema and is caused by the deficiency of the activated C1 esterase inhibitor protein (C1-INH or C1INH; approved gene symbol SERPING1). Published C1-INH mutations are represented in large universal databases (e.g., OMIM, HGMD), but these databases update their data rather infrequently, they are not interactive, and they do not allow searches according to different criteria. The HAEdb, a C1-INH gene mutation database (http://hae.biomembrane.hu) was created to contribute to the following expectations: 1) help the comprehensive collection of information on genetic alterations of the C1-INH gene; 2) create a database in which data can be searched and compared according to several flexible criteria; and 3) provide additional help in new mutation identification. The website uses MySQL, an open-source, multithreaded, relational database management system. The user-friendly graphical interface was written in the PHP web programming language. The website consists of two main parts, the freely browsable search function, and the password-protected data deposition function. Mutations of the C1-INH gene are divided in two parts: gross mutations involving DNA fragments >1 kb, and micro mutations encompassing all non-gross mutations. Several attributes (e.g., affected exon, molecular consequence, family history) are collected for each mutation in a standardized form. This database may facilitate future comprehensive analyses of C1-INH mutations and also provide regular help for molecular diagnostic testing of HAE patients in different centers.

  20. Baicalin and scutellarin are proteasome inhibitors that specifically target chymotrypsin-like catalytic activity.

    Science.gov (United States)

    Wu, Yi-Xin; Sato, Eiji; Kimura, Wataru; Miura, Naoyuki

    2013-09-01

    Baicalin and scutellarin are the major active principal flavonoids extracted from the Chinese herbal medicines Scutellaria baicalensis and Erigeron breviscapus (Vant.) Hand-Mazz. It has recently been reported that baicalin and scutellarin have antitumor activity. However, the mechanisms of action are unknown. We previously reported that some flavonoids have a specific role in the inhibition of the activity of proteasome subunits and induced apoptosis in tumor cells. To further investigate these pharmacological effects, we examined the inhibitory activity of baicalin and scutellarin on the extracted proteasomes from mice and cancer cells. Using fluorogenic substrates for proteasome catalytic subunits, we found that baicalin and scutellarin specifically inhibited chymotrypsin-like activity but did not inhibit trypsin-like and peptidyl-glutamyl peptide hydrolyzing activities. These data suggested that baicalin and scutellarin specifically inhibit chymotrypsin-like catalytic activity in the proteasome. Copyright © 2012 John Wiley & Sons, Ltd.

  1. Strategies to overcome HBV-specific T cell exhaustion: checkpoint inhibitors and metabolic re-programming.

    Science.gov (United States)

    Fisicaro, Paola; Boni, Carolina; Barili, Valeria; Laccabue, Diletta; Ferrari, Carlo

    2018-01-29

    HBV-specific T cells play a key role in antiviral protection and failure to control HBV is associated with severely dysfunctional T cell responses. Therefore, functional T cell reconstitution represents a potential way to treat chronically infected patients. The growing understanding of the dysregulated transcriptional/epigenetic and metabolic programs underlying T cell exhaustion allows to envisage functional T cell reconstitution strategies based on the combined/sequential use of compounds able to induce decline of antigen load, checkpoint modulation, metabolic and epigenetic reprogramming with possible boosting of functionally restored responses by specific vaccines. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Caracemide, a site-specific irreversible inhibitor of protein R1 of Escherichia coli ribonucleotide reductase

    DEFF Research Database (Denmark)

    Larsen, I. K.; Cornett, Claus; Karlsson, M.

    1992-01-01

    The anticancer drug caracemide, N-acetyl-N,O-di(methylcarbamoyl)hydroxylamine, and one of its degradation products, N-acetyl-O-methylcarbamoyl-hydroxylamine, were found to inhibit the enzyme ribonucleotide reductase of Escherichia coli by specific interaction with its larger component protein R1....

  3. A new generation of topical chronic wound treatments containing specific MMP inhibitors

    Directory of Open Access Journals (Sweden)

    Shrivastava R

    2014-09-01

    Full Text Available Ravi Shrivastava, Nathalie Cucuat, Monika Rousse, Thomas Weigand, Pedro Neto, Claire Janicot, Christiane Shrivastava VITROBIO Research Institute, Issoire, France Purpose: Incidence of chronic wounds is constantly rising worldwide, but all currently available treatments are intended either to provide symptomatic relief or to assist cicatrization to some extent, but not to directly stimulate cellular growth. Physiologically, chronic wound healing simply requires cell growth to fill the injured cavity. To grow, our cells need to attach onto a cushion, called extracellular matrix (ECM, secreted by the mother cells and composed of multiple proteins. Recent scientific works prove that the concentration of certain matrix metalloproteinases (MMPs is extremely high in all chronic wounds and, because of their proteolytic nature, some MMPs completely degrade the ECM, hindering cell attachment and cell growth. The aim of this study was to identify, neutralize, and eliminate these MMPs from the wound surface so as to design an effective treatment for chronic wounds. Methods: Acute and chronic models of human epithelial and fibroblast cells were prepared on a defined ECM cushion in vitro and MMPs were added in the culture medium to identify the MMPs causing ECM disintegration for each cell type. ECM-degrading MMPs were then incubated with selected procyanidin-rich plant extracts (PCDs and cell growth was reanalyzed. Results: It was observed that: 1 multiple MMPs are involved in cellular matrix destruction; 2 ECM-destroying MMPs are specific with respect to cell type; and 3 specific PCDs may bind and neutralize selected MMPs. Conclusion: Topical application of specific plant PCDs to selectively neutralize ECM-destroying MMPs in acute and chronic wounds represents a novel approach for the treatment of superficial and deep skin wounds. Keywords: extra cellular matrix (ECM, matrix metalloproteinases, procyanidins (PCDs, tannins, ulcers

  4. Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases

    Directory of Open Access Journals (Sweden)

    Daniel Decker

    2017-09-01

    Full Text Available UDP-sugars are essential precursors for glycosylation reactions producing cell wall polysaccharides, sucrose, glycoproteins, glycolipids, etc. Primary mechanisms of UDP sugar formation involve the action of at least three distinct pyrophosphorylases using UTP and sugar-1-P as substrates. Here, substrate specificities of barley and Arabidopsis (two isozymes UDP-glucose pyrophosphorylases (UGPase, Arabidopsis UDP-sugar pyrophosphorylase (USPase and Arabidopsis UDP-N-acetyl glucosamine pyrophosphorylase2 (UAGPase2 were investigated using a range of sugar-1-phosphates and nucleoside-triphosphates as substrates. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Fru-1-P and Fru-2-P (Km values over 10 mM. Contrary to an earlier report, their activity with Gal-1-P was extremely low. USPase reacted with a range of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P (Km of 1.3 mM, β-L-Ara-1-P and α-D-Fuc-1-P (Km of 3.4 mM, but not β-L-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P (Km of 1 mM and, to some extent, D-Glc-1-P (Km of 3.2 mM. Generally, different conformations/substituents at C2, C4, and C5 of the pyranose ring of a sugar were crucial determinants of substrate specificity of a given pyrophosphorylase. Homology models of UDP-sugar binding to UGPase, USPase and UAGPase2 revealed more common amino acids for UDP binding than for sugar binding, reflecting differences in substrate specificity of these proteins. UAGPase2 was inhibited by a salicylate derivative that was earlier shown to affect UGPase and USPase activities, consistent with a common structural architecture of the three pyrophosphorylases. The results are discussed with respect to the role of the pyrophosphorylases in sugar activation for glycosylated end-products.

  5. Seed-Specific Stable Expression of the ?-AI1 Inhibitor in Coffee Grains and the In Vivo Implications for the Development of the Coffee Berry Borer

    OpenAIRE

    Albuquerque, ?rika V. S.; Bezerra, Caroline A.; Romero, Juan V.; Valencia, Jorge W. A.; Valencia-Jim?nez, Arnubio; Pimenta, Lucas M.; Barbosa, Aulus E. A. D.; Silva, Maria C. M.; Meneguim, Ana M.; S?, Maria Eug?nia L.; Engler, Gilbert; de Almeida-Engler, Janice; Fernandez, Diana; Grossi-de-S?, Maria F.

    2015-01-01

    Genetic transformation of coffee (Coffea spp.), the second most traded commodity worldwide, is an alternative approach to introducing features that cannot be introgressed by traditional crossings. The transgenic stability, heritability and quantitative and spatial expression patterns of the seed-specific promoter phytohemagglutinin (PHA-L) from Phaseolus vulgaris were characterized in genetically modified C. arabica expressing the ?-amylase inhibitor-1 (?-AI1) gene. The ?-AI1 inhibitor shows ...

  6. 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.

  7. Enzyme immunoassay for measurement of murine plasminogen activator inhibitor-1, employing a specific antibody produced by the DNA vaccine method.

    Science.gov (United States)

    Yamada, Takayuki; Takagi, Akira; Takeshita, Kyosuke; Yamamoto, Koji; Ito, Masafumi; Matsushita, Tadashi; Murate, Takashi; Saito, Hidehiko; Kojima, Tetsuhito

    2003-01-01

    We developed a sensitive immunoassay to determine the concentration of mouse plasminogen activator inhibitor-1. The assay was a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) based on the production of a specific polyclonal antibody against mouse plasminogen activator inhibitor type-1 (PAI-1) used both as a trapping and detecting antibody. This antibody was raised in a rabbit by direct introduction of the expression vector plasmid DNA encoding mouse PAI-1, instead of conventional immunization with the purified protein. The standard curve was constructed with a recombinant glutathione S-transferase (GST)-mouse PAI-1 fusion protein (GST-mPAI-1) and dose-response of the assay was linear for GST-mPAI-1 between 6.25 and 100 pM. In order to assess the consistency of the assay, we measured PAI-1 antigen in normal mouse pooled plasma several times. We found that the intra-assay and inter-assay coefficients of variation (CV) were 4.8% and 9.2%, respectively, indicating that the ELISA would be sufficiently repeatable and reproducible. In this assay, lipopolysaccharide (LPS)-injected mice showed substantially higher levels (22-fold) of plasma PAI-1 antigen than did control mice (12.5+/-2.4 vs. 0.58+/-0.16 nM), similar to results reported elsewhere. Taken together, the DNA vaccine method is extremely useful for preparing specific antibodies against mouse PAI-1, which can be utilized to establish the ELISA and analyze the profile of PAI-1 distributions in mice under various conditions. This approach might also be useful for immunological investigation of other coagulation factors and related proteins.

  8. 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.

  9. Peptide aptamers: The versatile role of specific protein function inhibitors in plant biotechnology.

    Science.gov (United States)

    Colombo, Monica; Mizzotti, Chiara; Masiero, Simona; Kater, Martin M; Pesaresi, Paolo

    2015-11-01

    In recent years, peptide aptamers have emerged as novel molecular tools that have attracted the attention of researchers in various fields of basic and applied science, ranging from medicine to analytical chemistry. These artificial short peptides are able to specifically bind, track, and inhibit a given target molecule with high affinity, even molecules with poor immunogenicity or high toxicity, and represent a remarkable alternative to antibodies in many different applications. Their use is on the rise, driven mainly by the medical and pharmaceutical sector. Here we discuss the enormous potential of peptide aptamers in both basic and applied aspects of plant biotechnology and food safety. The different peptide aptamer selection methods available both in vivo and in vitro are introduced, and the most important possible applications in plant biotechnology are illustrated. In particular, we discuss the generation of broad-based virus resistance in crops, "reverse genetics" and aptasensors in bioassays for detecting contaminations in food and feed. Furthermore, we suggest an alternative to the transfer of peptide aptamers into plant cells via genetic transformation, based on the use of cell-penetrating peptides that overcome the limits imposed by both crop transformation and Genetically Modified Organism commercialization. © 2015 Institute of Botany, Chinese Academy of Sciences.

  10. Tofogliflozin, a potent and highly specific sodium/glucose cotransporter 2 inhibitor, improves glycemic control in diabetic rats and mice.

    Science.gov (United States)

    Suzuki, Masayuki; Honda, Kiyofumi; Fukazawa, Masanori; Ozawa, Kazuharu; Hagita, Hitoshi; Kawai, Takahiro; Takeda, Minako; Yata, Tatsuo; Kawai, Mio; Fukuzawa, Taku; Kobayashi, Takamitsu; Sato, Tsutomu; Kawabe, Yoshiki; Ikeda, Sachiya

    2012-06-01

    Sodium/glucose cotransporter 2 (SGLT2) is the predominant mediator of renal glucose reabsorption and is an emerging molecular target for the treatment of diabetes. We identified a novel potent and selective SGLT2 inhibitor, tofogliflozin (CSG452), and examined its efficacy and pharmacological properties as an antidiabetic drug. Tofogliflozin competitively inhibited SGLT2 in cells overexpressing SGLT2, and K(i) values for human, rat, and mouse SGLT2 inhibition were 2.9, 14.9, and 6.4 nM, respectively. The selectivity of tofogliflozin toward human SGLT2 versus human SGLT1, SGLT6, and sodium/myo-inositol transporter 1 was the highest among the tested SGLT2 inhibitors under clinical development. Furthermore, no interaction with tofogliflozin was observed in any of a battery of tests examining glucose-related physiological processes, such as glucose uptake, glucose oxidation, glycogen synthesis, hepatic glucose production, glucose-stimulated insulin secretion, and glucosidase reactions. A single oral gavage of tofogliflozin increased renal glucose clearance and lowered the blood glucose level in Zucker diabetic fatty rats. Tofogliflozin also improved postprandial glucose excursion in a meal tolerance test with GK rats. In db/db mice, 4-week tofogliflozin treatment reduced glycated hemoglobin and improved glucose tolerance in the oral glucose tolerance test 4 days after the final administration. No blood glucose reduction was observed in normoglycemic SD rats treated with tofogliflozin. These findings demonstrate that tofogliflozin inhibits SGLT2 in a specific manner, lowers blood glucose levels by increasing renal glucose clearance, and improves pathological conditions of type 2 diabetes with a low hypoglycemic potential.

  11. Regulation of the number of cell division rounds by tissue-specific transcription factors and Cdk inhibitor during ascidian embryogenesis.

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    Mami Kuwajima

    Full Text Available Mechanisms that regulate the number of cell division rounds during embryogenesis have remained largely elusive. To investigate this issue, we used the ascidian, which develops into a tadpole larva with a small number of cells. The embryonic cells divide 11.45 times on average from fertilization to hatching. The number of cell division rounds varies depending on embryonic lineages. Notochord and muscle consist of large postmitotic cells and stop dividing early in developing embryos. Here we show that conversion of mesenchyme to muscle cell fates by inhibition of inductive FGF signaling or mis-expression of a muscle-specific key transcription factor for muscle differentiation, Tbx6, changed the number of cell divisions in accordance with the altered fate. Tbx6 likely activates a putative mechanism to halt cell division at a specific stage. However, precocious expression of Tbx6 has no effect on progression of the developmental clock itself. Zygotic expression of a cyclin-dependent kinase inhibitor, CKI-b, is initiated in muscle and then in notochord precursors. CKI-b is possibly downstream of tissue-specific key transcription factors of notochord and muscle. In the two distinct muscle lineages, postmitotic muscle cells are generated after 9 and 8 rounds of cell division depending on lineage, but the final cell divisions occur at a similar developmental stage. CKI-b gene expression starts simultaneously in both muscle lineages at the 110-cell stage, suggesting that CKI-b protein accumulation halts cell division at a similar stage. The difference in the number of cell divisions would be due to the cumulative difference in cell cycle length. These results suggest that muscle cells do not count the number of cell division rounds, and that accumulation of CKI-b protein triggered by tissue-specific key transcription factors after cell fate determination might act as a kind of timer that measures elapsed time before cell division termination.

  12. High resolution crystal structure of rat long chain hydroxy acid oxidase in complex with the inhibitor 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1, 2, 3-thiadiazole. Implications for inhibitor specificity and drug design

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zhi-wei; Vignaud, Caroline; Jaafar, Adil; Lévy, Bernard; Guéritte, Françoise; Guénard, Daniel; Lederer, Florence; Mathews, F. Scott (CNRS-UMR); (WU-MED)

    2012-05-24

    Long chain hydroxy acid oxidase (LCHAO) is responsible for the formation of methylguanidine, a toxic compound with elevated serum levels in patients with chronic renal failure. Its isozyme glycolate oxidase (GOX), has a role in the formation of oxalate, which can lead to pathological deposits of calcium oxalate, in particular in the disease primary hyperoxaluria. Inhibitors of these two enzymes may have therapeutic value. These enzymes are the only human members of the family of FMN-dependent L-2-hydroxy acid-oxidizing enzymes, with yeast flavocytochrome b{sub 2} (Fcb2) among its well studied members. We screened a chemical library for inhibitors, using in parallel rat LCHAO, human GOX and the Fcb2 flavodehydrogenase domain (FDH). Among the hits was an inhibitor, CCPST, with an IC{sub 50} in the micromolar range for all three enzymes. We report here the crystal structure of a complex between this compound and LCHAO at 1.3 {angstrom} resolution. In comparison with a lower resolution structure of this enzyme, binding of the inhibitor induces a conformational change in part of the TIM barrel loop 4, as well as protonation of the active site histidine. The CCPST interactions are compared with those it forms with human GOX and those formed by two other inhibitors with human GOX and spinach GOX. These compounds differ from CCPST in having the sulfur replaced with a nitrogen in the five-membered ring as well as different hydrophobic substituents. The possible reason for the {approx}100-fold difference in affinity between these two series of inhibitors is discussed. The present results indicate that specificity is an issue in the quest for therapeutic inhibitors of either LCHAO or GOX, but they may give leads for this quest.

  13. The role of secretory leukocyte proteinase inhibitor and elafin (elastase-specific inhibitor/skin-derived antileukoprotease as alarm antiproteinases in inflammatory lung disease

    Directory of Open Access Journals (Sweden)

    Sallenave Jean-Michel

    2000-08-01

    Full Text Available Abstract Secretory leukocyte proteinase inhibitor and elafin are two low-molecular-mass elastase inhibitors that are mainly synthesized locally at mucosal sites. It is thought that their physicochemical properties allow them to efficiently inhibit target enzymes, such as neutrophil elastase, released into the interstitium. Historically, in the lung, these inhibitors were first purified from secretions of patients with chronic obstructive pulmonary disease and cystic fibrosis. This suggested that they might be important in controlling excessive neutrophil elastase release in these pathologies. They are upregulated by 'alarm signals' such as bacterial lipopolysaccharides, and cytokines such as interleukin-1 and tumor necrosis factor and have been shown to be active against Gram-positive and Gram-negative bacteria, so that they have joined the growing list of antimicrobial 'defensin-like' peptides produced by the lung. Their site of synthesis and presumed functions make them very attractive candidates as potential therapeutic agents under conditions in which the excessive release of elastase by neutrophils might be detrimental. Because of its natural tropism for the lung, the use of adenovirus-mediated gene transfer is extremely promising in such applications.

  14. Effect of catalase-specific inhibitor 3-amino-1,2,4-triazole on yeast peroxisomal catalase in vivo.

    Science.gov (United States)

    Ueda, Mitsuyoshi; Kinoshita, Hiroshi; Yoshida, Tomoko; Kamasawa, Naomi; Osumi, Masako; Tanaka, Atsuo

    2003-02-14

    3-Amino-1,2,4-triazole (3-AT) is known as an inhibitor of catalase to whose active center it specifically and covalently binds. Subcellular fractionation and immunoelectronmicroscopic observation of the yeast Candida tropicalis revealed that, in 3-AT-treated cells in which the 3-AT was added to the n-alkane medium from the beginning of cultivation, catalase transported into peroxisomes was inactivated and was present as insoluble aggregated forms in the organelle. The aggregation of catalase in peroxisomes occurred only in these 3-AT-treated cells and not in cells in which 3-AT was added at the late exponential growth phase. Furthermore, 3-AT did not affect the transportation of catalase into peroxisomes. The appearance of aggregation only in cells to which 3-AT was added from the beginning of cultivation suggests that, in the process of catalase transportation into yeast peroxisomes, some conformational change may take place and that correct folding may be inhibited by the binding of 3-AT to the active center of catalase. Accordingly, 3-AT will be an interesting compound for investigation of the transport machinery of the peroxisomal tetrameric catalase.

  15. Placenta-specific novel splice variants of Rho GDP dissociation inhibitor β are highly expressed in cancerous cells

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    Hatakeyama Keiichi

    2012-12-01

    Full Text Available Abstract Background Alternative splicing of pre-mRNA transcripts not only plays a role in normal molecular processes but is also associated with cancer development. While normal transcripts are ubiquitously expressed in normal tissues, splice variants created through abnormal alternative splicing events are often expressed in cancer cells. Although the Rho GDP dissociation inhibitor β (ARHGDIB gene has been found to be ubiquitously expressed in normal tissues and involved in cancer development, the presence of splice variants of ARHGDIB has not yet been investigated. Results Validation analysis for the presence of and exon structures of splice variants of ARHGDIB, performed using reverse-transcriptase polymerase chain reaction and DNA sequencing, successfully identified novel splice variants of ARHGDIB, that is, 6a, 6b, and 6c, in colon, pancreas, stomach, and breast cancer cell lines. Quantitative real-time polymerase chain reaction analysis showed that these variants were also highly expressed in normal placental tissue but not in other types of normal tissue. Conclusions Expression of ARHGDIB variants 6a, 6b, and 6c appears to be restricted to cancer cells and normal placental tissue, suggesting that these variants possess cancer-specific functions and, as such, are potential cancer-related biomarkers.

  16. Proton Pump Inhibitors and the Risk for Fracture at Specific Sites: Data Mining of the FDA Adverse Event Reporting System.

    Science.gov (United States)

    Wang, Liwei; Li, Mei; Cao, Yuying; Han, Zhengqi; Wang, Xueju; Atkinson, Elizabeth J; Liu, Hongfang; Amin, Shreyasee

    2017-07-17

    Proton pump inhibitors (PPIs) are widely used to treat gastric acid-related disorders. Concerns have been raised about potential fracture risk, especially at the hip, spine and wrist. However, fracture risk at other bone sites has not been as well studied. We investigated the association between PPIs and specific fracture sites using an aggregated knowledge-enhanced database, the Food and Drug Administration Adverse Event Reporting System Data Mining Set (AERS-DM). Proportional reporting ratio (PRR) was used to detect statistically significant associations (signals) between PPIs and fractures. We analyzed both high level terms (HLT) and preferred terms (PT) for fracture sites, defined by MedDRA (Medical Dictionary for Regulatory Activities). Of PPI users reporting fractures, the mean age was 65.3 years and the female to male ratio was 3.4:1. Results revealed signals at multiple HLT and PT fracture sites, consistent for both sexes. These included fracture sites with predominant trabecular bone, not previously reported as being associated with PPIs, such as 'rib fractures', where signals were detected for overall PPIs as well as for each of 5 generic ingredients (insufficient data for dexlansoprazole). Based on data mining from AERS-DM, PPI use appears to be associated with an increased risk for fractures at multiple sites.

  17. Ca2+-dependent proteolytic activity in crab claw muscle: effects of inhibitors and specificity for myofibrillar proteins

    International Nuclear Information System (INIS)

    Mykles, D.L.; Skinner, D.M.

    1983-01-01

    The claw closer muscle of the Bermuda land crab, Gecarcinus lateralis, undergoes a sequential atrophy and restoration during each molting cycle. The role of Ca 2+ -dependent proteinases in the turn-over of myofibrillar protein in normal anecdysial (intermolt) claw muscle is described. Crab Ca 2+ -dependent proteinase degrades the myofibrillar proteins actin, myosin heavy and light chains, paramyosin, tropomyosin, and troponin-T and -I. Ca 2+ -dependent proteinase activity in whole homogenates and 90,000 x g supernatant fractions from muscle homogenates has been characterized with respect to Ca 2+ requirement, substrate specificity, and effects of proteinase inhibitors. The enzyme is inhibited by antipain, leupeptin, E-64, and iodoacetamide; it is insensitive to pepstatin A. The specificity of crab Ca 2+ -dependent proteinase was examined with native myosin with normal ATPase activity as well as with radioiodinated myosin and radioiodinated hemolymph proteins. Hydrolysis of 125 I-myosin occurs in two phases, both Ca 2+ -dependent: (1) heavy chain (M/sub r/ = 200,000) is cleaved into four large fragments (M/sub r/ = 160,000, 110,000, 73,000, 60,000) and numerous smaller fragments; light chain (M/sub r/ = 18,000) is cleaved to a 15,000-Da fragment; (2) the fragments produced in the first phase are hydrolyzed to acid-soluble material. Although radioiodinated native hemolymph proteins are not susceptible to the Ca 2+ -dependent proteinase, those denatured by carboxymethylation are degraded. These data suggest that crab Ca 2+ -dependent proteinase is involved in turnover of myofibrillar protein in normal muscle and muscle undergoing proecdysial atrophy

  18. CD8+ T Cells Specific to Apoptosis-Associated Antigens Predict the Response to Tumor Necrosis Factor Inhibitor Therapy in Rheumatoid Arthritis.

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    Alessandra Citro

    Full Text Available CD8+ T cells specific to caspase-cleaved antigens derived from apoptotic T cells (apoptotic epitopes represent a principal player in chronic immune activation, which is known to amplify immunopathology in various inflammatory diseases. The purpose of the present study was to investigate the relationship involving these autoreactive T cells, the rheumatoid arthritis immunopathology, and the response to tumor necrosis factor-α inhibitor therapy. The frequency of autoreactive CD8+ T cells specific to various apoptotic epitopes, as detected by both enzyme-linked immunospot assay and dextramers of major histocompatibility complex class I molecules complexed with relevant apoptotic epitopes, was longitudinally analyzed in the peripheral blood of rheumatoid arthritis patients who were submitted to etanercept treatment (or other tumor necrosis factor inhibitors as a control. The percentage of apoptotic epitope-specific CD8+ T cells was significantly higher in rheumatoid arthritis patients than in healthy donors, and correlated with the disease activity. More important, it was significantly more elevated in responders to tumor necrosis factor-α inhibitor therapy than in non-responders before the start of therapy; it significantly dropped only in the former following therapy. These data indicate that apoptotic epitope-specific CD8+ T cells may be involved in rheumatoid arthritis immunopathology through the production of inflammatory cytokines and that they may potentially represent a predictive biomarker of response to tumor necrosis factor-α inhibitor therapy to validate in a larger cohort of patients.

  19. Pharmacokinetics of gene recombined angiogenesis inhibitor Kringle 5 in vivo using 131I specific markers and SPECT/CT

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    Ge Yan

    2016-10-01

    Full Text Available The previous pharmacokinetic methods can be only limited to drug analysis in vitro, which provide less information on the distribution and metabolismof drugs, and limit the interpretation and assessment of pharmacokinetics, the determination of metabolic principles, and evaluation of treatment effect. The objective of the study was to investigate the pharmacokinetic characteristics of gene recombination angiogenesis inhibitor Kringle 5 in vivo. The SPECT/CT and specific 131I-Kringle 5 marked by Iodogen method were both applied to explore the pharmacokinetic characteristics of 131I-Kringle 5 in vivo, and to investigate the dynamic distributions of 131I-Kringle 5 in target organs. Labeling recombinant angiogenesis inhibitor Kringle 5 using 131I with longer half-life and imaging in vivo using SPECT instead of PET, could overcome the limitations of previous methods. When the doses of 131I-Kringle 5 were 10.0, 7.5 and 5.0 g/kg, respectively, the two-compartment open models can be determined within all the metabolic process in vivo. There were no significant differences in t1/2α, t1/2β, apparent volume of distribution and CL between those three levels. The ratio of AUC(0~∞ among three different groups of 10.0, 7.5 and 5.0 g/kg was 2.56:1.44:1.0, which was close to the ratio (2:1.5:1.0. It could be clear that in the range of 5.0–10.0 g/kg, Kringle 5 was characterized by the first-order pharmacokinetics. Approximately 30 min after 131I-Kringle 5 was injected, 131I-Kringle 5 could be observed to concentrate in the heart, kidneys, liver and other organs by means of planar imaging and tomography. After 1 h of being injected, more radionuclide retained in the bladder, but not in intestinal. It could be concluded that 131I-Kringle 5 is mainly excreted through the kidneys. About 2 h after the injection of 131I-Kringle 5, the radionuclide in the heart, kidneys, liver and other organs was gradually reduced, while more radionuclide was concentrated

  20. 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.

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    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

  1. 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

  2. IMD-4690, a novel specific inhibitor for plasminogen activator inhibitor-1, reduces allergic airway remodeling in a mouse model of chronic asthma via regulating angiogenesis and remodeling-related mediators.

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    Toshifumi Tezuka

    Full Text Available Plasminogen activator inhibitor (PAI-1 is the principal inhibitor of plasminogen activators, and is responsible for the degradation of fibrin and extracellular matrix. IMD-4690 is a newly synthesized inhibitor for PAI-1, whereas the effect on allergic airway inflammation and remodeling is still unclear. We examined the in vivo effects by using a chronic allergen exposure model of bronchial asthma in mice. The model was generated by an immune challenge for 8 weeks with house dust mite antigen, Dermatophagoides pteronyssinus (Dp. IMD-4690 was intraperitoneally administered during the challenge. Lung histopathology, hyperresponsiveness and the concentrations of mediators in lung homogenates were analyzed. The amount of active PAI-1 in the lungs was increased in mice treated with Dp. Administration with IMD-4690 reduced an active/total PAI-1 ratio. IMD-4690 also reduced the number of bronchial eosinophils in accordance with the decreased expressions of Th2 cytokines in the lung homogenates. Airway remodeling was inhibited by reducing subepithelial collagen deposition, smooth muscle hypertrophy, and angiogenesis. The effects of IMD-4690 were partly mediated by the regulation of TGF-β, HGF and matrix metalloproteinase. These results suggest that PAI-1 plays crucial roles in airway inflammation and remodeling, and IMD-4690, a specific PAI-1 inhibitor, may have therapeutic potential for patients with refractory asthma due to airway remodeling.

  3. IMD-4690, a novel specific inhibitor for plasminogen activator inhibitor-1, reduces allergic airway remodeling in a mouse model of chronic asthma via regulating angiogenesis and remodeling-related mediators.

    Science.gov (United States)

    Tezuka, Toshifumi; Ogawa, Hirohisa; Azuma, Masahiko; Goto, Hisatsugu; Uehara, Hisanori; Aono, Yoshinori; Hanibuchi, Masaki; Yamaguchi, Yoichi; Fujikawa, Tomoyuki; Itai, Akiko; Nishioka, Yasuhiko

    2015-01-01

    Plasminogen activator inhibitor (PAI)-1 is the principal inhibitor of plasminogen activators, and is responsible for the degradation of fibrin and extracellular matrix. IMD-4690 is a newly synthesized inhibitor for PAI-1, whereas the effect on allergic airway inflammation and remodeling is still unclear. We examined the in vivo effects by using a chronic allergen exposure model of bronchial asthma in mice. The model was generated by an immune challenge for 8 weeks with house dust mite antigen, Dermatophagoides pteronyssinus (Dp). IMD-4690 was intraperitoneally administered during the challenge. Lung histopathology, hyperresponsiveness and the concentrations of mediators in lung homogenates were analyzed. The amount of active PAI-1 in the lungs was increased in mice treated with Dp. Administration with IMD-4690 reduced an active/total PAI-1 ratio. IMD-4690 also reduced the number of bronchial eosinophils in accordance with the decreased expressions of Th2 cytokines in the lung homogenates. Airway remodeling was inhibited by reducing subepithelial collagen deposition, smooth muscle hypertrophy, and angiogenesis. The effects of IMD-4690 were partly mediated by the regulation of TGF-β, HGF and matrix metalloproteinase. These results suggest that PAI-1 plays crucial roles in airway inflammation and remodeling, and IMD-4690, a specific PAI-1 inhibitor, may have therapeutic potential for patients with refractory asthma due to airway remodeling.

  4. Tissue- and agonist-specific regulation of human and murine plasminogen activator inhibitor-1 promoters in transgenic mice.

    Science.gov (United States)

    Eren, M; Painter, C A; Gleaves, L A; Schoenhard, J A; Atkinson, J B; Brown, N J; Vaughan, D E

    2003-11-01

    Numerous studies have described regulatory factors and sequences that control transcriptional responses in vitro. However, there is a paucity of information on the qualitative and quantitative regulation of heterologous promoters using transgenic strategies. In order to investigate the physiological regulation of human plasminogen activator inhibitor type-1 (hPAI-1) expression in vivo compared to murine PAI-1 (mPAI-1) and to test the physiological relevance of regulatory mechanisms described in vitro, we generated transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the proximal -2.9 kb of the hPAI-1 promoter. Transgenic animals were treated with Ang II, TGF-beta1 and lipopolysaccharide (LPS) to compare the relative activation of the human and murine PAI-1 promoters. Ang II increased EGFP expression most effectively in brain, kidney and spleen, while mPAI-1 expression was quantitatively enhanced most prominently in heart and spleen. TGF-beta1 failed to induce activation of the hPAI-1 promoter but potently stimulated mPAI-1 in kidney and spleen. LPS administration triggered robust expression of mPAI-1 in liver, kidney, pancreas, spleen and lung, while EGFP was induced only modestly in heart and kidney. These results indicate that the transcriptional response of the endogenous mPAI-1 promoter varies widely in terms of location and magnitude of response to specific stimuli. Moreover, the physiological regulation of PAI-1 expression likely involves a complex interaction of transcription factors and DNA sequences that are not adequately replicated by in vitro functional studies focused on the proximal -2.9 kb promoter.

  5. RAGE-Specific Inhibitor FPS-ZM1 Attenuates AGEs-Induced Neuroinflammation and Oxidative Stress in Rat Primary Microglia.

    Science.gov (United States)

    Shen, Chao; Ma, Yingjuan; Zeng, Ziling; Yin, Qingqing; Hong, Yan; Hou, Xunyao; Liu, Xueping

    2017-10-01

    Advanced glycation end products (AGEs) enhance microglial activation and intensify the inflammatory response and oxidative stress in the brain. This process may occur due to direct cytotoxicity or interacting with AGEs receptors (RAGE), which are expressed on the surface of microglia. FPS-ZM1 is a high-affinity but nontoxic RAGE-specific inhibitor that has been recently shown to attenuate the Aβ-induced inflammatory response by blocking the ligation of Aβ to RAGE. In this study, we further investigated the effect of FPS-ZM1 on the AGEs/RAGE interaction and downstream elevation of neuroinflammation and oxidative stress in primary microglia cells. The results suggested that FPS-ZM1 significantly suppressed AGEs-induced RAGE overexpression, RAGE-dependent microglial activation, nuclear translocation of nuclear factor kappaB p65 (NF-κB p65), and the expression of downstream inflammatory mediators such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) and inducible nitric oxide synthase (iNOS)/nitric oxide (NO). Furthermore, FPS-ZM1 attenuated AGEs-stimulated NADPH oxidase (NOX) activation and reactive oxygen species (ROS) expression. Finally, FPS-ZM1 elevated the levels of transcription factors nuclear-factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1), as well as decreased antioxidant capacity and increased production of oxidative species. Our results suggest that FPS-ZM1 may be neuroprotective through attenuating microglial activation, oxidative stress and inflammation by blocking RAGE.

  6. Prostate-specific membrane antigen targeted imaging and therapy of prostate cancer using a PSMA inhibitor as a homing ligand.

    Science.gov (United States)

    Kularatne, Sumith A; Wang, Kevin; Santhapuram, Hari-Krishna R; Low, Philip S

    2009-01-01

    Prostate cancer (PCa) is a major cause of mortality and morbidity in Western society today. Current methods for detecting PCa are limited, leaving most early malignancies undiagnosed and sites of metastasis in advanced disease undetected. Major deficiencies also exist in the treatment of PCa, especially metastatic disease. In an effort to improve both detection and therapy of PCa, we have developed a PSMA-targeted ligand that delivers attached imaging and therapeutic agents selectively to PCa cells without targeting normal cells. The PSMA-targeted radioimaging agent (DUPA-(99m)Tc) was found to bind PSMA-positive human PCa cells (LNCaP cell line) with nanomolar affinity (K(D) = 14 nM). Imaging and biodistribution studies revealed that DUPA-(99m)Tc localizes primarily to LNCaP cell tumor xenografts in nu/nu mice (% injected dose/gram = 11.3 at 4 h postinjection; tumor-to-muscle ratio = 75:1). Two PSMA-targeted optical imaging agents (DUPA-FITC and DUPA-rhodamine B) were also shown to efficiently label PCa cells and to internalize and traffic to intracellular endosomes. A PSMA-targeted chemotherapeutic agent (DUPA-TubH) was demonstrated to kill PSMA-positive LNCaP cells in culture (IC(50) = 3 nM) and to eliminate established tumor xenografts in nu/nu mice with no detectable weight loss. Blockade of tumor targeting upon administration of excess PSMA inhibitor (PMPA) and the absence of targeting to PSMA-negative tumors confirmed the specificity of each of the above targeted reagents for PSMA. Tandem use of the imaging and therapeutic agents targeted to the same receptor could allow detection, staging, monitoring, and treatment of PCa with improved accuracy and efficacy.

  7. Analysis of binding properties and specificity through identification of the interface forming residues (IFR for serine proteases in silico docked to different inhibitors

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    da Silveira Carlos H

    2010-10-01

    Full Text Available Abstract Background Enzymes belonging to the same super family of proteins in general operate on variety of substrates and are inhibited by wide selection of inhibitors. In this work our main objective was to expand the scope of studies that consider only the catalytic and binding pocket amino acids while analyzing enzyme specificity and instead, include a wider category which we have named the Interface Forming Residues (IFR. We were motivated to identify those amino acids with decreased accessibility to solvent after docking of different types of inhibitors to sub classes of serine proteases and then create a table (matrix of all amino acid positions at the interface as well as their respective occupancies. Our goal is to establish a platform for analysis of the relationship between IFR characteristics and binding properties/specificity for bi-molecular complexes. Results We propose a novel method for describing binding properties and delineating serine proteases specificity by compiling an exhaustive table of interface forming residues (IFR for serine proteases and their inhibitors. Currently, the Protein Data Bank (PDB does not contain all the data that our analysis would require. Therefore, an in silico approach was designed for building corresponding complexes The IFRs are obtained by "rigid body docking" among 70 structurally aligned, sequence wise non-redundant, serine protease structures with 3 inhibitors: bovine pancreatic trypsin inhibitor (BPTI, ecotine and ovomucoid third domain inhibitor. The table (matrix of all amino acid positions at the interface and their respective occupancy is created. We also developed a new computational protocol for predicting IFRs for those complexes which were not deciphered experimentally so far, achieving accuracy of at least 0.97. Conclusions The serine proteases interfaces prefer polar (including glycine residues (with some exceptions. Charged residues were found to be uniquely prevalent at the

  8. Analysis of binding properties and specificity through identification of the interface forming residues (IFR) for serine proteases in silico docked to different inhibitors.

    Science.gov (United States)

    Ribeiro, Cristina; Togawa, Roberto C; Neshich, Izabella A P; Mazoni, Ivan; Mancini, Adauto L; Minardi, Raquel C de Melo; da Silveira, Carlos H; Jardine, José G; Santoro, Marcelo M; Neshich, Goran

    2010-10-20

    Enzymes belonging to the same super family of proteins in general operate on variety of substrates and are inhibited by wide selection of inhibitors. In this work our main objective was to expand the scope of studies that consider only the catalytic and binding pocket amino acids while analyzing enzyme specificity and instead, include a wider category which we have named the Interface Forming Residues (IFR). We were motivated to identify those amino acids with decreased accessibility to solvent after docking of different types of inhibitors to sub classes of serine proteases and then create a table (matrix) of all amino acid positions at the interface as well as their respective occupancies. Our goal is to establish a platform for analysis of the relationship between IFR characteristics and binding properties/specificity for bi-molecular complexes. We propose a novel method for describing binding properties and delineating serine proteases specificity by compiling an exhaustive table of interface forming residues (IFR) for serine proteases and their inhibitors. Currently, the Protein Data Bank (PDB) does not contain all the data that our analysis would require. Therefore, an in silico approach was designed for building corresponding complexes. The IFRs are obtained by "rigid body docking" among 70 structurally aligned, sequence wise non-redundant, serine protease structures with 3 inhibitors: bovine pancreatic trypsin inhibitor (BPTI), ecotine and ovomucoid third domain inhibitor. The table (matrix) of all amino acid positions at the interface and their respective occupancy is created. We also developed a new computational protocol for predicting IFRs for those complexes which were not deciphered experimentally so far, achieving accuracy of at least 0.97. The serine proteases interfaces prefer polar (including glycine) residues (with some exceptions). Charged residues were found to be uniquely prevalent at the interfaces between the "miscellaneous-virus" subfamily

  9. Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor

    Directory of Open Access Journals (Sweden)

    Kala Mrinalini

    2005-12-01

    Full Text Available Abstract Background The isozymes of alkaline phosphatase, the tissue non-specific, intestinal and placental, have similar properties and a high degree of identity. The placental isozyme (PLAP is an oncofetal antigen expressed in several malignancies including choriocarcinoma, seminoma and ovarian carcinoma. We had earlier attempted to isolate PLAP-specific scFv from a synthetic human immunoglobulin library but were unable to do so, presumably because of the similarity between the isozymes. In this work, we have employed a PLAP-specific uncompetitive inhibitor, L-Phe-Gly-Gly, to select isozyme specific scFvs. An uncompetitive inhibitor binds to the enzyme in the presence of substrate and stabilizes the enzyme-substrate complex. Several uncompetitive inhibitors have varying degrees of isozyme specificity for human alkaline phosphatase isozymes. A specific uncompetitive inhibitor would be able to unmask conformational differences between the otherwise very similar molecules. Also, such inhibitors would be directed to regions at/close to the active site of the enzyme. In this work, the library was first incubated with PLAP and the bound clones then eluted by incubation with L-Phe-Gly-Gly along with the substrate, para-nitro phenyl phosphate (pNPP. The scFvs were then studied with regard to the biochemical modulation of their binding, isozyme specificity and effect on enzyme activity. Results Of 13 clones studied initially, the binding of 9 was inhibited by L-Phe-Gly-Gly (with pNPP and 2 clones were inhibited by pNPP alone. Two clones had absolute and 2 clones had partial specificity to PLAP. Two clones were cross-reactive with only one other isozyme. Three scFv clones, having an accessible His6-tag, were purified and studied for their modulation of enzyme activity. All the three scFvs inhibited PLAP activity with the kinetics of competitive inhibition. Cell ELISA could demonstrate binding of the specific scFvs to the cell surface expressed PLAP

  10. Thymoquinone-rich fraction nanoemulsion (TQRFNE) decreases Aβ40 and Aβ42 levels by modulating APP processing, up-regulating IDE and LRP1, and down-regulating BACE1 and RAGE in response to high fat/cholesterol diet-induced rats.

    Science.gov (United States)

    Ismail, Norsharina; Ismail, Maznah; Azmi, Nur Hanisah; Bakar, Muhammad Firdaus Abu; Yida, Zhang; Abdullah, Maizaton Atmadini; Basri, Hamidon

    2017-11-01

    Though the causes of Alzheimer's disease (AD) are yet to be understood, much evidence has suggested that excessive amyloid-β (Aβ) accumulation due to abnormal amyloid-β precursor protein (APP) processing and Aβ metabolism are crucial processes towards AD pathogenesis. Hence, approaches aiming at APP processing and Aβ metabolism are currently being actively pursued for the management of AD. Studies suggest that high cholesterol and a high fat diet have harmful effects on cognitive function and may instigate the commencement of AD pathogenesis. Despite the neuropharmacological attributes of black cumin seed (Nigella sativa) extracts and its main active compound, thymoquinone (TQ), limited records are available in relation to AD research. Nanoemulsion (NE) is exploited as drug delivery systems due to their capacity of solubilising non-polar active compounds and is widely examined for brain targeting. Herewith, the effects of thymoquinone-rich fraction nanoemulsion (TQRFNE), thymoquinone nanoemulsion (TQNE) and their counterparts' conventional emulsion in response to high fat/cholesterol diet (HFCD)-induced rats were investigated. Particularly, the Aβ generation; APP processing, β-secretase 1 (BACE1), γ-secretases of presenilin 1 (PSEN1) and presenilin 2 (PSEN2), Aβ degradation; insulin degrading enzyme (IDE), Aβ transportation; low density lipoprotein receptor-related protein 1 (LRP1) and receptor for advanced glycation end products (RAGE) were measured in brain tissues. TQRFNE reduced the brain Aβ fragment length 1-40 and 1-42 (Aβ40 and Aβ42) levels, which would attenuate the AD pathogenesis. This reduction could be due to the modulation of β- and γ-secretase enzyme activity, and the Aβ degradation and transportation in/out of the brain. The findings show the mechanistic actions of TQRFNE in response to high fat and high cholesterol diet associated to Aβ generation, degradation and transportation in the rat's brain tissue. Copyright © 2017 Elsevier

  11. 99mTc-labeling and evaluation of a HYNIC modified small-molecular inhibitor of prostate-specific membrane antigen

    International Nuclear Information System (INIS)

    Xu, Xiaoping; Zhang, Jianping; Hu, Silong; He, Simin; Bao, Xiao; Ma, Guang; Luo, Jianmin; Cheng, Jingyi; Zhang, Yingjian

    2017-01-01

    Introduction: Prostate-specific membrane antigen (PSMA) is a well-established target in the development of radiopharmaceuticals for the diagnosis and therapy of prostate cancer (PCa). In this study, we evaluated a novel 99m Tc-labeled small molecular inhibitor of PSMA. Methods: This new small-molecular inhibitor of PSMA, 6-hydrazinonicotinate-Aminocaproic acid-Lysine-Urea-Glutamate (HYNIC-ALUG) was radiolabeled by 99m Tc and was evaluated both in vitro and in vivo using PCa models (PC-3 and LNCaP). Radiation dosimetry was assessed in mice. Results: 99m Tc-HYNIC-ALUG showed excellent stability in different media. A cell assay preliminarily displayed its specificity for PSMA. The inhibitor showed good pharmacokinetics making it suitable for in vivo imaging. PC-3-derived tumors showed no obvious radioactive uptake; however, the LNCaP-derived tumors showed very high radioactive uptake which was significantly decreased by the selective PSMA inhibitor 2-PMPA. Biodistribution in LNCaP xenografts showed an optimum tumor-to-blood ratio of 24.23 ± 3.54 at 2 h. Tumor uptake was also decreased in the inhibition experiment with 2-PMPA (19.45 ± 2.14%ID/g versus 1.42 ± 0.15%ID/g at 2 h). The effective dose of the 99m Tc-HYNIC-ALUG was 8.4E-04 mSv/MBq. Conclusions: A new 99m Tc-labeled PSMA inhibitor with specific accumulation in PSMA-positive tumors and low background in other organs was synthesized. The radiopharmaceutical also showed very low radiation dosimetry. This agent may significantly improve the diagnosis, staging, and subsequent monitoring of therapeutic effects in PCa patients.

  12. Subtype-specific suppression of Shiga toxin 2 released from Escherichia coli upon exposure to protein synthesis inhibitors

    DEFF Research Database (Denmark)

    Pedersen, Malene Gantzhorn; Hansen, Claus; Riise, Erik

    2008-01-01

    Shiga toxins (Stx) are important virulence factors in the pathogenesis of severe disease including hemolytic-uremic syndrome, caused by Stx-producing Escherichia coli (STEC). STEC strains increase the release of Stx in vitro following the addition of fluoroquinolones, whereas protein synthesis...... inhibitors previously have been reported to suppress the release of Stx. The amount of Stx released from wild-type STEC strains incubated with protein synthesis inhibitors was examined by a Vero cell cytotoxicity assay. The amounts released were compared to the Stx type (Stx1 or Stx2) and additionally...... to the individual subtypes and toxin variants of Stx2. In general, Stx2 release was suppressed significantly upon exposure to protein synthesis inhibitors at MICs, which was not observed in the case of Stx1. Also, the average amount of different Stx2 toxin variants released was suppressed to various levels ranging...

  13. Substrate and Inhibitor-Specific Conformational Changes in the Human Serotonin Transporter Revealed by Voltage-Clamp Fluorometry

    DEFF Research Database (Denmark)

    Söderhielm, Pella C; Andersen, Jacob; Munro, Lachlan

    2015-01-01

    of TM6, Ala419 in the interface between TM8 and extracellular loop (EL) 4, and Leu481 in EL5. The reporter positions were used for time-resolved measurement of conformational changes during 5-HT transport and binding of cocaine and the selective serotonin reuptake inhibitors fluoxetine and escitalopram...... changes overall, which included movements within or around TM1b, EL4, and EL5. Taken together, our data lead us to suggest that competitive inhibitors stabilize hSERT in a state that is different from the apo outward-open conformation as well as inward-facing conformations....

  14. Isozyme-specific enzyme inhibitors. 14. 5'(R)-C-[(L-homocystein-S-yl)methyl]adenosine 5'-(beta,gamma-imidotriphosphate), a potent inhibitor of rat methionine adenosyltransferases.

    Science.gov (United States)

    Kappler, F; Vrudhula, V M; Hampton, A

    1987-09-01

    The title compound is a covalent adduct of L-methionine (Met) and beta,gamma-imido-ATP. In its synthesis the N-Boc derivative of 5'(R)-C-(aminomethyl)-N6-benzoyl-5'-O-tosyl-2',3'-O- isopropylidenadenosine was converted by the successive actions of CF3CO2H and HNO2 into the corresponding 5'(R)-C-hydroxymethyl derivative. Treatment of this with disodium L-homocysteinate led to attack of sulfur at C6', apparently via a 5',6'-epoxide, and to total stereoselective inversion at C5' to furnish, after debenzoylation, 5'(R)-C-(L-homocystein-S-ylmethyl)-2',3'-O-isopropylidene ade nosine. The 5' configuration was established by conversion of this into the known 5'(S)-C-methyl-2',3'-O-isopropylidene adenosine with Raney nickel. The alpha-amino acid residue was protected as an N-Boc methyl ester, after which the 5'-hydroxyl was phosphorylated with benzyl phosphate and dicyclohexylcarbodiimide. The phosphoanhydride bond with inorganic imidodiphosphate was then created by established methods. Finally, blocking groups were removed under conditions that gave the desired adduct with no racemization of its L-methionine residue. It was a potent inhibitor [KM(ATP)/Ki = 1080; KM(Met)/Ki = 7.7] of the M-2 (normal tissue) form of rat methionine adenosyltransferase and of the M-T (hepatoma tissue) form [KM(ATP)/Ki = 670; KM(Met)/Ki = 22]. Inhibitions were competitive with respect to ATP or to L-methionine, indicating a dual substrate site mode of binding to the enzyme forms.

  15. Inhibitors of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) exert a strong anti-herpesviral activity.

    Science.gov (United States)

    Hutterer, Corina; Milbradt, Jens; Hamilton, Stuart; Zaja, Mirko; Leban, Johann; Henry, Christophe; Vitt, Daniel; Steingruber, Mirjam; Sonntag, Eric; Zeitträger, Isabel; Bahsi, Hanife; Stamminger, Thomas; Rawlinson, William; Strobl, Stefan; Marschall, Manfred

    2017-07-01

    Infection with human cytomegalovirus (HCMV) is a serious medical problem, particularly in immunocompromised individuals and neonates. The success of (val)ganciclovir therapy is hampered by low drug compatibility and induction of viral resistance. A novel strategy of antiviral treatment is based on the exploitation of cell-directed signaling, e. g. pathways with a known relevance for carcinogenesis and tumor drug development. Here we describe a principle for putative antiviral drugs based on targeting dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). DYRKs constitute an evolutionarily conserved family of protein kinases with key roles in the control of cell proliferation and differentiation. Members of the DYRK family are capable of phosphorylating a number of substrate proteins, including regulators of the cell cycle, e.g. DYRK1B can induce cell cycle arrest, a critical step for the regulation of HCMV replication. Here we provide first evidence for a critical role of DYRKs during viral replication and the high antiviral potential of DYRK inhibitors (SC84227, SC97202 and SC97208, Harmine and AZ-191). Using established replication assays for laboratory and clinically relevant strains of HCMV, concentration-dependent profiles of inhibition were obtained. Mean inhibitory concentrations (EC50) of 0.98 ± 0.08 μM/SC84227, 0.60 ± 0.02 μM/SC97202, 6.26 ± 1.64 μM/SC97208, 0.71 ± 0.019 μM/Harmine and 0.63 ± 0.23 μM/AZ-191 were determined with HCMV strain AD169-GFP for the infection of primary human fibroblasts. A first analysis of the mode of antiviral action suggested a block of viral replication at the early-late stage of HCMV gene expression. Moreover, rhesus macaque cytomegalovirus (RhCMV), varicella-zoster virus (VZV) and herpes simplex virus (HSV-1) showed a similarly high sensitivity to these compounds. Thus, we conclude that DYRK signaling represents a promising target pathway for the development of novel anti

  16. 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.

  17. Seed-specific stable expression of the alpha-AI1 inhibitor in coffee grains and the in vivo implications for the development of the coffee berry borer

    OpenAIRE

    Albuquerque, E. V. S.; Bezerra, C. A.; Romero, J. V.; Valencia, J. W. A.; Valencia-Jimenez, A.; Pimenta, L. M.; Barbosa, Aead; Silva, M. C. M.; Meneguim, A. M.; Sa, M. E. L.; Engler, G.; de Almeida-Engler, J.; Fernandez, Diana; Grossi-de-Sa, M. F.

    2015-01-01

    Genetic transformation of coffee (Coffea spp.), the second most traded commodity worldwide, is an alternative approach to introducing features that cannot be introgressed by traditional crossings. The transgenic stability, heritability and quantitative and spatial expression patterns of the seed-specific promoter phytohemagglutinin (PHA-L) from Phaseolus vulgaris were characterized in genetically modified C. arabica expressing the alpha-amylase inhibitor-1 (alpha-AI1) gene. The alpha-AI1 inhi...

  18. Structure of ‘linkerless’ hydroxamic acid inhibitor-HDAC8 complex confirms the formation of an isoform-specific subpocket

    Energy Technology Data Exchange (ETDEWEB)

    Tabackman, Alexa A.; Frankson, Rochelle; Marsan, Eric S.; Perry, Kay; Cole, Kathryn E. (Ithaca); (Cornell); (Christopher Newport U)

    2016-11-04

    Histone deacetylases (HDACs) catalyze the hydrolysis of acetylated lysine side chains in histone and non-histone proteins, and play a critical role in the regulation of many biological processes, including cell differentiation, proliferation, senescence, and apoptosis. Aberrant HDAC activity is associated with cancer, making these enzymes important targets for drug design. In general, HDAC inhibitors (HDACi) block the proliferation of tumor cells by inducing cell differentiation, cell cycle arrest, and/or apoptosis, and comprise some of the leading therapies in cancer treatments. To date, four HDACi have been FDA approved for the treatment of cancers: suberoylanilide hydroxamic acid (SAHA, Vorinostat, Zolinza®), romidepsin (FK228, Istodax®), belinostat (Beleodaq®), and panobinostat (Farydak®). Most current inhibitors are pan-HDACi, and non-selectively target a number of HDAC isoforms. Six previously reported HDACi were rationally designed, however, to target a unique sub-pocket found only in HDAC8. While these inhibitors were indeed potent against HDAC8, and even demonstrated specificity for HDAC8 over HDACs 1 and 6, there were no structural data to confirm the mode of binding. Here we report the X-ray crystal structure of Compound 6 complexed with HDAC8 to 1.98 Å resolution. We also describe the use of molecular docking studies to explore the binding interactions of the other 5 related HDACi. Our studies confirm that the HDACi induce the formation of and bind in the HDAC8-specific subpocket, offering insights into isoform-specific inhibition.

  19. The dual specificity PI3K/mTOR inhibitor PKI-587 displays efficacy against T-cell acute lymphoblastic leukemia (T-ALL).

    Science.gov (United States)

    Gazi, Mohiuddin; Moharram, Sausan A; Marhäll, Alissa; Kazi, Julhash U

    2017-04-28

    Although significant improvements have been made in the treatment of acute lymphoblastic leukemia (ALL), there is a substantial subset of high-risk T-cell ALL (T-ALL) patients with relatively poor prognosis. Like in other leukemia types, alterations of the PI3K/mTOR pathway are predominant in ALL which is also responsible for treatment failure and relapse. In this study, we show that relapsed T-ALL patients display an enrichment of the PI3K/mTOR pathway. Using a panel of inhibitors targeting multiple components of the PI3K/mTOR pathway, we observed that the dual-specific PI3K/mTOR inhibitor PKI-587 was the most selective inhibitor for T-ALL cells dependent on the PI3K/mTOR pathway. Furthermore, we observed that PKI-587 blocked proliferation and colony formation of T-ALL cell lines. Additionally, PKI-587 selectively abrogated PI3K/mTOR signaling without affecting MAPK signaling both in in vitro and in vivo. Inhibition of the PI3K/mTOR pathway using PKI-587 delayed tumor progression, reduced tumor load and enhanced the survival rate in immune-deficient mouse xenograft models without inducing weight loss in the inhibitor treated mice. This preclinical study shows beneficial effects of PKI-587 on T-ALL that warrants further investigation in the clinical setting. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Bayesian approach to the assessment of the population-specific risk of inhibitors in hemophilia A patients: a case study

    Directory of Open Access Journals (Sweden)

    Cheng J

    2016-10-01

    Full Text Available Ji Cheng,1,2 Alfonso Iorio,2,3 Maura Marcucci,4 Vadim Romanov,5 Eleanor M Pullenayegum,6,7 John K Marshall,3,8 Lehana Thabane1,2 1Biostatistics Unit, St Joseph’s Healthcare Hamilton, 2Department of Clinical Epidemiology and Biostatistics, 3Department of Medicine, McMaster University, Hamilton, ON, Canada; 4Geriatrics, Fondazione Ca’ Granda Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy; 5Baxter HealthCare, Global Medical Affairs, Westlake Village, CA, USA; 6Child Health Evaluation Sciences, Hospital for Sick Children, 7Dalla Lana School of Public Health, University of Toronto, Toronto, 8Division of Gastroenterology, Hamilton Health Science, Hamilton, ON, Canada Background: Developing inhibitors is a rare event during the treatment of hemophilia A. The multifacets and uncertainty surrounding the development of inhibitors further complicate the process of estimating inhibitor rate from the limited data. Bayesian statistical modeling provides a useful tool in generating, enhancing, and exploring the evidence through incorporating all the available information.Methods: We built our Bayesian analysis using three study cases to estimate the inhibitor rates of patients with hemophilia A in three different scenarios: Case 1, a single cohort of previously treated patients (PTPs or previously untreated patients; Case 2, a meta-analysis of PTP cohorts; and Case 3, a previously unexplored patient population – patients with baseline low-titer inhibitor or history of inhibitor development. The data used in this study were extracted from three published ADVATE (antihemophilic factor [recombinant] is a product of Baxter for treating hemophilia A post-authorization surveillance studies. Noninformative and informative priors were applied to Bayesian standard (Case 1 or random-effects (Case 2 and Case 3 logistic models. Bayesian probabilities of satisfying three meaningful thresholds of the risk of developing a clinical

  1. Bayesian approach to the assessment of the population-specific risk of inhibitors in hemophilia A patients: a case study.

    Science.gov (United States)

    Cheng, Ji; Iorio, Alfonso; Marcucci, Maura; Romanov, Vadim; Pullenayegum, Eleanor M; Marshall, John K; Thabane, Lehana

    2016-01-01

    Developing inhibitors is a rare event during the treatment of hemophilia A. The multifacets and uncertainty surrounding the development of inhibitors further complicate the process of estimating inhibitor rate from the limited data. Bayesian statistical modeling provides a useful tool in generating, enhancing, and exploring the evidence through incorporating all the available information. We built our Bayesian analysis using three study cases to estimate the inhibitor rates of patients with hemophilia A in three different scenarios: Case 1, a single cohort of previously treated patients (PTPs) or previously untreated patients; Case 2, a meta-analysis of PTP cohorts; and Case 3, a previously unexplored patient population - patients with baseline low-titer inhibitor or history of inhibitor development. The data used in this study were extracted from three published ADVATE (antihemophilic factor [recombinant] is a product of Baxter for treating hemophilia A) post-authorization surveillance studies. Noninformative and informative priors were applied to Bayesian standard (Case 1) or random-effects (Case 2 and Case 3) logistic models. Bayesian probabilities of satisfying three meaningful thresholds of the risk of developing a clinical significant inhibitor (10/100, 5/100 [high rates], and 1/86 [the Food and Drug Administration mandated cutoff rate in PTPs]) were calculated. The effect of discounting prior information or scaling up the study data was evaluated. Results based on noninformative priors were similar to the classical approach. Using priors from PTPs lowered the point estimate and narrowed the 95% credible intervals (Case 1: from 1.3 [0.5, 2.7] to 0.8 [0.5, 1.1]; Case 2: from 1.9 [0.6, 6.0] to 0.8 [0.5, 1.1]; Case 3: 2.3 [0.5, 6.8] to 0.7 [0.5, 1.1]). All probabilities of satisfying a threshold of 1/86 were above 0.65. Increasing the number of patients by two and ten times substantially narrowed the credible intervals for the single cohort study (1.4 [0.7, 2

  2. Semisynthesis and Structure-Activity Studies of Uncarinic Acid C Isolated from Uncaria rhynchophylla as a Specific Inhibitor of the Nucleation Phase in Amyloid β42 Aggregation.

    Science.gov (United States)

    Yoshioka, Takuya; Murakami, Kazuma; Ido, Kyohei; Hanaki, Mizuho; Yamaguchi, Kanoko; Midorikawa, Satohiro; Taniwaki, Shinji; Gunji, Hiroki; Irie, Kazuhiro

    2016-10-28

    Oligomers of the 42-mer amyloid-β protein (Aβ42), rather than fibrils, cause synaptic dysfunction in the pathology of Alzheimer's disease (AD). The nucleation phase in a nucleation-dependent aggregation model of Aβ42 is related to the formation of oligomers. Uncaria rhynchophylla is one component of "Yokukansan", a Kampo medicine, which is widely used for treating AD symptoms. Previously, an extract of U. rhynchophylla was found to reduce the aggregation of Aβ42, but its active principles have yet to be identified. In the present work, uncarinic acid C (3) was identified as an inhibitor of Aβ42 aggregation that is present in U. rhynchophylla. Moreover, compound 3 acted as a specific inhibitor of the nucleation phase of Aβ42 aggregation. Compound 3 was synthesized from saponin A (10), an abundant byproduct of rutin purified from Uncaria elliptica. Comprehensive structure-activity studies on 3 suggest that both a C-27 ferulate and a C-28 carboxylic acid group are required for its inhibitory activity. These findings may aid the development of oligomer-specific inhibitors for AD therapy.

  3. Immunological changes in human immunodeficiency virus (HIV)-infected individuals during HIV-specific protease inhibitor treatment

    DEFF Research Database (Denmark)

    Ullum, H; Katzenstein, T; Aladdin, H

    1999-01-01

    The present study examines the influence of effective anti-retroviral treatment on immune function, evaluated by a broad array of immunological tests. We followed 12 individuals infected with human immunodeficiency virus (HIV) for 6 months after initiation of combination anti-retroviral treatment...... including a protease inhibitor. Unstimulated and pokeweed mitogen (PWM)-, interleukin (IL)-2- and phytohaemagglutinin (PHA)-stimulated lymphocyte proliferative responses increased during follow-up reaching average levels from 1.3-fold (PHA) to 3.7-fold (PWM) above baseline values. The total CD4+ lymphocyte...

  4. Total synthesis and structural revision of TMG-chitotriomycin, a specific inhibitor of insect and fungal beta-N-acetylglucosaminidases.

    Science.gov (United States)

    Yang, You; Li, Yao; Yu, Biao

    2009-09-02

    TMG-chitotriomycin, a potent and selective inhibitor of the beta-N-acetylglucosaminidases that possesses an unique N,N,N-trimethyl-d-glucosamine (TMG) residue, is revised to be the TMG-beta-(1-->4)-chitotriose instead of the originally proposed alpha-anomer via its total synthesis, for which a highly convergent approach was developed in which the sterically demanding (1-->4)-glycosidic linkages are efficiently constructed by the Au(I)-catalyzed glycosylation protocol with glycosyl o-hexynylbenzoates as donors.

  5. The potency and specificity of the interaction between the IA3 inhibitor and its target aspartic proteinase from Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Phylip, L H; Lees, W E; Brownsey, B G

    2001-01-01

    The yeast IA3 polypeptide consists of only 68 residues, and the free inhibitor has little intrinsic secondary structure. IA3 showed subnanomolar potency toward its target, proteinase A from Saccharomyces cerevisiae, and did not inhibit any of a large number of aspartic proteinases with similar...... by the nontarget aspartic proteinases, it was not cleaved by proteinase A. The random coil IA3 polypeptide escapes cleavage by being stabilized in a helical conformation upon interaction with the active site of proteinase A. This results, paradoxically, in potent selective inhibition of the target enzyme....

  6. Extracellular concentration of homocysteine in human cell lines is influenced by specific inhibitors of cyst(e)ine transport.

    Science.gov (United States)

    Hultberg, Björn

    2004-04-01

    Despite the growing evidence that plasma homocysteine is a cardiovascular risk factor, the mechanism behind the vascular injuries is still unknown. Studies of the cellular uptake systems for homocysteine are scarce, but membrane transporters of cyst(e)ine seem to be involved. In the present study the cellular uptake of extracellular homocysteine in HeLa and hepatoma cell lines is investigated by using several different transport inhibitors for cellular uptake of cyst(e)ine. It is shown that systems A and Xc- are the main transport systems for homocysteine uptake in HeLa cells. It is also confirmed that the magnitude of homocysteine uptake in hepatoma cells is lower than in HeLa cells. However, in the presence of high amounts of extracellular homocysteine both cell types exhibited a high elimination of homocysteine, which was inhibited by the presence of inhibitors of systems A or Xc-. It is possible that there is normally a high turnover of homocysteine in cell cultures, which is not detected by occasional determinations of homocysteine concentrations. The complex pattern of homocysteine production, release, uptake and distribution between different cells in the body is important to examine further in order to possibly be able to modulate the elimination of homocysteine from circulation and thereby lower the risk of cardiovascular disease.

  7. Anti-adipogenic effects of KD025 (SLx-2119), a ROCK2-specific inhibitor, in 3T3-L1 cells.

    Science.gov (United States)

    Diep, Duy Trong Vien; Hong, Kyungki; Khun, Triyeng; Zheng, Mei; Ul-Haq, Asad; Jun, Hee-Sook; Kim, Young-Bum; Chun, Kwang-Hoon

    2018-02-06

    Adipose tissue is a specialized organ that synthesizes and stores fat. During adipogenesis, Rho and Rho-associated kinase (ROCK) 2 are inactivated, which enhances the expression of pro-adipogenic genes and induces the loss of actin stress fibers. Furthermore, pan ROCK inhibitors enhance adipogenesis in 3T3-L1 cells. Here, we show that KD025 (formerly known as SLx-2119), a ROCK2-specific inhibitor, suppresses adipogenesis in 3T3-L1 cells partially through a ROCK2-independent mechanism. KD025 downregulated the expression of key adipogenic transcription factors PPARγ and C/EBPα during adipogenesis in addition to lipogenic factors FABP4 and Glut4. Interestingly, adipogenesis was blocked by KD025 during days 1~3 of differentiation; after differentiation terminated, lipid accumulation was unaffected. Clonal expansion occurred normally in KD025-treated cells. These results suggest that KD025 could function during the intermediate stage after clonal expansion. Data from depletion of ROCKs showed that KD025 suppressed cell differentiation partially independent of ROCK's activity. Furthermore, no further loss of actin stress fibers emerged in KD025-treated cells during and after differentiation compared to control cells. These results indicate that in contrast to the pro-adipogenic effect of pan-inhibitors, KD025 suppresses adipogenesis in 3T3-L1 cells by regulating key pro-adipogenic factors. This outcome further implies that KD025 could be a potential anti-adipogenic/obesity agent.

  8. Discovery of core-structurally novel PTP1B inhibitors with specific selectivity containing oxindole-fused spirotetrahydrofurochroman by one-pot reaction.

    Science.gov (United States)

    Dong, Suzhen; Lei, Yubing; Jia, Shikun; Gao, Lixin; Li, Jia; Zhu, Tong; Liu, Shunying; Hu, Wenhao

    2017-02-15

    Protein tyrosine phosphatase 1B (PTP1B) has been proposed to be an ideal target for treatment of type II diabetes and obesity. However, no druggable PTP1B inhibitor has been established and there is still an urgent demand for the development of structurally novel PTPIB inhibitor. Herein, we reported core-structurally novel PTP1B inhibitors with low micromole-ranged inhibitory activity by one-pot reaction from simple starting materials. Further studies demonstrated some of these active compounds had a specific selectivity over other PTPs. The structure and activity relationship was also described. The best active and selective compound 5e inhibited PTP1B activity with an IC 50 of 4.53μM. Molecular docking analysis further demonstrated that compound 5e bound to the active pocket of PTP1B. The results might provide some insights for further development of new drugs for type II diabetes and obesity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Immune activation and HIV-specific T cell responses are modulated by a cyclooxygenase-2 inhibitor in untreated HIV-infected individuals: An exploratory clinical trial.

    Directory of Open Access Journals (Sweden)

    Christian Prebensen

    Full Text Available Pathologically elevated immune activation and inflammation contribute to HIV disease progression and immunodeficiency, potentially mediated by elevated levels of prostaglandin E2, which suppress HIV-specific T cell responses. We have previously shown that a high dose of the cyclooxygenase-2 inhibitor celecoxib can reduce HIV-associated immune activation and improve IgG responses to T cell-dependent vaccines. In this follow-up study, we included 56 HIV-infected adults, 28 antiretroviral therapy (ART-naïve and 28 on ART with undetectable plasma viremia but CD4 counts below 500 cells/μL. Patients in each of the two study groups were randomized to receive 90 mg qd of the cyclooxygenase-2 inhibitor etoricoxib for six months, two weeks or to a control arm, respectively. T cell activation status, HIV Gag-specific T cell responses and plasma inflammatory markers, tryptophan metabolism and thrombin generation were analyzed at baseline and after four months. In addition, patients received tetanus toxoid, conjugated pneumococcal and seasonal influenza vaccines, to which IgG responses were determined after four weeks. In ART-naïve patients, etoricoxib reduced the density of the activation marker CD38 in multiple CD8+ T cell subsets, improved Gag-specific T cell responses, and reduced in vitro plasma thrombin generation, while no effects were seen on plasma markers of inflammation or tryptophan metabolism. No significant immunological effects of etoricoxib were observed in ART-treated patients. Patients receiving long-term etoricoxib treatment had poorer tetanus toxoid and conjugated pneumococcal vaccine responses than those receiving short-course etoricoxib. Cyclooxygenase-2 inhibitors may attenuate harmful immune activation in HIV-infected patients without access to ART.

  10. pKa modulation of the acid/base catalyst within GH32 and GH68: a role in substrate/inhibitor specificity?

    Directory of Open Access Journals (Sweden)

    Shuguang Yuan

    Full Text Available Glycoside hydrolases of families 32 (GH32 and 68 (GH68 belong to clan GH-J, containing hydrolytic enzymes (sucrose/fructans as donor substrates and fructosyltransferases (sucrose/fructans as donor and acceptor substrates. In GH32 members, some of the sugar substrates can also function as inhibitors, this regulatory aspect further adding to the complexity in enzyme functionalities within this family. Although 3D structural information becomes increasingly available within this clan and huge progress has been made on structure-function relationships, it is not clear why some sugars bind as inhibitors without being catalyzed. Conserved aspartate and glutamate residues are well known to act as nucleophile and acid/bases within this clan. Based on the available 3D structures of enzymes and enzyme-ligand complexes as well as docking simulations, we calculated the pKa of the acid-base before and after substrate binding. The obtained results strongly suggest that most GH-J members show an acid-base catalyst that is not sufficiently protonated before ligand entrance, while the acid-base can be fully protonated when a substrate, but not an inhibitor, enters the catalytic pocket. This provides a new mechanistic insight aiming at understanding the complex substrate and inhibitor specificities observed within the GH-J clan. Moreover, besides the effect of substrate entrance on its own, we strongly suggest that a highly conserved arginine residue (in the RDP motif rather than the previously proposed Tyr motif (not conserved provides the proton to increase the pKa of the acid-base catalyst.

  11. [COMPARATIVE SENSITIVITY OF CHOLINESTERASES IN VERTEBRATES AND INVERTEBRATES TO HIGHLY SPECIFIC ORGANOPHOSPHORUS INHIBITORS DIISOPROPYL FLUOROPHOSPHATE (DFP) AND (2-ETHOXYMETHYL PHOSPHORYL THIOETHYL) ETHYL (METHYL) SULPHONIUM SULPHOMETHYLAT (GD-42)].

    Science.gov (United States)

    Basova, N E; Kormilitsyn, B N; Perchenok, A Yu; Rozengart, E V; Saakov, V S; Suvorov, A A

    2015-01-01

    The review presents data on comparative reactivity of 68 cholinesterase preparation from various organs and tissues in a number of vertebrates and invertebrates based on sensitivity to two highly specific and most studied organophosphorus inhibitors--diisopropyl fluorophosphates (DFP) and (2-ethoxymethyl phosphoryl thioethyl) ethyl (methyl) sulphonium sulphomethylat (GD-42). Analysis of these data suggests a great diversity in enzymologic characteristics of cholinesterase preparation in representatives of vertebrates and invertebrates, this variety observed even for closely related enzymes in animals of almost the same level of development.

  12. Rapid and individual-specific glycoprofiling of the low abundance N-glycosylated protein tissue inhibitor of metalloproteinases-1

    DEFF Research Database (Denmark)

    Thaysen-Andersen, Morten; Thøgersen, Ida B.; Nielsen, Hans Jørgen

    2007-01-01

    A gel-based method for a mass spectrometric site-specific glycoanalysis was developed using a recombinant glycoprotein expressed in two different cell lines. Hydrophilic interaction liquid chromatography at nanoscale level was used to enrich for glycopeptides prior to MS. The glycoprofiling...... glycoprofiling of a low abundance glycoprotein performed in an individual-specific manner allows for future studies of glycosylated biomarkers for person-specific detection of altered glycosylation and may thus allow early detection and monitoring of diseases....

  13. Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures.

    Science.gov (United States)

    Du, Zhimei; Treiber, David; McCarter, John D; Fomina-Yadlin, Dina; Saleem, Ramsey A; McCoy, Rebecca E; Zhang, Yuling; Tharmalingam, Tharmala; Leith, Matthew; Follstad, Brian D; Dell, Brad; Grisim, Brent; Zupke, Craig; Heath, Carole; Morris, Arvia E; Reddy, Pranhitha

    2015-01-01

    The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in

  14. PF-4708671, a specific inhibitor of p70 ribosomal S6 kinase 1, activates Nrf2 by promoting p62-dependent autophagic degradation of Keap1

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jeong Su [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Kang, Dong Hoon [Department of Life Science and Ewha Research Center for Systems Biology (Korea, Republic of); The Research Center for Cell Homeostasis, Ewha Womans University, Seoul 127-750 (Korea, Republic of); Lee, Da Hyun [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Bae, Soo Han, E-mail: soohanbae@yuhs.ac [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of)

    2015-10-23

    p70 ribosomal S6 kinase 1 (S6K1) is an important serine/threonine kinase and downstream target of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. PF-4708671 is a specific inhibitor of S6K1, and prevents S6K1-mediated phosphorylation of the S6 protein. PF-4708671 treatment often leads to apoptotic cell death. However, the protective mechanism against PF-4708671-induced cell death has not been elucidated. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway is essential for protecting cells against oxidative stress. p62, an adaptor protein in the autophagic process, enhances Nrf2 activation through the impairment of Keap1 activity. In this study, we showed that PF-4708671 induces autophagic Keap1 degradation-mediated Nrf2 activation in p62-dependent manner. Furthermore, p62-dependent Nrf2 activation plays a crucial role in protecting cells from PF-4708671-mediated apoptosis. - Highlights: • PF-4708671, a S6K1-specific inhibitor, prevents S6K1-mediated S6 phosphorylation. • However, PF-4708671 treatment often leads to apoptotic cell death. • Protective mechanism against PF-4708671-induced cell death remains to be elucidated. • PF-4708671 induced p62-dependent, autophagic Keap1 degradation-mediated Nrf2 activation. • p62-dependent Nrf2 activation protects cells from PF-4708671-mediated apoptosis.

  15. Crystal structure of the N domain of human somatic angiotensin I-converting enzyme provides a structural basis for domain-specific inhibitor design.

    Science.gov (United States)

    Corradi, Hazel R; Schwager, Sylva L U; Nchinda, Aloysius T; Sturrock, Edward D; Acharya, K Ravi

    2006-03-31

    Human somatic angiotensin I-converting enzyme (sACE) is a key regulator of blood pressure and an important drug target for combating cardiovascular and renal disease. sACE comprises two homologous metallopeptidase domains, N and C, joined by an inter-domain linker. Both domains are capable of cleaving the two hemoregulatory peptides angiotensin I and bradykinin, but differ in their affinities for a range of other substrates and inhibitors. Previously we determined the structure of testis ACE (C domain); here we present the crystal structure of the N domain of sACE (both in the presence and absence of the antihypertensive drug lisinopril) in order to aid the understanding of how these two domains differ in specificity and function. In addition, the structure of most of the inter-domain linker allows us to propose relative domain positions for sACE that may contribute to the domain cooperativity. The structure now provides a platform for the design of "domain-specific" second-generation ACE inhibitors.

  16. Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high-throughput screening of Wnt inhibitors.

    Science.gov (United States)

    Yamaguchi, Kiyoshi; Zhu, Chi; Ohsugi, Tomoyuki; Yamaguchi, Yuko; Ikenoue, Tsuneo; Furukawa, Yoichi

    2017-12-01

    Constitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. Cell-based assays using synthetic TCF/LEF (T-cell factor/lymphoid enhancer factor) reporters, as readouts of β-catenin/TCF-dependent transcriptional activity, have contributed greatly to the discovery of small molecules that modulate Wnt signaling. In the present study, we report a novel screening method, called a bidirectional dual reporter assay. Integrated transcriptome analysis identified a histidine ammonia-lyase gene (HAL) that was negatively regulated by β-catenin/TCF-dependent transcriptional activity. We leveraged a promoter region of the HAL gene as another transcriptional readout of Wnt signaling. Cells stably expressing both an optimized HAL reporter and the TCF/LEF reporter enabled bidirectional reporter activities in response to Wnt signaling. Increased HAL reporter activity and decreased TCF/LEF reporter activity were observed simultaneously in the cells when β-catenin/TCF7L2 was inhibited. Notably, this method could decrease the number of false positives observed when screening an inhibitor library compared with the conventional TCF/LEF assay. We found that Brefeldin A, a disruptor of the Golgi apparatus, inhibited the Wnt/β-catenin signaling pathway. The utility of our system could be expanded to examine other disease-associated pathways beyond the Wnt/β-catenin signaling pathway. © 2017 Wiley Periodicals, Inc.

  17. TGF-b and a specific TGF-b inhibitor regulate pericentrin B and MYH9 in glioma cell lines

    Directory of Open Access Journals (Sweden)

    Óscar Álzate

    2006-01-01

    Full Text Available Malignant gliomas are heterogeneous, highly invasive vascular tumours. The multifunctional cytokine, transforming growth factor-beta (TGF-P, is expressed by grade III/IV gliomas and promotes tumour angiogenesis, invasión and immune escape. It has been shown previously that a small TGF-P receptor type I (TGF-(3-RI molecule inhibitor (SB-431542 blocks TGF-(3-mediated signal transduction, induction of angiogenic factor expression and cellular motility. As glioma cell lines display differential sensitivity to TGF-P, it was expected that they would also be differentially impacted by disruption of TGF-P signalling. Differential in gel expression (DIGE analysis and mass spectrometry was used in this work for determining protein regulation effects of both TGF-P and SB-431542 on human glioma cell lines. It was found that pericentrin B and non muscle myosin were differentially expressed in fragments which likely resulted from protease activation by the tumour growth mechanism. These results suggest that both pericentrin B and non-muscle myosin might be potential glioma biomarkers. Key words: DIGE, proteomics, glioma, TGF-P, mass spectrometry, non muscle myosin, pericentrin B.

  18. Biochemical detection of E-ADA on Neospora caninum tachyzoites and the effects of a specific enzymatic inhibitor

    Directory of Open Access Journals (Sweden)

    Alexandre A. Tonin

    2015-01-01

    Full Text Available Objective. This study aimed to investigate the presence and activity of the ecto adenosine deaminase (E-ADA enzyme in tachyzoites of Neospora caninum (Nc-1 strain, as well as to assess the activity of a well-known E-ADA inhibitor, the deoxycoformycin. Materials and methods. The parasites were grown in cell culture, being subsequently separated in a pellet of tachyzoites, on which the E-ADA activity was tested using the concentrations 0 (control, 0.2, 0.4 and 0.8 mg mL-1. Results. The E-ADA showed high activity, progressively increasing its activity according to the enhancement of the protein concentration. The test was carried out with different concentrations of deoxycoformycin, showing that it was able to inhibit the E-ADA present on the free form of the parasite. Conclusions. Based on these results we conclude that the E-ADA is present on tachyzoites of N. caninum, and deoxycoformycin is able to inhibit this enzyme. In this sense, knowing the negative impact of N. caninum on reproductive issue in cattle (mainly abortion, might it is an alternative in order to deal with this parasitic infection.

  19. Oncogenic PKC-ι activates Vimentin during epithelial-mesenchymal transition in melanoma; a study based on PKC-ι and PKC-ζ specific inhibitors.

    Science.gov (United States)

    Ratnayake, Wishrawana S; Apostolatos, Christopher A; Apostolatos, André H; Schutte, Ryan J; Huynh, Monica A; Ostrov, David A; Acevedo-Duncan, Mildred

    2018-05-21

    Melanoma is one of the fastest growing cancers in the United States and is accompanied with a poor prognosis owing to tumors being resistant to most therapies. Atypical protein kinase Cs (aPKC) are involved in malignancy in many cancers. We previously reported that aPKCs play a key role in melanoma's cell motility by regulating cell signaling pathways which induce epithelial-mesenchymal Transition (EMT). We tested three novel inhibitors; [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1T) along with its nucleoside analog 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) which are specific to protein kinase C-iota (PKC-ι) and 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) which is specific to PKC-zeta (PKC-ζ) on cell proliferation, apoptosis, migration and invasion of two malignant melanoma cell lines compared to normal melanocytes. Molecular modeling was used to identify potential binding sites for the inhibitors and to predict selectivity. Kinase assay showed >50% inhibition for specified targets beyond 5 μM for all inhibitors. Both ICA-1 and ζ-Stat significantly reduced cell proliferation and induced apoptosis, while ICA-1 also significantly reduced migration and melanoma cell invasion. PKC-ι stimulated EMT via TGFβ/Par6/RhoA pathway and activated Vimentin by phosphorylation at S39. Both ICA-1 and ζ-Stat downregulate TNF-α induced NF-κB translocation to the nucleus there by inducing apoptosis. Results suggest that PKC-ι is involved in melanoma malignancy than PKC-ζ. Inhibitors proved to be effective under in-vitro conditions and need to be tested in-vivo for the validity as effective therapeutics. Overall, results show that aPKCs are essential for melanoma progression and metastasis and that they could be used as effective therapeutic targets for malignant melanoma.

  20. Protective effects of phosphodiesterase-4-specific inhibitor rolipram on acute ischemia-reperfusion injury in rat kidney.

    Science.gov (United States)

    Mammadov, Emin; Aridogan, Ibrahim Atilla; Izol, Volkan; Acikalin, Arbil; Abat, Deniz; Tuli, Abdullah; Bayazit, Yildirim

    2012-12-01

    To investigate the effect of Rolipram, a phosphodiesterase-4-inhibitor, on renal ischemia-reperfusion injury (IRI) in rats. Thirty rats were divided into 5 different groups of 6 rats. Nothing was done to the control group. In the second group, the renal pedicle was clamped for 30 minutes. In the third group, 1 mg/kg of Rolipram was given by intraperitoneal injection 30 minutes before clamping. The fourth group received the same injection when the clamp was placed, as did the fifth group 30 minutes after the clamp was opened. Clamping time was set at 30 minutes. Twenty-four hours later, nephrectomy was performed in all the groups. Half of each kidney was examined histopathologically. Levels of biochemical agents, such as malondialdehyde, superoxide dismutase, and catalase, were measured in the other half. The malondialdehyde (MDA) levels significantly decreased and reached control levels in the group in which Rolipram was administered 30 minutes after reperfusion (P = .07). The catalase and superoxide dismutase activities obtained from renal homogentisates of the ischemia groups were evaluated; there were striking increases in tissue levels of these 2 enzymes in the groups in which Rolipram was administered during ischemia and 30 minutes after ischemia (P < .001). Histopathologically, there was no significant difference in inflammation between the Rolipram-administrated groups compared with group 1 (control) and group 2 (IRI). Tubular necrosis and apoptosis was significantly lower in group 5 than the other groups, except group 1 (P < .001). We suggest that in surgical procedures that can lead to renal IRI, the administration of Rolipram can decrease oxidative renal tissue damage and the severe deterioration of renal function. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma.

    Directory of Open Access Journals (Sweden)

    Peter Y Yu

    Full Text Available STAT3 is a transcription factor involved in cytokine and receptor kinase signal transduction that is aberrantly activated in a variety of sarcomas, promoting metastasis and chemotherapy resistance. The purpose of this work was to develop and test a novel putative STAT3 inhibitor, LY5.An in silico fragment-based drug design strategy was used to create LY5, a small molecule inhibitor that blocks the STAT3 SH2 domain phosphotyrosine binding site, inhibiting homodimerization. LY5 was evaluated in vitro demonstrating good biologic activity against rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma cell lines at high nanomolar/low micromolar concentrations, as well as specific inhibition of STAT3 phosphorylation without effects on other STAT3 family members. LY5 exhibited excellent oral bioavailability in both mice and healthy dogs, and drug absorption was enhanced in the fasted state with tolerable dosing in mice at 40 mg/kg BID. However, RNAi-mediated knockdown of STAT3 did not phenocopy the biologic effects of LY5 in sarcoma cell lines. Moreover, concentrations needed to inhibit ex vivo metastasis growth using the PuMA assay were significantly higher than those needed to inhibit STAT3 phosphorylation in vitro. Lastly, LY5 treatment did not inhibit the growth of sarcoma xenografts or prevent pulmonary metastasis in mice.LY5 is a novel small molecule inhibitor that effectively inhibits STAT3 phosphorylation and cell proliferation at nanomolar concentrations. LY5 demonstrates good oral bioavailability in mice and dogs. However LY5 did not decrease tumor growth in xenograft mouse models and STAT3 knockdown did not induce concordant biologic effects. These data suggest that the anti-cancer effects of LY5 identified in vitro were not mediated through STAT3 inhibition.

  2. Target specificity, in vivo pharmacokinetics, and efficacy of the putative STAT3 inhibitor LY5 in osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma.

    Science.gov (United States)

    Yu, Peter Y; Gardner, Heather L; Roberts, Ryan; Cam, Hakan; Hariharan, Seethalakshmi; Ren, Ling; LeBlanc, Amy K; Xiao, Hui; Lin, Jiayuh; Guttridge, Denis C; Mo, Xiaokui; Bennett, Chad E; Coss, Christopher C; Ling, Yonghua; Phelps, Mitch A; Houghton, Peter; London, Cheryl A

    2017-01-01

    STAT3 is a transcription factor involved in cytokine and receptor kinase signal transduction that is aberrantly activated in a variety of sarcomas, promoting metastasis and chemotherapy resistance. The purpose of this work was to develop and test a novel putative STAT3 inhibitor, LY5. An in silico fragment-based drug design strategy was used to create LY5, a small molecule inhibitor that blocks the STAT3 SH2 domain phosphotyrosine binding site, inhibiting homodimerization. LY5 was evaluated in vitro demonstrating good biologic activity against rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma cell lines at high nanomolar/low micromolar concentrations, as well as specific inhibition of STAT3 phosphorylation without effects on other STAT3 family members. LY5 exhibited excellent oral bioavailability in both mice and healthy dogs, and drug absorption was enhanced in the fasted state with tolerable dosing in mice at 40 mg/kg BID. However, RNAi-mediated knockdown of STAT3 did not phenocopy the biologic effects of LY5 in sarcoma cell lines. Moreover, concentrations needed to inhibit ex vivo metastasis growth using the PuMA assay were significantly higher than those needed to inhibit STAT3 phosphorylation in vitro. Lastly, LY5 treatment did not inhibit the growth of sarcoma xenografts or prevent pulmonary metastasis in mice. LY5 is a novel small molecule inhibitor that effectively inhibits STAT3 phosphorylation and cell proliferation at nanomolar concentrations. LY5 demonstrates good oral bioavailability in mice and dogs. However LY5 did not decrease tumor growth in xenograft mouse models and STAT3 knockdown did not induce concordant biologic effects. These data suggest that the anti-cancer effects of LY5 identified in vitro were not mediated through STAT3 inhibition.

  3. [New Radiopharmaceuticals Based on Prostate-Specific Inhibitors of Membrane Antigen for Diagnostics and Therapy of Metastatic Prostate Cancer].

    Science.gov (United States)

    Vlasova, O P; German, K E; Krilov, V V; Petriev, V M; Epstein, N B

    2015-01-01

    About 10.7% cases of prostate cancer were registered in Russia in 2011 (40,000 patients). More than half of cancer cases were revealed in advanced (III-IV) stages when metastases inevitably developed quickly. Clinical problem of early diagnostics and treatment of metastatic prostate cancer is still not solved. Anatomical imaging techniques have low sensitivity and specificity for the detection of this disease. Metabolic visualization methods which use prostate specific antigen (PSA) as a marker are also ineffective. This article describes prostate-specific membrane antigens (PSMA) that are proposed as a marker for diagnostics and therapy of prostate cancer. The most promising PSMA-based radiopharmaceutical agent for diagnostics has been developed and clinically tested in the European countries. These pharmaceuticals are based on small peptide molecules modified with urea, and have the highest affinity to PSMA. Favorable phannacokinetics, rapid accumulation in the tumor and rapid excretion from the body are beneficial features of these pharmaceuticals.

  4. Seed-Specific Stable Expression of the α-AI1 Inhibitor in Coffee Grains and the In Vivo Implications for the Development of the Coffee Berry Borer.

    Science.gov (United States)

    Albuquerque, Érika V S; Bezerra, Caroline A; Romero, Juan V; Valencia, Jorge W A; Valencia-Jiménez, Arnubio; Pimenta, Lucas M; Barbosa, Aulus E A D; Silva, Maria C M; Meneguim, Ana M; Sá, Maria Eugênia L; Engler, Gilbert; de Almeida-Engler, Janice; Fernandez, Diana; Grossi-de-Sá, Maria F

    Genetic transformation of coffee ( Coffea spp.), the second most traded commodity worldwide, is an alternative approach to introducing features that cannot be introgressed by traditional crossings. The transgenic stability, heritability and quantitative and spatial expression patterns of the seed-specific promoter phytohemagglutinin (PHA-L) from Phaseolus vulgaris were characterized in genetically modified C. arabica expressing the α-amylase inhibitor-1 ( α-AI1 ) gene. The α-AI1 inhibitor shows considerable activity toward digestive enzymes of the coffee berry borer (CBB) Hypothenemus hampei . This insect pest expends its life cycle almost entirely in coffee berries. Transgene containment in the fruit is important to meeting food and environmental safety requirements for releasing genetically modified (GM) crops. PCR analysis of T2 coffee plants showed a Mendelian single-copy segregation pattern. Ectopic transgene expression was only detected in coffee grains, as demonstrated by reverse transcription-PCR analysis of different plant tissues. An intense immunocytochemical signal associated with α-AI1 protein expression was localized to endospermic cells. In addition, a delay in the larval development of CBB was observed after challenging transgenic coffee seeds with the insect. These results indicate that the PHA-L promoter might be a useful tool in coffee for the seed-specific expression of genes related to coffee bean productivity, quality and pest protection. The biotechnological applicability of the α-AI1 gene for controlling CBB is also discussed. This work is the first report showing a seed-specific transgene expression in coffee plants.

  5. Delphinidin, a specific inhibitor of histone acetyltransferase, suppresses inflammatory signaling via prevention of NF-κB acetylation in fibroblast-like synoviocyte MH7A cells

    International Nuclear Information System (INIS)

    Seong, Ah-Reum; Yoo, Jung-Yoon; Choi, KyungChul; Lee, Mee-Hee; Lee, Yoo-Hyun; Lee, Jeongmin; Jun, Woojin; Kim, Sunoh; Yoon, Ho-Geun

    2011-01-01

    Highlights: → Delphinidin is a novel inhibitor of p300/CBP histone acetyltransferase. → Delphinidin prevents the hyperacetylation of p65 by inhibiting the HAT activity of p300/CBP. → Delphinidin efficiently suppresses the expression of inflammatory cytokines in MH7A cells via hypoacetylation of NF-κB. → Delphinidin inhibits cytokine release in the Jurkat T lymphocyte cell line. -- Abstract: Histone acetyltransferase (HAT) inhibitors (HATi) isolated from dietary compounds have been shown to suppress inflammatory signaling, which contributes to rheumatoid arthritis. Here, we identified a novel HATi in Punica granatum L. known as delphinidin (DP). DP did not affect the activity of other epigenetic enzymes (histone deacetylase, histone methyltransferase, or sirtuin1). DP specifically inhibited the HAT activities of p300/CBP. It also inhibited p65 acetylation in MH7A cells, a human rheumatoid arthritis synovial cell line. DP-induced hypoacetylation was accompanied by cytosolic accumulation of p65 and nuclear localization of IKBα. Accordingly, DP treatment inhibited TNFα-stimulated increases in NF-κB function and expression of NF-κB target genes in these cells. Importantly, DP suppressed lipopolysaccharide-induced pro-inflammatory cytokine expression in Jurkat T lymphocytes, demonstrating that HATi efficiently suppresses cytokine-mediated immune responses. Together, these results show that the HATi activity of DP counters anti-inflammatory signaling by blocking p65 acetylation and that this compound may be useful in preventing inflammatory arthritis.

  6. Delphinidin, a specific inhibitor of histone acetyltransferase, suppresses inflammatory signaling via prevention of NF-{kappa}B acetylation in fibroblast-like synoviocyte MH7A cells

    Energy Technology Data Exchange (ETDEWEB)

    Seong, Ah-Reum; Yoo, Jung-Yoon; Choi, KyungChul [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, College of Medicine, Yonsei University, Seoul (Korea, Republic of); Lee, Mee-Hee [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, College of Medicine, Yonsei University, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University, College of Medicine, Seoul (Korea, Republic of); Lee, Yoo-Hyun [Department of Food Science and Nutrition, The University of Suwon, Kyunggi-do (Korea, Republic of); Lee, Jeongmin [Department of Medical Nutrition, Kyung Hee University, Kyunggi-do (Korea, Republic of); Jun, Woojin [Department of Food and Nutrition, Chonnam National University, Gwangju (Korea, Republic of); Kim, Sunoh, E-mail: sunoh@korea.ac.kr [Jeollanamdo Institute of Natural Resources Research, Jeonnam (Korea, Republic of); Yoon, Ho-Geun, E-mail: yhgeun@yuhs.ac [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, College of Medicine, Yonsei University, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University, College of Medicine, Seoul (Korea, Republic of)

    2011-07-08

    Highlights: {yields} Delphinidin is a novel inhibitor of p300/CBP histone acetyltransferase. {yields} Delphinidin prevents the hyperacetylation of p65 by inhibiting the HAT activity of p300/CBP. {yields} Delphinidin efficiently suppresses the expression of inflammatory cytokines in MH7A cells via hypoacetylation of NF-{kappa}B. {yields} Delphinidin inhibits cytokine release in the Jurkat T lymphocyte cell line. -- Abstract: Histone acetyltransferase (HAT) inhibitors (HATi) isolated from dietary compounds have been shown to suppress inflammatory signaling, which contributes to rheumatoid arthritis. Here, we identified a novel HATi in Punica granatum L. known as delphinidin (DP). DP did not affect the activity of other epigenetic enzymes (histone deacetylase, histone methyltransferase, or sirtuin1). DP specifically inhibited the HAT activities of p300/CBP. It also inhibited p65 acetylation in MH7A cells, a human rheumatoid arthritis synovial cell line. DP-induced hypoacetylation was accompanied by cytosolic accumulation of p65 and nuclear localization of IKB{alpha}. Accordingly, DP treatment inhibited TNF{alpha}-stimulated increases in NF-{kappa}B function and expression of NF-{kappa}B target genes in these cells. Importantly, DP suppressed lipopolysaccharide-induced pro-inflammatory cytokine expression in Jurkat T lymphocytes, demonstrating that HATi efficiently suppresses cytokine-mediated immune responses. Together, these results show that the HATi activity of DP counters anti-inflammatory signaling by blocking p65 acetylation and that this compound may be useful in preventing inflammatory arthritis.

  7. 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

  8. Gambogic Acid Is a Tissue-Specific Proteasome Inhibitor In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Xiaofen Li

    2013-01-01

    Full Text Available Gambogic acid (GA is a natural compound derived from Chinese herbs that has been approved by the Chinese Food and Drug Administration for clinical trials in cancer patients; however, its molecular targets have not been thoroughly studied. Here, we report that GA inhibits tumor proteasome activity, with potency comparable to bortezomib but much less toxicity. First, GA acts as a prodrug and only gains proteasome-inhibitory function after being metabolized by intracellular CYP2E1. Second, GA-induced proteasome inhibition is a prerequisite for its cytotoxicity and anticancer effect without off-targets. Finally, because expression of the CYP2E1 gene is very high in tumor tissues but low in many normal tissues, GA could therefore produce tissue-specific proteasome inhibition and tumor-specific toxicity, with clinical significance for designing novel strategies for cancer treatment.

  9. Rational design of urea-based glutamate carboxypeptidase II (GCPII) inhibitors as versatile tools for specific drug targeting and delivery

    Czech Academy of Sciences Publication Activity Database

    Tykvart, Jan; Schimer, Jiří; Bařinková, Jitka; Pachl, Petr; Poštová Slavětínská, Lenka; Majer, Pavel; Konvalinka, Jan; Šácha, Pavel

    2014-01-01

    Roč. 22, č. 15 (2014), s. 4099-4108 ISSN 0968-0896 R&D Projects: GA ČR GBP208/12/G016 Grant - others:OPPK(CZ) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 Keywords : GCPII * PSMA * structure-aided drug design * specific drug targeting Subject RIV: CE - Biochemistry Impact factor: 2.793, year: 2014

  10. Development of Tethered Hsp90 Inhibitors Carrying Radioiodinated Probes to Specifically Discriminate and Kill Malignant Breast Tumor Cells

    Science.gov (United States)

    2017-05-01

    properties and the causes of poor solubility and poor permeability . Journal of pharmacological and toxicological methods 44, 235-249. Liu, S., Li, D., Zhang...250,000 women respectively. Unfortunately, the sensitivity but low specificity of screening has led to concerns about over treatment of indolent...cancer deaths annually. Clinical data indicate a strong link between high expression/activation of Heat shock protein 90 (Hsp90) with poor prognosis

  11. Development of a monoclonal antibody that specifically detects tissue inhibitor of metalloproteinase-4 (TIMP-4) in formalin-fixed, paraffin-embedded human tissues.

    Science.gov (United States)

    Donover, P Scott; Wojciechowski, Brian S; Thirumaran, Rajesh; Zemba-Palko, Vlasta; Prendergast, George C; Wallon, U Margaretha

    2010-08-01

    Overexpression of the extracellular metalloproteinase inhibitor TIMP-4 in estrogen receptor-negative breast cancers was found recently to be associated with a poor prognosis for survival. To pursue exploration of the theranostic applications of TIMP-4, specific antibodies with favorable properties for immunohistochemical use and other clinical assays are needed. Here we report the characterization of a monoclonal antibody (clone 9:4-7) specific for full-length human TIMP-4 with suitable qualities. The antibody was determined to be an IgG(2b) immunoglobulin. In enzyme-linked immunosorbent assay (ELISA) and immunoblotting assays, it did not exhibit any detectable crossreactivity with recombinant forms of the other human TIMPs 1, 2, and 3. In contrast, the antibody displayed high specificity and sensitivity for TIMP-4 including in formalin-fixed and paraffin-embedded specimens of human breast specimens. An analysis of tissue microarrays of human cancer and corresponding normal tissues revealed specific staining patterns with excellent signal-to-noise ratios. This study documents TIMP-4 monoclonal antibody clone 9:4-7 as an effective tool for preclinical and clinical investigations. Published 2010 Wiley-Liss, Inc.

  12. A New Assay for Measurement of Acetylcholinesterase and Butyrylcholinesterase in Canine Whole Blood Combining Specific Substrates and Ethopropazine Hydrochloride as a Selective Butyrylcholinesterase Inhibitor

    Directory of Open Access Journals (Sweden)

    F Tecles, A Tvarijonaviciute and JJ Cerón*

    2013-11-01

    Full Text Available In the present report, a new assay combining specific substrates and a selective BChE inhibitor (ethopropazine hydrochloride was used to measure both AChE and BChE in canine whole blood samples. Acetylthiocholine iodide (ATCI and butyrylthiocholine iodide (BTCI were used as substrates, whereas 2,2’-dithiodipiridine was used as chromophore. Ethopropazine concentration inhibiting over 95% BChE with minimum AChE inhibition was fixed at 0.3mM. The results confirmed that whole blood cholinesterase activity measured with BTCI in absence of ethopropazine corresponded with serum BChE, whereas whole blood cholinesterase analysed with ATCI in presence of ethopropazine reflected mainly erythrocytes and plasma AChE activity. This procedure showed good repeatability, it was easy and fast, and can be routinely used in veterinary laboratories.

  13. Inhibition of X-ray and doxorubicin-induced apoptosis by butyrolactone I, a CDK-specific inhibitor, in human tumor cells

    International Nuclear Information System (INIS)

    Lu Yanjun; Takebe, Hiraku; Yagi, Takashi

    2000-01-01

    Cell-cycle progression is coordinately regulated by cyclin-dependent kinases (CDKs). The inhibition of CDKs by p21 wafl/Cipl/Sdil prevents the apoptosis of cells treated with DNA-damaging agents. In this study, we found that butyrolactone I, a specific inhibitor of CDC2 family kinases, blocks the X-ray- or doxorubicin-induced apoptosis of DLD1 (p21 +/+) human colorectal carcinoma cells in a dose-dependent manner. We also found that butyrolactone I inhibits the CDK2 activity and enhances cell survival after an X-ray irradiation or doxorubicin treatment in both DLD1 (p21 -/-) and DLD1 (p21 +/+) cells. These findings suggest that butyrolactone I prevents apoptosis by the direct inhibition of CDK and also, possibly, by CDK-inhibition through p53-independent p21-induction. Our findings indicate that CDK activity is required for DNA-damaging agent-induced apoptosis. (author)

  14. Total levels of tissue inhibitor of metalloproteinases 1 in plasma yield high diagnostic sensitivity and specificity in patients with colon cancer

    DEFF Research Database (Denmark)

    Holten-Andersen, Mads N; Christensen, Ib Jarle; Nielsen, Hans Jørgen

    2002-01-01

    : Total TIMP-1 plasma levels were measured by ELISA in blood samples from two different blood donor populations from IBD patients, and preoperative samples from patients with primary colon cancer (CC), rectal cancer (RC), or breast cancer. RESULTS: There were no significant differences in plasma TIMP-1......PURPOSE: The purpose of this study was to measure total levels of tissue inhibitor of metalloproteinases (TIMP-1) by ELISA in plasma from blood donors, patients with inflammatory bowel disease (IBD), and patients with cancer and to correlate the results to patient diagnosis. EXPERIMENTAL DESIGN...... levels between healthy donors and IBD or breast cancer patients, whereas patients with CC or RC had significantly elevated TIMP-1 levels. Total TIMP-1 levels identified patients with CC with a sensitivity of 63% at 98% specificity, patients with early CC (Dukes' A+B) with a sensitivity of 56% at 98...

  15. Tissue-specific expression and regulation by 1,25(OH)2D3 of chick protein kinase inhibitor (PKI) mRNA.

    Science.gov (United States)

    Marchetto, G S; Henry, H L

    1997-02-01

    The heat-stable protein kinase inhibitor (PKI) protein is a specific and potent competitive inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKA). Previously, it has been shown that vitamin D status affects chick kidney PKI activity: a 5- to 10-fold increase in PKI activity was observed in kidneys of chronically vitamin D-deficient chicks and treatment with 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) in cultured kidney cells resulted in a 95% decrease in PKI activity. The authors have recently cloned the cDNA for chick kidney PKI and have used the coding sequence to study the regulation of PKI mRNA. Northern analysis showed the expression of two PKI messages, which are 2.7 and 3.3 kb in size. These mRNAs are expressed in brain, muscle, testis, and kidney, but not in pancreas, liver, or intestine. PKI mRNA steady-state levels are downregulated by 47% in kidneys from vitamin D-replete chicks as compared to vitamin D-deficient chicks. PKI mRNA levels in brain, muscle, and testis are not affected by vitamin D status. Treatment of primary chick kidney cultures treated with 10(-7) M 1,25(OH)2D3 for 24h resulted in a 20-30% decrease in PKI mRNA. 1,25(OH)2D3 treatment does not affect the stability of PKI mRNA as determined by treatment of cell cultures with actinomycin D. This study shows that 1,25(OH)2D3 directly and tissue-specifically downregulates PKI mRNA in the chick kidney.

  16. Function-Oriented Synthesis of Marine Phidianidine Derivatives as Potential PTP1B Inhibitors with Specific Selectivity

    Directory of Open Access Journals (Sweden)

    Jin Liu

    2018-03-01

    Full Text Available Phidianidines A and B are two novel marine indole alkaloids bearing an uncommon 1,2,4-oxadiazole ring and exhibiting various biological activities. Our previous research showed that the synthesized phidianidine analogs had the potential to inhibit the activity of protein tyrosine phosphatase 1B (PTP1B, a validated target for Type II diabetes, which indicates that these analogs are worth further structural modification. Therefore, in this paper, a series of phidianidine derivatives were designed and rapidly synthesized with a function-oriented synthesis (FOS strategy. Their inhibitory effects on PTP1B and T-cell protein tyrosine phosphatase (TCPTP were evaluated, and several compounds displayed significant inhibitory potency and specific selectivity over PTP1B. The structure–activity relationship (SAR and molecular docking analyses are also described.

  17. Function-Oriented Synthesis of Marine Phidianidine Derivatives as Potential PTP1B Inhibitors with Specific Selectivity.

    Science.gov (United States)

    Liu, Jin; Chen, Yu; Li, Jing-Ya; Luo, Cheng; Li, Jia; Chen, Kai-Xian; Li, Xu-Wen; Guo, Yue-Wei

    2018-03-20

    Phidianidines A and B are two novel marine indole alkaloids bearing an uncommon 1,2,4-oxadiazole ring and exhibiting various biological activities. Our previous research showed that the synthesized phidianidine analogs had the potential to inhibit the activity of protein tyrosine phosphatase 1B (PTP1B), a validated target for Type II diabetes, which indicates that these analogs are worth further structural modification. Therefore, in this paper, a series of phidianidine derivatives were designed and rapidly synthesized with a function-oriented synthesis (FOS) strategy. Their inhibitory effects on PTP1B and T-cell protein tyrosine phosphatase (TCPTP) were evaluated, and several compounds displayed significant inhibitory potency and specific selectivity over PTP1B. The structure-activity relationship (SAR) and molecular docking analyses are also described.

  18. Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains.

    Science.gov (United States)

    Sha, Fern; Gencer, Emel Basak; Georgeon, Sandrine; Koide, Akiko; Yasui, Norihisa; Koide, Shohei; Hantschel, Oliver

    2013-09-10

    The dysregulated tyrosine kinase BCR-ABL causes chronic myelogenous leukemia in humans and forms a large multiprotein complex that includes the Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2). The expression of SHP2 is necessary for BCR-ABL-dependent oncogenic transformation, but the precise signaling mechanisms of SHP2 are not well understood. We have developed binding proteins, termed monobodies, for the N- and C-terminal SH2 domains of SHP2. Intracellular expression followed by interactome analysis showed that the monobodies are essentially monospecific to SHP2. Two crystal structures revealed that the monobodies occupy the phosphopeptide-binding sites of the SH2 domains and thus can serve as competitors of SH2-phosphotyrosine interactions. Surprisingly, the segments of both monobodies that bind to the peptide-binding grooves run in the opposite direction to that of canonical phosphotyrosine peptides, which may contribute to their exquisite specificity. When expressed in cells, monobodies targeting the N-SH2 domain disrupted the interaction of SHP2 with its upstream activator, the Grb2-associated binder 2 adaptor protein, suggesting decoupling of SHP2 from the BCR-ABL protein complex. Inhibition of either N-SH2 or C-SH2 was sufficient to inhibit two tyrosine phosphorylation events that are critical for SHP2 catalytic activity and to block ERK activation. In contrast, targeting the N-SH2 or C-SH2 revealed distinct roles of the two SH2 domains in downstream signaling, such as the phosphorylation of paxillin and signal transducer and activator of transcription 5. Our results delineate a hierarchy of function for the SH2 domains of SHP2 and validate monobodies as potent and specific antagonists of protein-protein interactions in cancer cells.

  19. Adapted J6/JFH1-based Hepatitis C virus recombinants with genotype-specific NS4A show similar efficacies against lead protease inhibitors, alpha interferon, and a putative NS4A inhibitor

    DEFF Research Database (Denmark)

    Gottwein, Judith M; Jensen, Sanne B; Serre, Stéphanie B N

    2013-01-01

    To facilitate studies of hepatitis C virus (HCV) NS4A, we aimed at developing J6/JFH1-based recombinants with genotype 1- to 7-specific NS4A proteins. We developed efficient culture systems expressing NS4A proteins of genotypes (isolates) 1a (H77 and TN), 1b (J4), 2a (J6), 4a (ED43), 5a (SA13), 6a...... (HK6a), and 7a (QC69), with peak infectivity titers of ∼3.5 to 4.5 log10 focus-forming units per ml. Except for genotype 2a (J6), growth depended on adaptive mutations identified in long-term culture. Genotype 1a, 1b, and 4a recombinants were adapted by amino acid substitutions F772S (p7) and V1663A...... (NS4A), while 5a, 6a, and 7a recombinants required additional substitutions in the NS3 protease and/or NS4A. We demonstrated applicability of the developed recombinants for study of antivirals. Genotype 1 to 7 NS4A recombinants showed similar responses to the protease inhibitors telaprevir (VX-950...

  20. Human leukocyte and porcine pancreatic elastase: X-ray crystal structures, mechanism, substrate specificity, and mechanism-based inhibitors

    International Nuclear Information System (INIS)

    Bode, W.; Meyer, E. Jr.; Powers, J.C.

    1989-01-01

    The serine protease family of enzymes is one of the most widely studied group of enzymes, as evidenced by the fact that more crystal structures are available for individuals of this superfamily than for any other homologous group of enzymes. These enzymes contain a conserved triad of catalytic residues including Ser-195, His-57, and Asp-102. The active-site serine is very nucleophilic, and serine proteases are inhibited by specific serine protease reagents such as diisopropyl phosphorofluoridate (DFP), phenylmethanesulfonyl fluoride, and 3,4-dichloroisocoumarin. Elastases are a group of proteases that possess the ability to cleave the important connective tissue protein elastin. Elastin has the unique property of elastic recoil, is widely distributed in vertebrate tissue, and is particularly abundant in the lungs, arteries, skin, and ligaments. Human neutrophil elastase and pancreatic elastase are two major serine proteases that cleave elastin. Neutrophil elastase is found in the dense granules of polymorphonuclear leukycytes and is essential for phagocytosis and defense against infection by invading microorganisms. Pancreatic elastase is stored as an inactive zymogen in the pancreas and is secreted into the intestines where it becomes activated by trypsin and then participates in digestion. Both elastases cleave substrates at peptide bonds where the P 1 residue is an amino acid residue with a small alkyl side chain

  1. Tissue-Specific Control of the Endocycle by the Anaphase Promoting Complex/Cyclosome Inhibitors UVI4 and DEL1.

    Science.gov (United States)

    Heyman, Jefri; Polyn, Stefanie; Eekhout, Thomas; De Veylder, Lieven

    2017-09-01

    The endocycle represents a modified mitotic cell cycle that in plants is often coupled to cell enlargement and differentiation. Endocycle onset is controlled by activity of the Anaphase Promoting Complex/Cyclosome (APC/C), a multisubunit E3 ubiquitin ligase targeting cell-cycle factors for destruction. CELL CYCLE SWITCH52 (CCS52) proteins represent rate-limiting activator subunits of the APC/C. In Arabidopsis ( Arabidopsis thaliana ), mutations in either CCS52A1 or CCS52A2 activators result in a delayed endocycle onset, whereas their overexpression triggers increased DNA ploidy levels. Here, the relative contribution of the APC/C CCS52A1 and APC/C CCS52A2 complexes to different developmental processes was studied through analysis of their negative regulators, being the ULTRAVIOLET-B-INSENSITIVE4 protein and the DP-E2F-Like1 transcriptional repressor, respectively. Our data illustrate cooperative activity of the APC/C CCS52A1 and APC/C CCS52A2 complexes during root and trichome development, but functional interdependency during leaf development. Furthermore, we found APC/C CCS52A1 activity to control CCS52A2 expression. We conclude that interdependency of CCS52A-controlled APC/C activity is controlled in a tissue-specific manner. © 2017 American Society of Plant Biologists. All Rights Reserved.

  2. A PKA survival pathway inhibited by DPT-PKI, a new specific cell permeable PKA inhibitor, is induced by T. annulata in parasitized B-lymphocytes.

    Science.gov (United States)

    Guergnon, Julien; Dessauge, Frederic; Traincard, François; Cayla, Xavier; Rebollo, Angelita; Bost, Pierre Etienne; Langsley, Gordon; Garcia, Alphonse

    2006-08-01

    T. annulata, an intracellular pathogenic parasite of the Aplicomplexa protozoan family infects bovine B-lymphocytes and macrophages. Parasitized cells that become transformed survive and proliferate independently of exogenous growth factors. In the present study, we used the isogenic non parasitized BL3 and parasitized TBL3 B cell lines, as a model to evaluate the contribution of two-major PI3-K- and PKA-dependent anti-apoptotic pathways in the survival of T. annulata parasitized B lymphocytes. We found that T. annulata increases PKA activity, induces over-expression of the catalytic subunit and down-regulates the pro-survival phosphorylation state of Akt/PKB. Consistent with a role of PKA activation in survival, two pharmacological inhibitors H89 and KT5720 ablate PKA-dependent survival of parasitized cells. To specifically inhibit PKA pro-survival pathways we linked the DPTsh1 peptide shuttle sequence to PKI(5-24) and we generated DPT-PKI, a cell permeable PKI. DPT-PKI specifically inhibited PKA activity in bovine cell extracts and, as expected, also inhibited the PKA-dependent survival of T. annulata parasitized TBL3 cells. Thus, parasite-dependent constitutive activation of PKA in TBL3 cells generates an anti-apoptotic pathway that can protect T. annulata-infected B cells from apoptosis. These results also indicate that DPT-PKI could be a powerful tool to inhibit PKA pathways in other cell types.

  3. Crystal Structure of the Homo sapiens Kynureninase-3-Hydroxyhippuric Acid Inhibitor Complex: Insights into the Molecular Basis Of Kynureninase Substrate Specificity

    Energy Technology Data Exchange (ETDEWEB)

    Lima,Santiago; Kumar,Sunil; Gawandi,Vijay; Momany,Cory; Phillips,Robert S.; (Georgia)

    2009-02-23

    Homo sapiens kynureninase is a pyridoxal-5'-phosphate dependent enzyme that catalyzes the hydrolytic cleavage of 3-hydroxykynurenine to yield 3-hydroxyanthranilate and L-alanine as part of the tryptophan catabolic pathway leading to the de novo biosynthesis of NAD{sup +}. This pathway results in quinolinate, an excitotoxin that is an NMDA receptor agonist. High levels of quinolinate have been correlated with the etiology of neurodegenerative disorders such as AIDS-related dementia and Alzheimer's disease. We have synthesized a novel kynureninase inhibitor, 3-hydroxyhippurate, cocrystallized it with human kynureninase, and solved the atomic structure. On the basis of an analysis of the complex, we designed a series of His-102, Ser-332, and Asn-333 mutants. The H102W/N333T and H102W/S332G/N333T mutants showed complete reversal of substrate specificity between 3-hydroxykynurenine and L-kynurenine, thus defining the primary residues contributing to substrate specificity in kynureninases.

  4. Icotinib, a potent and specific EGFR tyrosine kinase inhibitor, inhibits growth of squamous cell carcinoma cell line A431 through negatively regulating AKT signaling.

    Science.gov (United States)

    Gao, Zhenzhen; Chen, Wei; Zhang, Xiaohua; Cai, Peifen; Fang, Xianying; Xu, Qiang; Sun, Yang; Gu, Yanhong

    2013-06-01

    Icotinib is a potent and specific epidermal growth factor receptor tyrosine kinase inhibitor. In this study, we reported that icotinib had the antitumor activity on human squamous cell carcinoma cell line A431 in vitro. Meanwhile, adhesion to fibronectin and expression of integrin α3 and β1 were significantly reduced in a dose-dependent manner after the treatment of icotinib. Moreover, icotinib induced cell cycle arrested and affected expression of various cell cycle related proteins in squamous cancer cell line A431, whereas it did not cause apoptosis. Furthermore, icotinib remarkably down-regulated phosphorylation of protein kinase B (AKT) though blocking the interaction between 3-phosphoinositide-dependent protein kinase-1 (PDK1) and AKT in A431 cells. Taken together, it is shown that the small molecular compound, icotinib, has an anti-squamous cell carcinoma activity in vitro and its antitumor mechanism is associated with the blockage of the interaction between PDK1 and AKT. These results provide a novel strategy for anti-squamous cell carcinoma therapy. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  5. A multimodal RAGE-specific inhibitor reduces amyloid β–mediated brain disorder in a mouse model of Alzheimer disease

    Science.gov (United States)

    Deane, Rashid; Singh, Itender; Sagare, Abhay P.; Bell, Robert D.; Ross, Nathan T.; LaRue, Barbra; Love, Rachal; Perry, Sheldon; Paquette, Nicole; Deane, Richard J.; Thiyagarajan, Meenakshisundaram; Zarcone, Troy; Fritz, Gunter; Friedman, Alan E.; Miller, Benjamin L.; Zlokovic, Berislav V.

    2012-01-01

    In Alzheimer disease (AD), amyloid β peptide (Aβ) accumulates in plaques in the brain. Receptor for advanced glycation end products (RAGE) mediates Aβ-induced perturbations in cerebral vessels, neurons, and microglia in AD. Here, we identified a high-affinity RAGE-specific inhibitor (FPS-ZM1) that blocked Aβ binding to the V domain of RAGE and inhibited Aβ40- and Aβ42-induced cellular stress in RAGE-expressing cells in vitro and in the mouse brain in vivo. FPS-ZM1 was nontoxic to mice and readily crossed the blood-brain barrier (BBB). In aged APPsw/0 mice overexpressing human Aβ-precursor protein, a transgenic mouse model of AD with established Aβ pathology, FPS-ZM1 inhibited RAGE-mediated influx of circulating Aβ40 and Aβ42 into the brain. In brain, FPS-ZM1 bound exclusively to RAGE, which inhibited β-secretase activity and Aβ production and suppressed microglia activation and the neuroinflammatory response. Blockade of RAGE actions at the BBB and in the brain reduced Aβ40 and Aβ42 levels in brain markedly and normalized cognitive performance and cerebral blood flow responses in aged APPsw/0 mice. Our data suggest that FPS-ZM1 is a potent multimodal RAGE blocker that effectively controls progression of Aβ-mediated brain disorder and that it may have the potential to be a disease-modifying agent for AD. PMID:22406537

  6. Mechanism of action of hypoglycemic effects of an intestine-specific inhibitor of microsomal triglyceride transfer protein (MTP) in obese rats.

    Science.gov (United States)

    Sakata, Shohei; Katsumi, Sohei; Mera, Yasuko; Kuroki, Yukiharu; Nashida, Reiko; Kakutani, Makoto; Ohta, Takeshi

    2015-01-01

    Diminished insulin sensitivity in the peripheral tissues and failure of pancreatic beta cells to secrete insulin are known major determinants of type 2 diabetes mellitus. JTT-130, an intestine-specific microsomal transfer protein inhibitor, has been shown to suppress high fat-induced obesity and ameliorate impaired glucose tolerance while enhancing glucagon-like peptide-1 (GLP-1) secretion. We investigated the effects of JTT-130 on glucose metabolism and elucidated the mechanism of action, direct effects on insulin sensitivity and glucose-stimulated insulin secretion in a high fat diet-induced obesity rat model. Male Sprague Dawley rats fed a high-fat diet were treated with a single administration of JTT-130. Glucose tolerance, hyperglycemic clamp and hyperinsulinemic-euglycemic testing were performed to assess effects on insulin sensitivity and glucose-stimulated insulin secretion, respectively. Plasma GLP-1 and tissue triglyceride content were also determined under the same conditions. A single administration of JTT-130 suppressed plasma glucose elevations after oral glucose loading and increased the disposition index while elevating GLP-1. JTT-130 also enhanced glucose-stimulated insulin secretion in hyperglycemic clamp tests, whereas increased insulin sensitivity was observed in hyperinsulinemic-euglycemic clamp tests. Single-dose administration of JTT-130 decreased lipid content in the liver and skeletal muscle. JTT-130 demonstrated acute and direct hypoglycemic effects by enhancing insulin secretion and/or insulin sensitivity. Copyright © 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

  7. Insights into the molecular mechanism of action of Celastraceae sesquiterpenes as specific, non-transported inhibitors of human P-glycoprotein.

    Science.gov (United States)

    Muñoz-Martínez, Francisco; Reyes, Carolina P; Pérez-Lomas, Antonio L; Jiménez, Ignacio A; Gamarro, Francisco; Castanys, Santiago

    2006-01-01

    Dihydro-beta-agarofuran sesquiterpenes from Celastraceae have been recently shown to bind to human P-glycoprotein (Pgp), functioning as specific, mixed-type inhibitors of its drug transport activity, as well as multidrug resistance (MDR) modulators in vitro. However, nothing is known about whether such compounds are themselves transported by Pgp, or whether they affect Pgp expression as well as its activity, or about the location of their binding site within the protein. We performed transport experiments with a newly synthesized fluorescent sesquiterpene derivative, which retains the anti-Pgp activity of its natural precursor. This probe was poorly transported by Pgp, MRP1, MRP2 and BCRP transporters, compared with classical MDR substrates. Moreover, Pgp did not confer cross-resistance to the most potent dihydro-beta-agarofurans, which did not affect Pgp expression levels in several MDR cell lines. Finally, we observed competitive and non-competitive interactions between one of such dihydro-beta-agarofurans (Mama12) and classical Pgp modulators such as cyclosporin A, verapamil, progesterone, vinblastine and GF120918. These findings suggest that multidrug ABC transporters do not confer resistance to dihydro-beta-agarofurans and could not affect their absorption and biodistribution in the body. Moreover, we mapped their binding site(s) within Pgp, which may prove useful for the rational design of improved modulators based on the structure of dihydro-beta-agarofurans.

  8. 5α-Reductase inhibitor is less effective in men with small prostate volume and low serum prostatic specific antigen level.

    Science.gov (United States)

    Lin, Victor C; Liao, Chun-Hou; Wang, Chung-Cheng; Kuo, Hann-Chorng

    2015-09-01

    Large total prostate volumes (TPVs) or high serum prostate-specific antigen (PSA) levels indicate high-risk clinical progression of benign prostatic hyperplasia. This prospective study investigated the treatment outcome of combined 5α-reductase inhibitor and α-blocker in patients with and without large TPVs or high PSA levels. Men aged ≥ 45 years with International Prostate Symptom scores (IPSS) ≥ 8, TPV ≥ 20 mL, and maximum flow rate ≤ 15 mL/s received a combination therapy (dutasteride plus doxaben) for 2 years. Patients with baseline PSA ≥ 4 ng/mL underwent prostatic biopsy for excluding malignancy. The changes in the parameters from baseline to 24 months after combination therapy were compared in those with and without TPV ≥ 40 mL or PSA levels ≥ 1.5 ng/mL. A total of 285 patients (mean age 72 ± 9 years) completed the study. Combination therapy resulted in significant continuous improvement in IPSS, quality of life index, maximum flow rate, and postvoid residual (all p < 0.0001) regardless of baseline TPV or PSA levels. However, only patients with baseline TPV ≥ 40 mL had significant improvements in IPSS-storage subscore, voided volume, reduction in TPV, transitional zone index, and PSA levels. In addition, patients with baseline TPV < 40 mL and PSA < 1.5 ng/mL had neither a reduction in TPV nor a decrease in serum PSA level. A high TPV indicates more outlet resistance, whereas elevated serum PSA level reflects glandular proliferation. Thus, patients with TPV<40 mL and low PSA levels has less benefit from 5α-reductase inhibitor therapy. The therapeutic effect of combined treatment may arise mainly from the α-blocker in these patients. Copyright © 2013. Published by Elsevier B.V.

  9. 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.

  10. Differentiation stage-specific regulation of primitive human hematopoietic progenitor cycling by exogenous and endogenous inhibitors in an in vivo model.

    Science.gov (United States)

    Cashman, J D; Clark-Lewis, I; Eaves, A C; Eaves, C J

    1999-12-01

    Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice transplanted with human cord blood or adult marrow cells and injected 6 weeks posttransplant with 2 daily doses of transforming growth factor-beta(1) (TGF-beta(1)), monocyte chemoattractant protein-1 (MCP-1), or a nonaggregating form of macrophage inflammatory protein-1alpha (MIP-1alpha) showed unique patterns of inhibition of human progenitor proliferation 1 day later. TGF-beta(1) was active on long-term culture initiating cells (LTC-IC) and on primitive erythroid and granulopoietic colony-forming cells (HPP-CFC), but had no effect on mature CFC. MCP-1 inhibited the cycling of both types of HPP-CFC but not LTC-IC. MIP-1alpha did not inhibit either LTC-IC or granulopoietic HPP-CFC but was active on erythroid HPP-CFC and mature granulopoietic CFC. All of these responses were independent of the source of human cells transplanted. LTC-IC of either human cord blood or adult marrow origin continue to proliferate in NOD/SCID mice for many weeks, although the turnover of all types of human CFC in mice transplanted with adult human marrow (but not cord blood) is downregulated after 6 weeks. Interestingly, administration of either MIP-1beta, an antagonist of both MIP-1alpha and MCP-1 or MCP-1(9-76), an antagonist of MCP-1 (and MCP-2 and MCP-3), into mice in which human marrow-derived CFC had become quiescent, caused the rapid reactivation of these progenitors in vivo. These results provide the first definition of stage-specific inhibitors of human hematopoietic progenitor cell cycling in vivo. In addition they show that endogenous chemokines can contribute to late graft failure, which can be reversed by the administration of specific antagonists.

  11. The activity of non-specific esterase in the thyroid epithelial cells of the guinea pig as influenced by various inhibitors and activators. A histochemical study

    DEFF Research Database (Denmark)

    Kirkeby, S

    1976-01-01

    The action of various inhibitors and activators upon esterase activity in the thyroid epithelial cells is demonstrated. The agents used were triorthocresylphosphate (TOCP), parachloromercuribenzoate (PCMB), Arsanillic acid, p-nitrophenyl dimethyl carbamate and bis p-nitrophenyl phosphate. TOCP wa...

  12. Preclinical evaluation of BAY 1075553, a novel 18F-labelled inhibitor of prostate-specific membrane antigen for PET imaging of prostate cancer

    International Nuclear Information System (INIS)

    Lesche, Ralf; Kettschau, Georg; Gromov, Alexey V.; Boehnke, Niels; Borkowski, Sandra; Moenning, Ursula; Doehr, Olaf; Graham, Keith; Hegele-Hartung, Christa; Dinkelborg, Ludger M.

    2014-01-01

    Prostate-specific membrane antigen (PSMA) is a transmembrane protein overexpressed in prostate cancer and is therefore being explored as a biomarker for diagnosing and staging of the disease. Here we report preclinical data on BAY 1075553 (a 9:1 mixture of (2S,4S)- and (2R,4S)-2-[ 18 F]fluoro-4-phosphonomethyl-pentanedioic acid), a novel 18 F-labelled small molecule inhibitor of PSMA enzymatic activity, which can be efficiently synthesized from a direct radiolabelling precursor. The 18 F-radiolabelled stereoisomers of 2-[ 18 F]fluoro-4-(phosphonomethyl)-pentanedioic acid were synthesized from their respective isomerically pure precursors dimethyl 2-{[bis(benzyloxy)phosphoryl ]methyl}-4-(tosyloxy)pentanedioate. In vivo positron emission tomography (PET) imaging and biodistribution studies were conducted in mice bearing LNCaP, 22Rv1 and PC-3 tumours. Pharmacokinetic parameters and dosimetry estimates were calculated based on biodistribution studies in rodents. For non-clinical safety assessment (safety pharmacology, toxicology) to support a single-dose human microdose study, off-target effects in vitro, effects on vital organ functions (cardiovascular in dogs, nervous system in rats), mutagenicity screens and an extended single-dose study in rats were conducted with the non-radioactive racemic analogue of BAY 1075553. BAY 1075553 showed high tumour accumulation specific to PSMA-positive tumour-bearing mice and was superior to other stereoisomers tested. Fast clearance of BAY 1075553 resulted overall in low background signals in other organs except for high uptake into kidney and bladder which was mainly caused by renal elimination of BAY 1075553. A modest uptake into bone was observed which decreased over time indicating organ-specific uptake as opposed to defluorination of BAY 1075553 in vivo. Biodistribution studies found highest organ doses for kidneys and the urinary bladder wall resulting in a projected effective dose (ED) in humans of 0.0219 mSv/MBq. Non

  13. Preclinical evaluation of BAY 1075553, a novel {sup 18}F-labelled inhibitor of prostate-specific membrane antigen for PET imaging of prostate cancer

    Energy Technology Data Exchange (ETDEWEB)

    Lesche, Ralf; Kettschau, Georg; Gromov, Alexey V.; Boehnke, Niels; Borkowski, Sandra; Moenning, Ursula; Doehr, Olaf; Graham, Keith [Global Drug Discovery, Bayer Healthcare, Berlin, Germany, Berlin (Germany); Hegele-Hartung, Christa [Global Drug Discovery, Bayer Healthcare, Wuppertal, Germany, Wuppertal (Germany); Dinkelborg, Ludger M. [Global Drug Discovery, Bayer Healthcare, Berlin, Germany, Berlin (Germany); Piramal Imaging GmbH, Berlin (Germany)

    2014-01-15

    Prostate-specific membrane antigen (PSMA) is a transmembrane protein overexpressed in prostate cancer and is therefore being explored as a biomarker for diagnosing and staging of the disease. Here we report preclinical data on BAY 1075553 (a 9:1 mixture of (2S,4S)- and (2R,4S)-2-[{sup 18}F]fluoro-4-phosphonomethyl-pentanedioic acid), a novel {sup 18}F-labelled small molecule inhibitor of PSMA enzymatic activity, which can be efficiently synthesized from a direct radiolabelling precursor. The {sup 18}F-radiolabelled stereoisomers of 2-[{sup 18}F]fluoro-4-(phosphonomethyl)-pentanedioic acid were synthesized from their respective isomerically pure precursors dimethyl 2-{[bis(benzyloxy)phosphoryl ]methyl}-4-(tosyloxy)pentanedioate. In vivo positron emission tomography (PET) imaging and biodistribution studies were conducted in mice bearing LNCaP, 22Rv1 and PC-3 tumours. Pharmacokinetic parameters and dosimetry estimates were calculated based on biodistribution studies in rodents. For non-clinical safety assessment (safety pharmacology, toxicology) to support a single-dose human microdose study, off-target effects in vitro, effects on vital organ functions (cardiovascular in dogs, nervous system in rats), mutagenicity screens and an extended single-dose study in rats were conducted with the non-radioactive racemic analogue of BAY 1075553. BAY 1075553 showed high tumour accumulation specific to PSMA-positive tumour-bearing mice and was superior to other stereoisomers tested. Fast clearance of BAY 1075553 resulted overall in low background signals in other organs except for high uptake into kidney and bladder which was mainly caused by renal elimination of BAY 1075553. A modest uptake into bone was observed which decreased over time indicating organ-specific uptake as opposed to defluorination of BAY 1075553 in vivo. Biodistribution studies found highest organ doses for kidneys and the urinary bladder wall resulting in a projected effective dose (ED) in humans of 0.0219 m

  14. Three Dimensional Human Neuro-Spheroid Model of Alzheimer’s Disease Based on Differentiated Induced Pluripotent Stem Cells

    Science.gov (United States)

    Lee, Han-Kyu; Velazquez Sanchez, Clara; Chen, Mei; Morin, Peter J.; Wells, John M.; Hanlon, Eugene B.

    2016-01-01

    The testing of candidate drugs to slow progression of Alzheimer’s disease (AD) requires clinical trials that are lengthy and expensive. Efforts to model the biochemical milieu of the AD brain may be greatly facilitated by combining two cutting edge technologies to generate three-dimensional (3D) human neuro-spheroid from induced pluripotent stem cells (iPSC) derived from AD subjects. We created iPSC from blood cells of five AD patients and differentiated them into 3D human neuronal culture. We characterized neuronal markers of our 3D neurons by immunocytochemical staining to validate the differentiation status. To block the generation of pathologic amyloid β peptides (Aβ), the 3D-differentiated AD neurons were treated with inhibitors targeting β-secretase (BACE1) and γ-secretases. As predicted, both BACE1 and γ-secretase inhibitors dramatically decreased Aβ generation in iPSC-derived neural cells derived from all five AD patients, under standard two-dimensional (2D) differentiation conditions. However, BACE1 and γ-secretase inhibitors showed less potency in decreasing Aβ levels in neural cells differentiated under 3D culture conditions. Interestingly, in a single subject AD1, we found that BACE1 inhibitor treatment was not able to significantly reduce Aβ42 levels. To investigate underlying molecular mechanisms, we performed proteomic analysis of 3D AD human neuronal cultures including AD1. Proteomic analysis revealed specific reduction of several proteins that might contribute to a poor inhibition of BACE1 in subject AD1. To our knowledge, this is the first iPSC-differentiated 3D neuro-spheroid model derived from AD patients’ blood. Our results demonstrate that our 3D human neuro-spheroid model can be a physiologically relevant and valid model for testing efficacy of AD drug. PMID:27684569

  15. Identification of danthron as an isoform-specific inhibitor of HEME OXYGENASE-1/cytochrome P450 reductase interaction with anti-tumor activity.

    Science.gov (United States)

    Chou, Yi-Tai; Hsu, Fu-Fei; Hu, Dun-Yao; Chen, Ying-Chih; Hsu, Yuan-Hao; Hsu, John T-A; Chau, Lee-Young

    2018-01-23

    Heme oxygenase (HO) catalyzes NADPH-dependent degradation of heme to liberate iron, carbon monoxide and biliverdin. The interaction between HO and cytochrome P450 reductase (CPR), an electron donor, is essential for HO activity. HO-1 is a stress-inducible isoform whereas HO-2 is constitutively expressed. HO-1 induction is commonly seen in cancers and impacts disease progression, supporting the possibility of targeting HO-1 for cancer therapy. We employed a cell-based bioluminescence resonance energy transfer assay to screen compounds with ability to inhibit HO-1/CPR interaction. The effect of the identified compound on HO-1/CPR interaction was confirmed by pull down assay. Moreover, the anti-tumorigenic activity of the identified compound on HO-1-enhanced tumor growth and migration was assessed by trypan blue exclusion method and wound healing assay. Danthron was identified as an effective small molecule able to interfere with the interaction between HO-1 and CPR but not HO-2 and CPR. Additional experiments with structural analogues of danthron revealed that the positions of hydroxyl moieties significantly affected the potency of inhibition on HO-1/CPR interaction. Pull-down assay confirmed that danthron inhibited the interaction of CPR with HO-1 but not HO-2. Danthron suppressed growth and migration of HeLa cells with stable HO-1 overexpression but not mock cells. In contrast, anthrarufin, a structural analog with no ability to interfere HO-1/CPR interaction, exhibited no significant effect on HO-1-overexpressing HeLa cells. These findings demonstrate that danthron is an isoform-specific inhibitor for HO-1/CPR interaction and may serve as a lead compound for novel anticancer drug.

  16. A physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model of the histone deacetylase (HDAC) inhibitor vorinostat for pediatric and adult patients and its application for dose specification.

    Science.gov (United States)

    Moj, Daniel; Britz, Hannah; Burhenne, Jürgen; Stewart, Clinton F; Egerer, Gerlinde; Haefeli, Walter E; Lehr, Thorsten

    2017-11-01

    This study aimed at recommending pediatric dosages of the histone deacetylase (HDAC) inhibitor vorinostat and potentially more effective adult dosing regimens than the approved standard dosing regimen of 400 mg/day, using a comprehensive physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) modeling approach. A PBPK/PD model for vorinostat was developed for predictions in adults and children. It includes the maturation of relevant metabolizing enzymes. The PBPK model was expanded by (1) effect compartments to describe vorinostat concentration-time profiles in peripheral blood mononuclear cells (PBMCs), (2) an indirect response model to predict the HDAC inhibition, and (3) a thrombocyte model to predict the dose-limiting thrombocytopenia. Parameterization of drug and system-specific processes was based on published and unpublished in silico, in vivo, and in vitro data. The PBPK modeling software used was PK-Sim and MoBi. The PBPK/PD model suggests dosages of 80 and 230 mg/m 2 for children of 0-1 and 1-17 years of age, respectively. In comparison with the approved standard treatment, in silico trials reveal 11 dosing regimens (9 oral, and 2 intravenous infusion rates) increasing the HDAC inhibition by an average of 31%, prolonging the HDAC inhibition by 181%, while only decreasing the circulating thrombocytes to a tolerable 53%. The most promising dosing regimen prolongs the HDAC inhibition by 509%. Thoroughly developed PBPK models enable dosage recommendations in pediatric patients and integrated PBPK/PD models, considering PD biomarkers (e.g., HDAC activity and platelet count), are well suited to guide future efficacy trials by identifying dosing regimens potentially superior to standard dosing regimens.

  17. Molecular phylogeny of C1 inhibitor depicts two immunoglobulin-like domains fusion in fishes and ray-finned fishes specific intron insertion after separation from zebrafish

    International Nuclear Information System (INIS)

    Kumar, Abhishek; Bhandari, Anita; Sarde, Sandeep J.; Goswami, Chandan

    2014-01-01

    Highlights: • C1 inhibitors of fishes have two Ig domains fused in the N-terminal end. • Spliceosomal introns gain in two Ig domains of selected ray-finned fishes. • C1 inhibitors gene is maintained from 450 MY on the same locus. • C1 inhibitors gene is missing in frog and lampreys. • C1 inhibitors of tetrapod and fishes differ in the RCL region. - Abstract: C1 inhibitor (C1IN) is a multi-facet serine protease inhibitor in the plasma cascades, inhibiting several proteases, notably, regulates both complement and contact system activation. Despite huge advancements in the understanding of C1IN based on biochemical properties and its roles in the plasma cascades, the phylogenetic history of C1IN remains uncharacterized. To date, there is no comprehensive study illustrating the phylogenetic history of C1IN. Herein, we explored phylogenetic history of C1IN gene in vertebrates. Fishes have C1IN with two immunoglobulin like domains attached in the N-terminal region. The RCL regions of CIIN from fishes and tetrapod genomes have variations at the positions P2 and P1′. Gene structures of C1IN gene from selected ray-finned fishes varied in the Ig domain region with creation of novel intron splitting exon Im2 into Im2a and Im2b. This intron is limited to ray-finned fishes with genome size reduced below 1 Gb. Hence, we suggest that genome compaction and associated double-strand break repairs are behind this intron gain. This study reveals the evolutionary history of C1IN and confirmed that this gene remains the same locus for ∼450 MY in 52 vertebrates analysed, but it is not found in frogs and lampreys

  18. Molecular phylogeny of C1 inhibitor depicts two immunoglobulin-like domains fusion in fishes and ray-finned fishes specific intron insertion after separation from zebrafish

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Abhishek, E-mail: akumar@bot.uni-kiel.de [Department of Genetics and Molecular Biology in Botany, Institute of Botany, Christian-Albrechts-University at Kiel, Kiel (Germany); Bhandari, Anita [Molecular Physiology, Zoological Institute, Christian-Albrechts-University at Kiel, Kiel (Germany); Sarde, Sandeep J. [Department of Genetics and Molecular Biology in Botany, Institute of Botany, Christian-Albrechts-University at Kiel, Kiel (Germany); Goswami, Chandan [National Institute of Science Education and Research, Bhubaneswar, Orissa (India)

    2014-07-18

    Highlights: • C1 inhibitors of fishes have two Ig domains fused in the N-terminal end. • Spliceosomal introns gain in two Ig domains of selected ray-finned fishes. • C1 inhibitors gene is maintained from 450 MY on the same locus. • C1 inhibitors gene is missing in frog and lampreys. • C1 inhibitors of tetrapod and fishes differ in the RCL region. - Abstract: C1 inhibitor (C1IN) is a multi-facet serine protease inhibitor in the plasma cascades, inhibiting several proteases, notably, regulates both complement and contact system activation. Despite huge advancements in the understanding of C1IN based on biochemical properties and its roles in the plasma cascades, the phylogenetic history of C1IN remains uncharacterized. To date, there is no comprehensive study illustrating the phylogenetic history of C1IN. Herein, we explored phylogenetic history of C1IN gene in vertebrates. Fishes have C1IN with two immunoglobulin like domains attached in the N-terminal region. The RCL regions of CIIN from fishes and tetrapod genomes have variations at the positions P2 and P1′. Gene structures of C1IN gene from selected ray-finned fishes varied in the Ig domain region with creation of novel intron splitting exon Im2 into Im2a and Im2b. This intron is limited to ray-finned fishes with genome size reduced below 1 Gb. Hence, we suggest that genome compaction and associated double-strand break repairs are behind this intron gain. This study reveals the evolutionary history of C1IN and confirmed that this gene remains the same locus for ∼450 MY in 52 vertebrates analysed, but it is not found in frogs and lampreys.

  19. Affinity and specificity of serine endopeptidase-protein inhibitor interactions. Empirical free energy calculations based on X-ray crystallographic structures.

    Science.gov (United States)

    Krystek, S; Stouch, T; Novotny, J

    1993-12-05

    An empirical function was used to calculate free energy change (delta G) of complex formation between the following inhibitors and enzymes: Kunitz inhibitor (BPTI) with trypsin, trypsinogen and kallikrein; turkey ovomucoid 3rd domain (OMTKY3) with alpha-chymotrypsin and the Streptomyces griseus protease B; the potato chymotrypsin inhibitor with the protease B; and the barely chymotrypsin inhibitor and eglin-c with subtilisin and thermitase. Using X-ray coordinates of the nine complexes, we estimated the contributions that hydrophobic effect, electrostatic interactions and side-chain conformational entropy make towards the stability of the complexes. The calculated delta G values showed good agreement with the experimentally measured ones, the only exception being the kallikrein/BPTI complex whose X-ray structure was solved at an exceptionally low pH. In complexes with different enzymes, the same inhibitor residues contributed identically towards complex formation (delta G(residue) Spearman rank correlation coefficient 0.7 to 1.0). The most productive enzyme-contacting residues in OMTKY3, eglin-c, and the chymotrypsin inhibitors were found in analogous positions on their respective binding loops; thus, our calculations identified a functional (energetic) motif that parallels the well-known structural similarity of the binding loops. The delta G values calculated for BPTI complexed with trypsin (-21.7 kcal) and trypsinogen (-23.4 kcal) were similar and close to the experimental delta G value of the trypsin/BPTI complex (-18.1 kcal), lending support to the suggestion that the 10(7) difference in the observed stabilities (KA) of these two complexes reflects the energetic cost of conformational changes induced in trypsinogen during the pre-equilibrium stages of complex formation. In almost all of the complexes studied, the stabilization free energy contributed by the inhibitors was larger than that donated by the enzymes. In the trypsin-BPTI complex, the calculated

  20. A preclinical study on the rescue of normal tissue by nicotinic acid in high-dose treatment with APO866, a specific nicotinamide phosphoribosyltransferase inhibitor

    DEFF Research Database (Denmark)

    Olesen, Uffe Høgh; Thougaard, Annemette V; Jensen, Peter Buhl

    2010-01-01

    Inhibitor of nicotinamide phosphoribosyltransferase APO866 is a promising cancer drug currently in phase II clinical trials in oncology. Here, we present a strategy for increasing the therapeutic potential of APO866 through the rescue of normal tissues by coadministration of nicotinic acid (Vitam...

  1. Glu-Ureido-Based Inhibitors of Prostate-Specific Membrane Antigen: Lessons Learned During the Development of a Novel Class of Low-Molecular-Weight Theranostic Radiotracers

    Czech Academy of Sciences Publication Activity Database

    Kopka, K.; Benesova, M.; Bařinka, Cyril; Haberkorn, U.; Babich, J.

    2017-01-01

    Roč. 58, Suppl. 2 (2017), s. 17-26 ISSN 0161-5505 Institutional support: RVO:86652036 Keywords : small-molecule PSMA inhibitors * theranostic PSMA radioligands * PSMA radiotracers Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 6.646, year: 2016

  2. Elafin, an elastase-specific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis.

    LENUS (Irish Health Repository)

    Guyot, Nicolas

    2008-11-21

    Elafin is a neutrophil serine protease inhibitor expressed in lung and displaying anti-inflammatory and anti-bacterial properties. Previous studies demonstrated that some innate host defense molecules of the cystic fibrosis (CF) and chronic obstructive pulmonary disease airways are impaired due to increased proteolytic degradation observed during lung inflammation. In light of these findings, we thus focused on the status of elafin in CF lung. We showed in the present study that elafin is cleaved in sputum from individuals with CF. Pseudomonas aeruginosa-positive CF sputum, which was found to contain lower elafin levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective in cleaving recombinant elafin. NE plays a pivotal role in the process as only NE inhibitors are able to inhibit elafin degradation. Further in vitro studies demonstrated that incubation of recombinant elafin with excess of NE leads to the rapid cleavage of the inhibitor. Two cleavage sites were identified at the N-terminal extremity of elafin (Val-5-Lys-6 and Val-9-Ser-10). Interestingly, purified fragments of the inhibitor (Lys-6-Gln-57 and Ser-10-Gln-57) were shown to still be active for inhibiting NE. However, NE in excess was shown to strongly diminish the ability of elafin to bind lipopolysaccharide (LPS) and its capacity to be immobilized by transglutamination. In conclusion, this study provides evidence that elafin is cleaved by its cognate enzyme NE present at excessive concentration in CF sputum and that P. aeruginosa infection promotes this effect. Such cleavage may have repercussions on the innate immune function of elafin.

  3. Inherited phenotype instability of inflorescence and floral organ development in homeotic barley double mutants and its specific modification by auxin inhibitors and 2,4-D.

    Science.gov (United States)

    Šiukšta, Raimondas; Vaitkūnienė, Virginija; Kaselytė, Greta; Okockytė, Vaiva; Žukauskaitė, Justina; Žvingila, Donatas; Rančelis, Vytautas

    2015-03-01

    Barley (Hordeum vulgare) double mutants Hv-Hd/tw2, formed by hybridization, are characterized by inherited phenotypic instability and by several new features, such as bract/leaf-like structures, long naked gaps in the spike, and a wide spectrum of variations in the basic and ectopic flowers, which are absent in single mutants. Several of these features resemble those of mutations in auxin distribution, and thus the aim of this study was to determine whether auxin imbalances are related to phenotypic variations and instability. The effects of auxin inhibitors and 2,4-D (2,4-dichlorophenoxyacetic acid) on variation in basic and ectopic flowers were therefore examined, together with the effects of 2,4-D on spike structure. The character of phenotypic instability and the effects of auxin inhibitors and 2,4-D were compared in callus cultures and intact plants of single homeotic Hv-tw2 and Hv-Hooded/Kap (in the BKn3 gene) mutants and alternative double mutant lines: offspring from individual plants in distal hybrid generations (F9-F10) that all had the same BKn3 allele as determined by DNA sequencing. For intact plants, two auxin inhibitors, 9-hydroxyfluorene-9-carboxylic acid (HFCA) and p-chlorophenoxyisobutyric acid (PCIB), were used. Callus growth and flower/spike structures of the Hv-tw2 mutant differed in their responses to HFCA and PCIB. An increase in normal basic flowers after exposure to auxin inhibitors and a decrease in their frequencies caused by 2,4-D were observed, and there were also modifications in the spectra of ectopic flowers, especially those with sexual organs, but the effects depended on the genotype. Exposure to 2,4-D decreased the frequency of short gaps and lodicule transformations in Hv-tw2 and of long naked gaps in double mutants. The effects of auxin inhibitors and 2,4-D suggest that ectopic auxin maxima or deficiencies arise in various regions of the inflorescence/flower primordia. Based on the phenotypic instability observed, definite

  4. A cheap, simple high throughput method for screening native Helicobacter pylori urease inhibitors using a recombinant Escherichia coli, its validation and demonstration of Pistacia atlantica methanolic extract effectivity and specificity.

    Science.gov (United States)

    Amar, Natalie; Peretz, Avi; Gerchman, Yoram

    2017-02-01

    Helicobacter pylori is the most frequent and persistent bacterial infection worldwide, and a risk factor for active gastritis, peptic ulcers, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. Although combined antibiotics treatment is effective cases of antibiotic resistance are reported at an alarming rate. The H. pylori urease enzyme is essential for the bacteria establishment in the gastric mucosa, resulting urease inhibitors being sought after as effective and specific anti- H. pylori treatment. To-date, screening assays are based mostly on the analog plant urease enzyme but difference in properties of the plant and bacterial enzymes hamper these efforts. We have developed a screening assay based on recombinant Escherichia coli expressing native H. pylori urease, and validated this assay using thiourea and a methanolic extract of Pistacia atlantica. The assay demonstrated the thiourea and the extract to be potent urease inhibitors, with the extract having strong bacteriostatic activity against clinical isolates of H. pylori, including such with antibiotic resistance. The extract was also found to be neutral toward common probiotic bacteria, supporting its specificity and compatibility with digestive system desired microflora and suggesting it could be a good source for anti-H. pylori compounds. The assay has proven to be cheap, simple and native alternative to the plant enzyme based assay and could allow for high throughput screening for new urease inhibitors and could expedite screening and development of novel, better H. pylori remedies helping us to combat this infection. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. A Rhodium(III)-Based Inhibitor of Lysine-Specific Histone Demethylase 1 as an Epigenetic Modulator in Prostate Cancer Cells.

    Science.gov (United States)

    Yang, Chao; Wang, Wanhe; Liang, Jia-Xin; Li, Guodong; Vellaisamy, Kasipandi; Wong, Chun-Yuen; Ma, Dik-Lung; Leung, Chung-Hang

    2017-03-23

    We report herein a novel rhodium(III) complex 1 as a new LSD1 targeting agent and epigenetic modulator. Complex 1 disrupted the interaction of LSD1-H3K4me2 in human prostate carcinoma cells and enhanced the amplification of p21, FOXA2, and BMP2 gene promoters. Complex 1 was selective for LSD1 over other histone demethylases, such as KDM2b, KDM7, and MAO activities, and also showed antiproliferative activity toward human cancer cells. To date, complex 1 is the first metal-based inhibitor of LSD1 activity.

  6. Proposal for novel curcumin derivatives as potent inhibitors against Alzheimer's disease: Ab initio molecular simulations on the specific interactions between amyloid-beta peptide and curcumin

    Science.gov (United States)

    Ota, Shintaro; Fujimori, Mitsuki; Ishimura, Hiromi; Shulga, Sergiy; Kurita, Noriyuki

    2017-10-01

    Accumulation of amyloid-β (Aβ) peptides in a brain is closely related with the pathogenesis of Alzheimer's disease. To suppress the production of Aβ peptides, we propose novel curcumin derivatives and investigate their binding properties with the amyloid precursor protein (APP), using protein-ligand docking as well as ab initio molecular simulations. Our proposed derivative (curcumin XIV) is found to have a large binding energy with APP and interacts strongly with the cleavage site Ala19 by secretase. It is thus expected that curcumin XIV can protect APP from the secretase attack and be a potent inhibitor against the production of Aβ peptides.

  7. Identification of a novel polyprenylated acylphloroglucinol‑derived SIRT1 inhibitor with cancer‑specific anti-proliferative and invasion-suppressing activities.

    Science.gov (United States)

    Zhu, Lijia; Qi, Ji; Chiao, Christine Ya-Chi; Zhang, Qiang; Porco, John A; Faller, Douglas V; Dai, Yan

    2014-11-01

    SIRT1, a class III histone deacetylase, plays a critical role in regulating cancer cell growth, migration and invasion, which makes it a potential target for cancer therapeutics. In this study, we screened derivatives of several groups of natural products and identified a novel SIRT1 inhibitor JQ-101, a synthetic derivative of the polyprenylated acylphloroglucinol (PPAP) natural products, with an IC(50) for SIRT1 of 30 µM in vitro, with 5-fold higher activity for SIRT1 vs. SIRT2. Exposure of tumor cells to JQ-101 significantly enhanced acetylation of p53 and histone H4K16 at known sites of SIRT1 deacetylation, validating SIRT1 as its cellular target. JQ-101 suppressed cancer cell growth and survival by targeting SIRT1, and also exhibited selective cytotoxicity towards a panel of human tumor cell lines, while producing no toxicity in two normal human cell types at comparable concentrations. JQ-101 induced both apoptosis and cell senescence, and suppressed cancer cell invasion in vitro. In summary, we have identified JQ-101 as a new SIRT1 inhibitor which may have potential application in cancer treatment through its ability to induce tumor cell apoptosis and senescence and suppress cancer cell invasion.

  8. Identification of a novel polyprenylated acylphloroglucinol-derived SIRT1 inhibitor with cancer-specific anti-proliferative and invasion-suppressing activities

    Science.gov (United States)

    ZHU, LIJIA; QI, JI; CHIAO, CHRISTINE YA-CHI; ZHANG, QIANG; PORCO, JOHN A.; FALLER, DOUGLAS V.; DAI, YAN

    2014-01-01

    SIRT1, a class III histone deacetylase, plays a critical role in regulating cancer cell growth, migration and invasion, which makes it a potential target for cancer therapeutics. In this study, we screened derivatives of several groups of natural products and identified a novel SIRT1 inhibitor JQ-101, a synthetic derivative of the polyprenylated acylphloroglucinol (PPAP) natural products, with an IC50 for SIRT1 of 30 μM in vitro, with 5-fold higher activity for SIRT1 vs. SIRT2. Exposure of tumor cells to JQ-101 significantly enhanced acetylation of p53 and histone H4K16 at known sites of SIRT1 deacetylation, validating SIRT1 as its cellular target. JQ-101 suppressed cancer cell growth and survival by targeting SIRT1, and also exhibited selective cytotoxicity towards a panel of human tumor cell lines, while producing no toxicity in two normal human cell types at comparable concentrations. JQ-101 induced both apoptosis and cell senescence, and suppressed cancer cell invasion in vitro. In summary, we have identified JQ-101 as a new SIRT1 inhibitor which may have potential application in cancer treatment through its ability to induce tumor cell apoptosis and senescence and suppress cancer cell invasion. PMID:25189993

  9. Combination Treatment with PPARγ Ligand and Its Specific Inhibitor GW9662 Downregulates BIS and 14-3-3 Gamma, Inhibiting Stem-Like Properties in Glioblastoma Cells.

    Science.gov (United States)

    Im, Chang-Nim

    2017-01-01

    PPAR γ is a nuclear receptor that regulates differentiation and proliferation and is highly expressed in many cancer cells. Its synthetic ligands, such as rosiglitazone and ciglitazone, and its inhibitor GW9662, were shown to induce cellular differentiation, inhibit proliferation, and lead to apoptosis. Glioblastoma is a common brain tumor with poor survival prospects. Recently, glioblastoma stem cells (GSCs) have been examined as a potential target for anticancer therapy; however, little is known about the combined effect of various agents on GSCs. In this study, we found that cotreatment with PPAR γ ligands and GW9662 inhibited stem-like properties in GSC-like spheres, which significantly express SOX2. In addition, this treatment decreased the activation of STAT3 and AKT and decreased the amounts of 14-3-3 gamma and BIS proteins. Moreover, combined administration of small-interfering RNA (siRNA) transfection with PPAR γ ligands induced downregulation of SOX2 and MMP2 activity together with inhibition of sphere-forming activity regardless of poly(ADP-ribose) polymerase (PARP) cleavage. Taken together, our findings suggest that a combination therapy using PPAR γ ligands and its inhibitor could be a potential therapeutic strategy targeting GSCs.

  10. Combination Treatment with PPARγ Ligand and Its Specific Inhibitor GW9662 Downregulates BIS and 14-3-3 Gamma, Inhibiting Stem-Like Properties in Glioblastoma Cells

    Directory of Open Access Journals (Sweden)

    Chang-Nim Im

    2017-01-01

    Full Text Available PPARγ is a nuclear receptor that regulates differentiation and proliferation and is highly expressed in many cancer cells. Its synthetic ligands, such as rosiglitazone and ciglitazone, and its inhibitor GW9662, were shown to induce cellular differentiation, inhibit proliferation, and lead to apoptosis. Glioblastoma is a common brain tumor with poor survival prospects. Recently, glioblastoma stem cells (GSCs have been examined as a potential target for anticancer therapy; however, little is known about the combined effect of various agents on GSCs. In this study, we found that cotreatment with PPARγ ligands and GW9662 inhibited stem-like properties in GSC-like spheres, which significantly express SOX2. In addition, this treatment decreased the activation of STAT3 and AKT and decreased the amounts of 14-3-3 gamma and BIS proteins. Moreover, combined administration of small-interfering RNA (siRNA transfection with PPARγ ligands induced downregulation of SOX2 and MMP2 activity together with inhibition of sphere-forming activity regardless of poly(ADP-ribose polymerase (PARP cleavage. Taken together, our findings suggest that a combination therapy using PPARγ ligands and its inhibitor could be a potential therapeutic strategy targeting GSCs.

  11. TMG-chitotriomycin, an enzyme inhibitor specific for insect and fungal beta-N-acetylglucosaminidases, produced by actinomycete Streptomyces anulatus NBRC 13369.

    Science.gov (United States)

    Usuki, Hirokazu; Nitoda, Teruhiko; Ichikawa, Misato; Yamaji, Nahoko; Iwashita, Takashi; Komura, Hajime; Kanzaki, Hiroshi

    2008-03-26

    A novel beta-N-acetylglucosaminidase (GlcNAcase) inhibitor named TMG-chitotriomycin (1) was isolated from the culture filtrate of Streptomyces anulatus NBRC13369. The strain produced 1 only when colloidal chitin was used as the sole carbon source in the production medium. The structure of 1 was determined by spectral and constitutive sugar analyses of the corresponding alditol derivatives to be an equilibrated mixture of alpha-d-N,N,N-triMeGlcNH2-(1,4)-beta-d-GlcNAc-(1,4)-beta-d-GlcNAc-(1,4)-d-GlcNAc and its C-2 epimer of the reducing end residue. TMG-chitotriomycin (1) showed potent and selective inhibition of insect and fungal GlcNAcases with no inhibition of mammalian and plant GlcNAcases. In contrast, the known GlcNAcase inhibitor nagstatin potently inhibited all GlcNAcases. It should be emphasized that synthesized d-N,N,N-triMeGlcNH2, which is the component sugar of 1, showed no inhibition of the insect Spodoptera litura GlcNAcase. These results suggest that the (GlcNAc)3 unit positioned at the reducing end of 1 is essential for its enzyme inhibitory activity. The unique inhibitory spectrum of 1 will be useful to study chitinolytic systems and to develop selective fungicides or pesticides.

  12. A preclinical study on the rescue of normal tissue by nicotinic acid in high-dose treatment with APO866, a specific nicotinamide phosphoribosyltransferase inhibitor

    DEFF Research Database (Denmark)

    Olesen, Uffe Høgh; Thougaard, Annemette V; Jensen, Peter Buhl

    2010-01-01

    Inhibitor of nicotinamide phosphoribosyltransferase APO866 is a promising cancer drug currently in phase II clinical trials in oncology. Here, we present a strategy for increasing the therapeutic potential of APO866 through the rescue of normal tissues by coadministration of nicotinic acid (Vitamin...... B(3)). We examined the toxicity profile of APO866 in B6D2F1 mice and the effect of oral administration of nicotinic acid on tissue toxicity. Nicotinic acid (50 mg/kg) protects mice from death and severe toxicity from an APO866 dose (60 mg/kg) four times the monotherapy maximum tolerated dose (15 mg....../kg). In a panel of six cancer cell lines, we find that three (including ML-2 cells) are protected by nicotinic acid in vitro, whereas the cytotoxicity of APO866 remains unaffected in the remaining three (including A2780 cells). A selective biomarker for the protection by nicotinic acid was subsequently identified...

  13. Development of a disease-specific quality of life questionnaire for adult patients with hereditary angioedema due to C1 inhibitor deficiency (HAE-QoL): Spanish multi-centre research project.

    Science.gov (United States)

    Prior, Nieves; Remor, Eduardo; Gómez-Traseira, Carmen; López-Serrano, Concepción; Cabañas, Rosario; Contreras, Javier; Campos, Ángel; Cardona, Victoria; Cimbollek, Stefan; González-Quevedo, Teresa; Guilarte, Mar; de Rojas, Dolores Hernández Fernández; Marcos, Carmen; Rubio, María; Tejedor-Alonso, Miguel Ángel; Caballero, Teresa

    2012-07-20

    There is a need for a disease-specific instrument for assessing health-related quality of life in adults with hereditary angioedema due to C1 inhibitor deficiency, a rare, disabling and life-threatening disease. In this paper we report the protocol for the development and validation of a specific questionnaire, with details on the results of the process of item generation, domain selection, and the expert and patient rating phase. Semi-structured interviews were completed by 45 patients with hereditary angioedema and 8 experts from 8 regions in Spain. A qualitative content analysis of the responses was carried out. Issues raised by respondents were grouped into categories. Content analysis identified 240 different responses, which were grouped into 10 conceptual domains. Sixty- four items were generated. A total of 8 experts and 16 patients assessed the items for clarity, relevance to the disease, and correct dimension assignment. The preliminary version of the specific health-related quality of life questionnaire for hereditary angioedema (HAE-QoL v 1.1) contained 44 items grouped into 9 domains. To the best of our knowledge, this is the first multi-centre research project that aims to develop a specific health-related quality of life questionnaire for adult patients with hereditary angioedema due to C1 inhibitor deficiency. A preliminary version of the specific HAE-QoL questionnaire was obtained. The qualitative analysis of interviews together with the expert and patient rating phase helped to ensure content validity. A pilot study will be performed to assess the psychometric properties of the questionnaire and to decide on the final version.

  14. Melanoma inhibitor of apoptosis protein (ML-IAP) specific cytotoxic T lymphocytes cross-react with an epitope from the auto-antigen SS56

    DEFF Research Database (Denmark)

    Baek Sørensen, Rikke; Faurschou, Mikkel; Troelsen, Lone

    2009-01-01

    A large proportion of melanoma patients host a spontaneous T-cell response specifically against ML-IAP-derived peptides. In this study, we describe that some ML-IAP-specific cytotoxic T cells isolated from melanoma patients cross react with an epitope from the auto-antigen SS56. SS56 is a recentl...

  15. Efficacy and tolerability of naproxen/esomeprazole magnesium tablets compared with non-specific NSAIDs and COX-2 inhibitors: a systematic review and network analyses

    Directory of Open Access Journals (Sweden)

    Datto C

    2013-02-01

    Full Text Available Catherine Datto,1 Richard Hellmund,1 Mohd Kashif Siddiqui21AstraZeneca Pharmaceuticals LP, Wilmington, DE, USA; 2HERON PVT India, Chandigarh, UT, IndiaAbstract: Non-steroidal anti-inflammatory drugs (NSAIDs, such as non-selective NSAIDs (nsNSAIDs or selective cyclooxygenase-2 (COX-2 inhibitors, are commonly prescribed for arthritic pain relief in patients with osteoarthritis (OA, rheumatoid arthritis (RA, or ankylosing spondylitis (AS. Treatment guidelines for chronic NSAID therapy include the consideration for gastroprotection for those at risk of gastric ulcers (GUs associated with the chronic NSAID therapy. The United States Food and Drug Administration has approved naproxen/esomeprazole magnesium tablets for the relief of signs and symptoms of OA, RA, and AS, and to decrease the risk of developing GUs in patients at risk of developing NSAID-associated GUs. The European Medical Association has approved this therapy for the symptomatic treatment of OA, RA, and AS in patients who are at risk of developing NSAID-associated GUs and/or duodenal ulcers, for whom treatment with lower doses of naproxen or other NSAIDs is not considered sufficient. Naproxen/esomeprazole magnesium tablets have been compared with naproxen and celecoxib for these indications in head-to-head trials. This systematic literature review and network meta-analyses of data from randomized controlled trials was performed to compare naproxen/esomeprazole magnesium tablets with a number of additional relevant comparators. For this study, an original review examined MEDLINE®, Embase®, and the Cochrane Controlled Trials Register from database start to April 14, 2009. Using the same methodology, a review update was conducted to December 21, 2009. The systematic review and network analyses showed naproxen/esomeprazole magnesium tablets have an improved upper gastrointestinal tolerability profile (dyspepsia and gastric or gastroduodenal ulcers over several active comparators (naproxen

  16. Insect-specific irreversible inhibitors of acetylcholinesterase in pests including the bed bug, the eastern yellowjacket, German and American cockroaches, and the confused flour beetle.

    Science.gov (United States)

    Polsinelli, Gregory A; Singh, Sanjay K; Mishra, Rajesh K; Suranyi, Robert; Ragsdale, David W; Pang, Yuan-Ping; Brimijoin, Stephen

    2010-09-06

    Insecticides directed against acetylcholinesterase (AChE) are facing increased resistance among target species as well as increasing concerns for human toxicity. The result has been a resurgence of disease vectors, insects destructive to agriculture, and residential pests. We previously reported a free cysteine (Cys) residue at the entrance to the AChE active site in some insects but not higher vertebrates. We also reported Cys-targeting methanethiosulfonate molecules (AMTSn), which, under conditions that spared human AChE, caused total irreversible inhibition of aphid AChE, 95% inhibition of AChE from the malaria vector mosquito (Anopheles gambia), and >80% inhibition of activity from the yellow fever mosquito (Aedes aegypti) and northern house mosquito (Culex pipiens). We now find the same compounds inhibit AChE from cockroaches (Blattella germanica and Periplaneta americana), the flour beetle (Tribolium confusum), the multi-colored Asian ladybird beetle (Harmonia axyridis), the bed bug (Cimex lectularius), and a wasp (Vespula maculifrons), with IC(50) values of approximately 1-11muM. Our results support further study of Cys-targeting inhibitors as conceptually novel insecticides that may be free of resistance in a range of insect pests and disease vectors and, compared with current compounds, should demonstrate much lower toxicity to mammals, birds, and fish. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

  17. [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.

  18. A specific 15-lipoxygenase inhibitor limits the progression and monocyte-macrophage enrichment of hypercholesterolemia-induced atherosclerosis in the rabbit.

    Science.gov (United States)

    Bocan, T M; Rosebury, W S; Mueller, S B; Kuchera, S; Welch, K; Daugherty, A; Cornicelli, J A

    1998-02-01

    Oxidant signalling and lipoprotein oxidation may play important roles in atherosclerotic lesion development. Given coincident localization of 15-lipoxygenase (15-LO), stereospecific products of 15-LO and epitopes of modified LDL in atherosclerotic lesions, we hypothesized that inhibition of 15-LO by PD146176, an inhibitor of 15-LO with an IC50 in cells or isolated enzyme of 0.5-0.8 microM, may limit atherosclerotic lesion development through regulation of monocyte-macrophage enrichment. Rabbits exposed to chronic endothelial denudation of the iliac-femoral artery were meal-fed a 0.25% cholesterol (C), 3% peanut oil (PNO), 3% coconut oil (CNO) diet twice daily with and without 175 mg/kg PD146176 for 12 weeks. In a second study, atherosclerotic lesions were pre-established in rabbits through chronic endothelial denudation and meal-fed a 0.5% C, 3% PNO, 3% CNO diet for 9 weeks and a 0% C/fat diet for 6 weeks prior to an 8 week administration of PD146176 at 175 mg/kg, q.d. Plasma total and lipoprotein cholesterol exposure were similar in control and PD146176-treated animals in both studies but PD146176 increased plasma triglyceride exposure 2- to 4-fold. Plasma PD146176 concentrations ranged from 99 to 214 ng/ml at 2 h post-dose. In the progression study, the iliac-femoral monocyte-macrophage area was reduced 71%, cross-sectional lesion area was unchanged and cholesteryl ester (CE) content was reduced 63%. In the regression study, size and macrophage content of iliac-femoral, fibrous plaque-like lesions were decreased 34%, CE content was reduced 19% and gross extent of thoracic aortic lesions were reduced 41%. We conclude that PD146176 can limit monocyte macrophage enrichment of atherosclerotic lesions and can attenuate development of fibrofoamy and fibrous plaque lesions in the absence of changes in plasma total or lipoprotein cholesterol concentrations.

  19. 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.

  20. Gene expression-signature of belinostat in cell lines is specific for histone deacetylase inhibitor treatment, with a corresponding signature in xenografts

    DEFF Research Database (Denmark)

    Monks, A.; Hose, C.D.; Pezzoli, P.

    2009-01-01

    gene modulation were significantly correlated. A belinostat-gene profile was specific for HDACi in three cell lines when compared with equipotent concentrations of four mechanistically different chemotherapeutic agents: 5-fluorouracil, cisplatin, paclitaxel, and thiotepa. Belinostat- and trichostatin...... in a drug-sensitive tumor than a more resistant model. We have demonstrated a gene signature that is selectively regulated by HDACi when compared with other clinical agents allowing us to distinguish HDACi responses from those related to other mechanisms Udgivelsesdato: 2009/9...

  1. Fragment-Linking Approach Using (19)F NMR Spectroscopy To Obtain Highly Potent and Selective Inhibitors of β-Secretase.

    Science.gov (United States)

    Jordan, John B; Whittington, Douglas A; Bartberger, Michael D; Sickmier, E Allen; Chen, Kui; Cheng, Yuan; Judd, Ted

    2016-04-28

    Fragment-based drug discovery (FBDD) has become a widely used tool in small-molecule drug discovery efforts. One of the most commonly used biophysical methods in detecting weak binding of fragments is nuclear magnetic resonance (NMR) spectroscopy. In particular, FBDD performed with (19)F NMR-based methods has been shown to provide several advantages over (1)H NMR using traditional magnetization-transfer and/or two-dimensional methods. Here, we demonstrate the utility and power of (19)F-based fragment screening by detailing the identification of a second-site fragment through (19)F NMR screening that binds to a specific pocket of the aspartic acid protease, β-secretase (BACE-1). The identification of this second-site fragment allowed the undertaking of a fragment-linking approach, which ultimately yielded a molecule exhibiting a more than 360-fold increase in potency while maintaining reasonable ligand efficiency and gaining much improved selectivity over cathepsin-D (CatD). X-ray crystallographic studies of the molecules demonstrated that the linked fragments exhibited binding modes consistent with those predicted from the targeted screening approach, through-space NMR data, and molecular modeling.

  2. Development of a specific affinity-matured exosite inhibitor to MT1-MMP that efficiently inhibits tumor cell invasion in vitro and metastasis in vivo

    DEFF Research Database (Denmark)

    Botkjaer, Kenneth A; Kwok, Hang Fai; Terp, Mikkel G

    2016-01-01

    therapeutic target. Here, we report the identification of antibody fragments to MT1-MMP that potently and specifically inhibit its cell surface functions. Lead antibody clones displayed inhibitory activity towards pro-MMP-2 activation, collagen-film degradation and gelatin-film degradation, and were shown......The membrane-associated matrix metalloproteinase-14, MT1-MMP, has been implicated in pericellular proteolysis with an important role in cellular invasion of collagenous tissues. It is substantially upregulated in various cancers and rheumatoid arthritis, and has been considered as a potential...... to bind to the MT1-MMP catalytic domain outside the active site cleft, inhibiting binding to triple helical collagen. Affinity maturation using CDR3 randomization created a second generation of antibody fragments with dissociation constants down to 0.11 nM, corresponding to an improved affinity of 332...

  3. Investigation of specificity determinants in bacterial tRNA-guanine transglycosylase reveals queuine, the substrate of its eucaryotic counterpart, as inhibitor.

    Directory of Open Access Journals (Sweden)

    Inna Biela

    Full Text Available Bacterial tRNA-guanine transglycosylase (Tgt catalyses the exchange of the genetically encoded guanine at the wobble position of tRNAs(His,Tyr,Asp,Asn by the premodified base preQ1, which is further converted to queuine at the tRNA level. As eucaryotes are not able to synthesise queuine de novo but acquire it through their diet, eucaryotic Tgt directly inserts the hypermodified base into the wobble position of the tRNAs mentioned above. Bacterial Tgt is required for the efficient pathogenicity of Shigella sp, the causative agent of bacillary dysentery and, hence, it constitutes a putative target for the rational design of anti-Shigellosis compounds. Since mammalian Tgt is known to be indirectly essential to the conversion of phenylalanine to tyrosine, it is necessary to create substances which only inhibit bacterial but not eucaryotic Tgt. Therefore, it seems of utmost importance to study selectivity-determining features within both types of proteins. Homology models of Caenorhabditis elegans Tgt and human Tgt suggest that the replacement of Cys158 and Val233 in bacterial Tgt (Zymomonas mobilis Tgt numbering by valine and accordingly glycine in eucaryotic Tgt largely accounts for the different substrate specificities. In the present study we have created mutated variants of Z. mobilis Tgt in order to investigate the impact of a Cys158Val and a Val233Gly exchange on catalytic activity and substrate specificity. Using enzyme kinetics and X-ray crystallography, we gained evidence that the Cys158Val mutation reduces the affinity to preQ1 while leaving the affinity to guanine unaffected. The Val233Gly exchange leads to an enlarged substrate binding pocket, that is necessary to accommodate queuine in a conformation compatible with the intermediately covalently bound tRNA molecule. Contrary to our expectations, we found that a priori queuine is recognised by the binding pocket of bacterial Tgt without, however, being used as a substrate.

  4. The caspase-1 inhibitor CARD18 is specifically expressed during late differentiation of keratinocytes and its expression is lost in lichen planus.

    Science.gov (United States)

    Qin, Haihong; Jin, Jiang; Fischer, Heinz; Mildner, Michael; Gschwandtner, Maria; Mlitz, Veronika; Eckhart, Leopold; Tschachler, Erwin

    2017-08-01

    CARD18 contains a caspase recruitment domain (CARD) via which it binds to caspase-1 and thereby inhibits caspase-1-mediated activation of the pro-inflammatory cytokine interleukin (IL)-1β. To determine the expression profile and the role of CARD18 during differentiation of keratinocytes and to compare the expression of CARD18 in normal skin and in inflammatory skin diseases. Human keratinocytes were induced to differentiate in monolayer and in 3D skin equivalent cultures. In some experiments, CARD18-specific siRNAs were used to knock down expression of CARD18. CARD18 mRNA levels were determined by quantitative real-time PCR, and CARD18 protein was detected by Western blot and immunofluorescence analyses. In situ expression was analyzed in skin biopsies obtained from healthy donors and patients with psoriasis and lichen planus. CARD18 mRNA was expressed in the epidermis at more than 100-fold higher levels than in any other human tissue. Within the epidermis, CARD18 was specifically expressed in the granular layer. In vitro CARD18 was strongly upregulated at both mRNA and protein levels in keratinocytes undergoing terminal differentiation. In skin equivalent cultures the expression of CARD18 was efficiently suppressed by siRNAs without impairing stratum corneum formation. Epidermal expression of CARD18 was increased after ultraviolet (UV)B irradiation of skin explants. In skin biopsies of patients with psoriasis no consistent regulation of CARD18 expression was observed, however, in lesional epidermis of patients with lichen planus, CARD18 expression was either greatly diminished or entirely absent whereas in non-lesional areas expression was comparable to normal skin. Our results identify CARD18 as a differentiation-associated keratinocyte protein that is altered in abundance by UV stress. Its downregulation in lichen planus indicates a potential role in inflammatory reactions of the epidermis in this disease. Copyright © 2017 Japanese Society for Investigative

  5. BI-D1870 is a specific inhibitor of the p90 RSK (ribosomal S6 kinase) isoforms in vitro and in vivo

    DEFF Research Database (Denmark)

    Sapkota, Gopal P; Cummings, Lorna; Newell, Felicity S

    2007-01-01

    ), which then mediate many of the physiological processes that are regulated by these extracellular agonists. It can be difficult to assess the individual functions of each AGC kinase because their substrate specificities are similar. Here we describe the small molecule BI-D1870, which inhibits RSK1, RSK2......, RSK3 and RSK4 in vitro with an IC(50) of 10-30 nM, but does not signi-ficantly inhibit ten other AGC kinase members and over 40 other protein kinases tested at 100-fold higher concentrations. BI-D1870 is cell permeant and prevents the RSK-mediated phorbol ester- and EGF (epidermal growth factor......)-induced phosphoryl-ation of glycogen synthase kinase-3beta and LKB1 in human embry-onic kidney 293 cells and Rat-2 cells. In contrast, BI-D1870 does not affect the agonist-triggered phosphorylation of substrates for six other AGC kinases. Moreover, BI-D1870 does not suppress the phorbol ester- or EGF...

  6. Effects of RAGE-Specific Inhibitor FPS-ZM1 on Amyloid-β Metabolism and AGEs-Induced Inflammation and Oxidative Stress in Rat Hippocampus.

    Science.gov (United States)

    Hong, Yan; Shen, Chao; Yin, Qingqing; Sun, Menghan; Ma, Yingjuan; Liu, Xueping

    2016-05-01

    An increased level of advanced glycation end products (AGEs) is observed in brains of patients with Alzheimer's disease (AD). AGEs and receptor for AGEs (RAGE) play important roles in the pathogenesis of AD. FPS-ZM1 is a high-affinity RAGE-specific blocker that inhibits amyloid-β binding to RAGE, neurological damage and inflammation in the APP(sw/0) transgenic mouse model of AD. FPS-ZM1 is not toxic to mice and can easily cross the blood-brain barrier. In this study, an AGEs-RAGE-activated rat model were established by intrahippocampal injection of AGEs, then these rats were treated with intraperitoneal administration of FPS-ZM1 and the possible neuroprotective effects were investigated. We found that AGEs administration induced an-regulation of Abeta production, inflammation, and oxidative stress, and an increased escape latency of rats in the Morris water maze test, all of these are significantly reduced by FPS-ZM1 treatment. Our results suggest that the AGEs-RAGE pathway is responsible for cognitive deficits, and therefore may be a potential treatment target. FPS-ZM1 might be a novel therapeutic agent to treat AD patients.

  7. DNA sequence-specific dimeric bisbenzimidazoles DBP(n) and DBPA(n) as inhibitors of H-NS silencing in bacterial cells.

    Science.gov (United States)

    Melkina, Olga E; Koval, Vasilii S; Ivanov, Alexander A; Zhuze, Alexei L; Zavilgelsky, Gennadii B

    2018-03-01

    DNA sequence-specific fluorescent dimeric bisbenzimidazoles DBP(n) and DBPA(n), noncovalently interacting with A-T pairs in the minor groove of double-stranded DNA were used for studying and monitoring the expression of histone-like H-NS-dependent promoters. Histone-like H-NS selectively binds to AT-rich segments of DNA and silences a large number of genes in bacterial chromosomes. The H-NS-dependent promoters of Quorum Sensing (QS)-regulated lux operons of the marine bacteria mesophilic Aliivibrio fischeri, psychrophilic Aliivibrio logei were used. Escherichia coli lux biosensors were constructed by cloning fragments bearing QS-regulated promoters into the vector, thereby placing each fragment upstream of the promoterless Photorhabdus luminescens luxCDABE genes. It was shown that the dimeric bisbenzimidazoles DBP(n) and DBPA(n) counteract the H-NS silencing activity. Thus, the presence of DBP(n) or DBPA(n) in the medium leads to an approximately 10-100-fold increase in the level of transcription of QS promoters in E. coli hns + . The largest decrease in the level of H-NS repression was observed using ligands containing a linker with a length of ca. 18Å, such as DBP(2) and DBPA(2). Ligands containing linkers with n=1 and 3 are an order of magnitude less active; ligands with n=4 are inactive. DBPA(2) exhibits activity starting with a concentration of 0.5μM; the minimum concentration of DBP(2) is 5-7 times higher. It is suggested that A-T pairs located at five nucleotide pair intervals, which correspond to the linker length in highly active ligands with n=2, play a key role in the structure of H-NS-binding sites in QS-regulated promoters. Copyright © 2017 Elsevier GmbH. All rights reserved.

  8. Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003

    Science.gov (United States)

    Valenzuela, Nicole M.; Thomas, Kimberly A.; Mulder, Arend; Parry, Graham C.; Panicker, Sandip; Reed, Elaine F.

    2017-01-01

    Background Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling. Methods Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003). Results Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement. Conclusions Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR. PMID:28640789

  9. Green Ultrasound versus Conventional Synthesis and Characterization of Specific Task Pyridinium Ionic Liquid Hydrazones Tethering Fluorinated Counter Anions: Novel Inhibitors of Fungal Ergosterol Biosynthesis

    Directory of Open Access Journals (Sweden)

    Nadjet Rezki

    2017-11-01

    Full Text Available A series of specific task ionic liquids (ILs based on a pyridiniumhydrazone scaffold in combination with hexafluorophosphate (PF6−, tetrafluoroboron (BF4− and/or trifluoroacetate (CF3COO− counter anion, were designed and characterized by IR, NMR and mass spectrometry. The reactions were conducted under both conventional and green ultrasound procedures. The antifungal potential of the synthesized compounds 2–25 was investigated against 40 strains of Candida (four standard and 36 clinical isolates. Minimum inhibitory concentrations (MIC90 of the synthesized compounds were in the range of 62.5–2000 μg/mL for both standard and oral Candida isolates. MIC90 results showed that the synthesized 1-(2-(4-chlorophenyl-2-oxoethyl-4-(2-(4-fluorobenzylidenehydrazinecarbonyl-pyridin-1-ium hexafluorophosphate (11 was found to be most effective, followed by 4-(2-(4-fluorobenzylidenehydrazinecarbonyl-1-(2-(4-nitrophenyl-2-oxoethyl-pyridin-1-ium hexafluorophosphate (14 and 1-(2-ethoxy-2-oxoethyl-4-(2-(4-fluorobenzylidenehydrazinecarbonylpyridin-1-ium hexafluorophosphate (8. All the Candida isolates showed marked sensitivity towards the synthesized compounds. Ergosterol content was drastically reduced by more active synthesized compounds, and agreed well with MIC90 values. Confocal scanning laser microscopy (CLSM results showed that the red colored fluorescent dye enters the test agent treated cells, which confirms cell wall and cell membrane damage. The microscopy results obtained suggested membrane-located targets for the action of these synthesized compounds. It appears that the test compounds might be interacting with ergosterol in the fungal cell membranes, decreasing the membrane ergosterol content and ultimately leading to membrane disruption as visible in confocal results. The present study indicates that these synthesized compounds show significant antifungal activity against Candida which forms the basis to carry out further in vivo experiments

  10. Lu-177-PSMA-617 Prostate-Specific Membrane Antigen Inhibitor Therapy in Patients with Castration-Resistant Prostate Cancer: Stability, Bio-distribution and Dosimetry

    Directory of Open Access Journals (Sweden)

    Levent Kabasakal

    2017-06-01

    Full Text Available Objective: The aim of the study was to estimate the radiation-absorbed doses and to study the in vivo and in vitro stability as well as pharmacokinetic characteristics of lutetium-177 (Lu-177 prostate-specific membrane antigen (PSMA-617. Methods: For this purpose, 7 patients who underwent Lu-177-PSMA therapy were included into the study. The injected Lu-177-PSMA-617 activity ranged from 3.6 to 7.4 GBq with a mean of 5.2±1.8 GBq. The stability of radiotracer in saline was calculated up to 48 h. The stability was also calculated in blood and urine samples. Post-therapeutic dosimetry was performed based on whole body and single photon emission computed tomography/computed tomography (SPECT/CT scans on dual-headed SPECT/CT system. Results: The radiochemical yield of Lu-177-PSMA-617 was >99%. It remained stable in saline up to 48 h. Analyses of the blood and urine samples showed a single radioactivity peak even at 24 hours after injection. Half-life of the distribution and elimination phases were calculated to be 0.16±0.09 and 10.8±2.5 hours, respectively. The mean excretion rate was 56.5±8.8% ranging from 41.5% to 65.4% at 24 h. Highest radiation estimated doses were calculated for parotid glands and kidneys (1.90±1.19 and 0.82±0.25 Gy/GBq respectively. Radiation dose given to the bone marrow was significantly lower than those of kidney and parotid glands (p<0.05 (0.030±0.008 Gy/GBq. Conclusion: Lu-177-PSMA-617 is a highly stable compound both in vitro and in vivo. Lu-177-PSMA-617 therapy seems to be a safe method for the treatment of castration-resistant prostate cancer patients. The fractionation regime that enables the longest duration of tumor control and/or survival will have to be developed in further studies.

  11. Tau, APP, NCT and BACE1 in lymphocytes through cognitively normal ageing and neuropathology

    Directory of Open Access Journals (Sweden)

    MARISOL HERRERA-RIVERO

    2013-01-01

    Full Text Available Although Alzheimer's disease is a brain disorder, a number of peripheral alterations have been found in these patients; however, little is known about how the key genes involved in the pathophysiology express in peripheral cells such as lymphocytes during normal compared to neuropathological ageing. We analysed the expression of tau, of the amyloid precursor protein, of nicastrin and of the β-site APP cleaving enzyme genes by RT-PCR in lymphocytes from a small group of late-onset Alzheimer's disease patients, from aged patients suffering from neuropsychological conditions different from Alzheimer's and from cognitively healthy subjects divided in four groups by age. We also investigated correlations between gene expression and levels of blood pressure, glucose, total cholesterol and triglycerides as risk factors for Alzheimer's. Results show no tau expression in lymphocytes, a lack of detection of nicastrin expression in Alzheimer's patients and correlations between the medical conditions studied and gene expression in lymphocytes. We believe nicastrin gene expression in lymphocytes should be considered of interest for further analyses in a wider population to investigate whether it might represent a potential biomarker to differentiate Alzheimer's from other neuropsychological disorders.

  12. Histone deacetylase inhibitors (HDACIs): multitargeted anticancer agents.

    Science.gov (United States)

    Ververis, Katherine; Hiong, Alison; Karagiannis, Tom C; Licciardi, Paul V

    2013-01-01

    Histone deacetylase (HDAC) inhibitors are an emerging class of therapeutics with potential as anticancer drugs. The rationale for developing HDAC inhibitors (and other chromatin-modifying agents) as anticancer therapies arose from the understanding that in addition to genetic mutations, epigenetic changes such as dysregulation of HDAC enzymes can alter phenotype and gene expression, disturb homeostasis, and contribute to neoplastic growth. The family of HDAC inhibitors is large and diverse. It includes a range of naturally occurring and synthetic compounds that differ in terms of structure, function, and specificity. HDAC inhibitors have multiple cell type-specific effects in vitro and in vivo, such as growth arrest, cell differentiation, and apoptosis in malignant cells. HDAC inhibitors have the potential to be used as monotherapies or in combination with other anticancer therapies. Currently, there are two HDAC inhibitors that have received approval from the US FDA for the treatment of cutaneous T-cell lymphoma: vorinostat (suberoylanilide hydroxamic acid, Zolinza) and depsipeptide (romidepsin, Istodax). More recently, depsipeptide has also gained FDA approval for the treatment of peripheral T-cell lymphoma. Many more clinical trials assessing the effects of various HDAC inhibitors on hematological and solid malignancies are currently being conducted. Despite the proven anticancer effects of particular HDAC inhibitors against certain cancers, many aspects of HDAC enzymes and HDAC inhibitors are still not fully understood. Increasing our understanding of the effects of HDAC inhibitors, their targets and mechanisms of action will be critical for the advancement of these drugs, especially to facilitate the rational design of HDAC inhibitors that are effective as antineoplastic agents. This review will discuss the use of HDAC inhibitors as multitargeted therapies for malignancy. Further, we outline the pharmacology and mechanisms of action of HDAC inhibitors while

  13. Plasmids encoding PKI(1-31), a specific inhibitor of cAMP-stimulated gene expression, inhibit the basal transcriptional activity of some but not all cAMP-regulated DNA response elements in JEG-3 cells.

    Science.gov (United States)

    Grove, J R; Deutsch, P J; Price, D J; Habener, J F; Avruch, J

    1989-11-25

    encoding PKI(1-31) inhibit the expression that is stimulated by the addition of cAMP analogs in both cell lines; basal expression, however, is inhibited by PKI(1-31) only in the JEG-3 cell line and not in the CV-1 cells. These observations indicate that, in JEG-3 cells, PKI(1-31) is a specific inhibitor of kinase A-mediated gene transcription, but it does not modify kinase C-directed transcription.(ABSTRACT TRUNCATED AT 400 WORDS)

  14. A group-specific inhibitor of lysosomal cysteine proteinases selectively inhibits both proteolytic degradation and presentation of the antigen dinitrophenyl-poly-L-lysine by guinea pig accessory cells to T cells

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O

    1986-01-01

    of antigens by guinea pig accessory cells. The proteinase inhibitor benzyloxycarbonyl-phenylalanylalanine-diazomethyl-ketone, which selectively inhibits cysteine proteinases, was used to block this set of enzymes in cultured cells. We demonstrate that the selective inhibition of the cysteine proteinases...

  15. Histone deacetylase inhibitors (HDACIs: multitargeted anticancer agents

    Directory of Open Access Journals (Sweden)

    Ververis K

    2013-02-01

    Full Text Available Katherine Ververis,1 Alison Hiong,1 Tom C Karagiannis,1,* Paul V Licciardi2,*1Epigenomic Medicine, Alfred Medical Research and Education Precinct, 2Allergy and Immune Disorders, Murdoch Childrens Research Institute, Melbourne, VIC, Australia*These authors contributed equally to this workAbstract: Histone deacetylase (HDAC inhibitors are an emerging class of therapeutics with potential as anticancer drugs. The rationale for developing HDAC inhibitors (and other chromatin-modifying agents as anticancer therapies arose from the understanding that in addition to genetic mutations, epigenetic changes such as dysregulation of HDAC enzymes can alter phenotype and gene expression, disturb homeostasis, and contribute to neoplastic growth. The family of HDAC inhibitors is large and diverse. It includes a range of naturally occurring and synthetic compounds that differ in terms of structure, function, and specificity. HDAC inhibitors have multiple cell type-specific effects in vitro and in vivo, such as growth arrest, cell differentiation, and apoptosis in malignant cells. HDAC inhibitors have the potential to be used as monotherapies or in combination with other anticancer therapies. Currently, there are two HDAC inhibitors that have received approval from the US FDA for the treatment of cutaneous T-cell lymphoma: vorinostat (suberoylanilide hydroxamic acid, Zolinza and depsipeptide (romidepsin, Istodax. More recently, depsipeptide has also gained FDA approval for the treatment of peripheral T-cell lymphoma. Many more clinical trials assessing the effects of various HDAC inhibitors on hematological and solid malignancies are currently being conducted. Despite the proven anticancer effects of particular HDAC inhibitors against certain cancers, many aspects of HDAC enzymes and HDAC inhibitors are still not fully understood. Increasing our understanding of the effects of HDAC inhibitors, their targets and mechanisms of action will be critical for the

  16. 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.

  17. Squash inhibitor family of serine proteinases

    International Nuclear Information System (INIS)

    Otlewski, J.; Krowarsch, D.

    1996-01-01

    Squash inhibitors of serine proteinases form an uniform family of small proteins. They are built of 27-33 amino-acid residues and cross-linked with three disulfide bridges. The reactive site peptide bond (P1-P1') is between residue 5 (Lys, Arg or Leu) and 6 (always Ile). High resolution X-ray structures are available for two squash inhibitors complexed with trypsin. NMR solution structures have also been determined for free inhibitors. The major structural motif is a distorted, triple-stranded antiparallel beta-sheet. A similar folding motif has been recently found in a number of proteins, including: conotoxins from fish-hunting snails, carboxypeptidase inhibitor from potato, kalata B1 polypeptide, and in some growth factors (e.g. nerve growth factor, transforming growth factor β2, platelet-derived growth factor). Squash inhibitors are highly stable and rigid proteins. They inhibit a number of serine proteinases: trypsin, plasmin, kallikrein, blood clotting factors: X a and XII a , cathepsin G. The inhibition spectrum can be much broadened if specific amino-acid substitutions are introduced, especially at residues which contact proteinase. Squash inhibitors inhibit proteinases via the standard mechanism. According to the mechanism, inhibitors are substrates which exhibit at neutral pH a high k cat /K m index for hydrolysis and resynthesis of the reactive site, and a low value of the hydrolysis constant. (author)

  18. Potential anti-cholinesterase and β-site amyloid precursor protein cleaving enzyme 1 inhibitory activities of cornuside and gallotannins from Cornus officinalis fruits.

    Science.gov (United States)

    Bhakta, Himanshu Kumar; Park, Chan Hum; Yokozawa, Takako; Tanaka, Takashi; Jung, Hyun Ah; Choi, Jae Sue

    2017-07-01

    Cholinesterase (ChE) and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors are promising agents for the treatment of Alzheimer's disease (AD). In the present study, we examined the inhibitory activity of seven compounds isolated from the fruits of Cornus officinalis, cornuside, polymeric proanthocyanidins, 1,2,3-tri-O-galloyl-β-D-glucose, 1,2,3,6-tetra-O-galloyl-β-D-glucose, tellimagrandin I, tellimagrandin II, and isoterchebin, against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and BACE1. All of the compounds displayed concentration-dependent in vitro inhibitory activity toward the ChEs and BACE1. Among them, tellimagrandin II exhibited the best inhibitory activity toward ChEs, whereas the best BACE1 inhibitor was 1,2,3,6-tetra-O-galloyl-β-D-glucose. Isoterchebin and polymeric proanthocyanidins were also significant ChE inhibitors. The kinetic and docking studies demonstrated that all compounds interacted with both the catalytic active sites and the peripheral anionic sites of the ChEs and BACE1. Tellimagrandin II, isoterchebin, and the polymeric proanthocyanidins exhibited concentration-dependent inhibition of peroxynitrite-mediated protein tyrosine nitration. In conclusion, we identified significant ChE and BACE1 inhibitors from Corni Fructus that could have value as new multi-targeted compounds for anti-AD agents.

  19. Rapid, Automated, and Specific Immunoassay to Directly Measure Matrix Metalloproteinase-9–Tissue Inhibitor of Metalloproteinase-1 Interactions in Human Plasma Using AlphaLISA Technology: A New Alternative to Classical ELISA

    Directory of Open Access Journals (Sweden)

    Helena Pulido-Olmo

    2017-07-01

    Full Text Available The protocol describes a novel, rapid, and no-wash one-step immunoassay for highly sensitive and direct detection of the complexes between matrix metalloproteinases (MMPs and their tissue inhibitor of metalloproteinases (TIMPs based on AlphaLISA® technology. We describe two procedures: (i one approach is used to analyze MMP-9–TIMP-1 interactions using recombinant human MMP-9 with its corresponding recombinant human TIMP-1 inhibitor and (ii the second approach is used to analyze native or endogenous MMP-9–TIMP-1 protein interactions in samples of human plasma. Evaluating native MMP-9–TIMP-1 complexes using this approach avoids the use of indirect calculations of the MMP-9/TIMP-1 ratio for which independent MMP-9 and TIMP-1 quantifications by two conventional ELISAs are needed. The MMP-9–TIMP-1 AlphaLISA® assay is quick, highly simplified, and cost-effective and can be completed in less than 3 h. Moreover, the assay has great potential for use in basic and preclinical research as it allows direct determination of native MMP-9–TIMP-1 complexes in circulating blood as biofluid.

  20. Insights into the molecular evolution of peptidase inhibitors in arthropods.

    Science.gov (United States)

    Alonso, Joaquin; Martinez, Manuel

    2017-01-01

    Peptidase inhibitors are key proteins involved in the control of peptidases. In arthropods, peptidase inhibitors modulate the activity of peptidases involved in endogenous physiological processes and peptidases of the organisms with which they interact. Exploring available arthropod genomic sequences is a powerful way to obtain the repertoire of peptidase inhibitors in every arthropod species and to understand the evolutionary mechanisms involved in the diversification of this kind of proteins. A genomic comparative analysis of peptidase inhibitors in species belonging to different arthropod taxonomic groups was performed. The results point out: i) species or clade-specific presence is shown for several families of peptidase inhibitors; ii) multidomain peptidase inhibitors are commonly found in many peptidase inhibitor families; iii) several families have a wide range of members in different arthropod species; iv) several peptidase inhibitor families show species-specific (or clade-specific) gene family expansions; v) functional divergence may be assumed for particular clades; vi) passive expansions may be used by natural selection to fix adaptations. In conclusion, conservation and divergence of duplicated genes and the potential recruitment as peptidase inhibitors of proteins from other families are the main mechanisms used by arthropods to fix diversity. This diversity would be associated to the control of target peptidases and, as consequence, to adapt to specific environments.

  1. Monoamine Oxidase Inhibitors (MAOIs)

    Science.gov (United States)

    ... health-medications/index.shtml. Accessed May 16, 2016. Hirsch M, et al. Monoamine oxidase inhibitors (MAOIs) for ... www.uptodate.com/home. Accessed May 16, 2016. Hirsch M, et al. Discontinuing antidepressant medications in adults. ...

  2. ELISA analysis of soybean trypsin inhibitors in processed foods.

    Science.gov (United States)

    Brandon, D L; Bates, A H; Friedman, M

    1991-01-01

    Soybean proteins are widely used in human foods in a variety of forms, including infant formulas, flour, protein concentrates, protein isolates, soy sauces, textured soy fibers, and tofu. The presence of inhibitors of digestive enzymes in soy proteins impairs the nutritional quality and possibly the safety of soybeans and other legumes. Processing, based on the use of heat or fractionation of protein isolates, does not completely inactivate or remove these inhibitors, so that residual amounts of inhibitors are consumed by animals and humans. New monoclonal antibody-based immunoassays can measure low levels of the soybean Kunitz trypsin inhibitor (KTI) and the Bowman-Birk trypsin and chymotrypsin inhibitor (BBI) and the Bowman-Birk foods. The enzyme-linked immunosorbent assay (ELISA) was used to measure the inhibitor content of soy concentrates, isolates, and flours, both heated and unheated; a commercial soy infant formula; KTI and BBI with rearranged disulfide bonds; browning products derived from heat-treatment of KTI with glucose and starch; and KTI exposed to high pH. The results indicate that even low inhibitor isolates contain significant amounts of specific inhibitors. Thus, infants on soy formula consume about 10 mg of KTI plus BBI per day. The immunoassays complement the established enzymatic assays of trypsin and chymotrypsin inhibitors, and have advantages in (a) measuring low levels of inhibitors in processed foods; and (b) differentiating between the Kunitz and Bowman-Birk inhibitors. The significance of our findings for food safety are discussed.

  3. A complementary set of electrochemical and X-ray synchrotron techniques to determine the passivation mechanism of iron treated in a new corrosion inhibitor solution specifically developed for the preservation of metallic artefacts

    International Nuclear Information System (INIS)

    Mirambet, F.; Reguer, S.; Rocca, E.; Hollner, S.; Testemale, D.

    2010-01-01

    Metallic artefacts of the cultural heritage are often stored in uncontrolled environmental conditions. They are very sensitive to atmospheric corrosion caused by a succession of wet and dry periods due to variations of relative humidity and temperature. To avoid the complete degradation of the metallic artefacts, new preventive strategies must be developed. In this context, we have studied new compounds based on sodium carboxylates solutions CH 3 (CH 2 ) n-2 COO - , Na + hereafter called NaC n . They allow the formation of a passive layer at the metallic surface composed of a metal-carboxylate complex. To understand the action of those inhibitors in the passivation process of iron we have performed electrochemical measurements and surface characterisation. Moreover, to monitor in real time the growth of the coating, in situ X-ray absorption spectroscopy (XAS) experiments at iron K-edge were carried out in an electrochemical cell. These analyses have shown that in the case of NaC 10 solution, the protection of iron surface is correlated to the precipitation of a well-organised layer of FeC 10 . These experiments confirmed that this compound is a fully oxidised trinuclear Fe(III) complex containing decanoate anions as ligands. Such information concerning the passive layer is a key factor to evaluate its stability and finally the long-term efficiency of the protection treatment. (orig.)

  4. Vildagliptin: the first innovative DDP-4 inhibitor

    Directory of Open Access Journals (Sweden)

    Edvin Villkhauer

    2010-09-01

    Full Text Available A review of the main stages of investigation undertaken by Novartis Pharmaceuticals in search of a new molecule for the treatment of type 2 diabetesmellitus, dipeptidyl peptidase-4 (DPP-4 inhibitor (Vildaglyptin. The data on specificity and selectivity of the action of this molecule are presentedalong with the results of its comparison with another agent of this group (sitagliptin.

  5. Cysteine peptidases and their inhibitors in breast and genital cancer.

    Directory of Open Access Journals (Sweden)

    Magdalena Milan

    2010-11-01

    Full Text Available Cysteine proteinases and their inhibitors probably play the main role in carcinogenesis and metastasis. The metastasis process need external proteolytic activities that pass several barriers which are membranous structures of the connective tissue which includes, the basement membrane of blood vessels. Activities of the proteinases are regulated by endogenous inhibitors and activators. The imbalance between cysteine proteinases and cystatins seems to be associated with an increase in metastatic potential in some tumors. It has also been reported that proteinase inhibitors, specific antibodies for these enzymes and inhibition of the urokinase receptor may prevent cancer cell invasion. Some proteinase inhibitor could serve as agents for cancer treatment.

  6. SGLT2 inhibitors.

    Science.gov (United States)

    Dardi, I; Kouvatsos, T; Jabbour, S A

    2016-02-01

    Diabetes mellitus is a serious health issue and an economic burden, rising in epidemic proportions over the last few decades worldwide. Although several treatment options are available, only half of the global diabetic population achieves the recommended or individualized glycemic targets. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of antidiabetic agents with a novel insulin-independent action. SGLT2 is a transporter found in the proximal renal tubules, responsible for the reabsorption of most of the glucose filtered by the kidney. Inhibition of SGLT2 lowers the blood glucose level by promoting the urinary excretion of excess glucose. Due to their insulin-independent action, SGLT2 inhibitors can be used with any degree of beta-cell dysfunction or insulin resistance, related to a very low risk of hypoglycemia. In addition to improving glycemic control, SGLT2 inhibitors have been associated with a reduction in weight and blood pressure when used as monotherapy or in combination with other antidiabetic agents in patients with type 2 diabetes mellitus (T2DM). Treatment with SGLT2 inhibitors is usually well tolerated; however, they have been associated with an increased incidence of urinary tract and genital infections, although these infections are usually mild and easy to treat. SGLT2 inhibitors are a promising new option in the armamentarium of drugs for patients with T2DM. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Aromatase inhibitors in pediatrics.

    Science.gov (United States)

    Wit, Jan M; Hero, Matti; Nunez, Susan B

    2011-10-25

    Aromatase, an enzyme located in the endoplasmic reticulum of estrogen-producing cells, catalyzes the rate-limiting step in the conversion of androgens to estrogens in many tissues. The clinical features of patients with defects in CYP19A1, the gene encoding aromatase, have revealed a major role for this enzyme in epiphyseal plate closure, which has promoted interest in the use of inhibitors of aromatase to improve adult height. The availability of the selective aromatase inhibitors letrozole and anastrozole--currently approved as adjuvant therapy for breast cancer--have stimulated off-label use of aromatase inhibitors in pediatrics for the following conditions: hyperestrogenism, such as aromatase excess syndrome, Peutz-Jeghers syndrome, McCune-Albright syndrome and functional follicular ovarian cysts; hyperandrogenism, for example, testotoxicosis (also known as familial male-limited precocious puberty) and congenital adrenal hyperplasia; pubertal gynecomastia; and short stature and/or pubertal delay in boys. Current data suggest that aromatase inhibitors are probably effective in the treatment of patients with aromatase excess syndrome or testotoxicosis, partially effective in Peutz-Jeghers and McCune-Albright syndrome, but probably ineffective in gynecomastia. Insufficient data are available in patients with congenital adrenal hyperplasia or functional ovarian cysts. Although aromatase inhibitors appear effective in increasing adult height of boys with short stature and/or pubertal delay, safety concerns, including vertebral deformities, a decrease in serum HDL cholesterol levels and increase of erythrocytosis, are reasons for caution.

  8. Synergistic apoptosis induction in leukemic cells by the phosphatase inhibitor salubrinal and proteasome inhibitors.

    Directory of Open Access Journals (Sweden)

    Hannes C A Drexler

    Full Text Available Cells adapt to endoplasmic reticulum (ER-stress by arresting global protein synthesis while simultaneously activating specific transcription factors and their downstream targets. These processes are mediated in part by the phosphorylation-dependent inactivation of the translation initiation factor eIF2alpha. Following restoration of homeostasis protein synthesis is resumed when the serine/threonine-protein phosphatase PP1 dephosphorylates and reactivates eIF2alpha. Proteasome inhibitors, used to treat multiple myeloma patients evoke ER-stress and apoptosis by blocking the ER-associated degradation of misfolded proteins (ERAD, however, the role of eIF2alpha phosphorylation in leukemic cells under conditions of proteasome inhibitor-mediated ER stress is currently unclear.Bcr-Abl-positive and negative leukemic cell lines were used to investigate the functional implications of PP1-related phosphatase activities on eIF2alpha phosphorylation in proteasome inhibitor-mediated ER stress and apoptosis. Rather unexpectedly, salubrinal, a recently identified PP1 inhibitor capable to protect against ER stress in various model systems, strongly synergized with proteasome inhibitors to augment apoptotic death of different leukemic cell lines. Salubrinal treatment did not affect the phosphorlyation status of eIF2alpha. Furthermore, the proapoptotic effect of salubrinal occurred independently from the chemical nature of the proteasome inhibitor, was recapitulated by a second unrelated phosphatase inhibitor and was unaffected by overexpression of a dominant negative eIF2alpha S51A variant that can not be phosphorylated. Salubrinal further aggravated ER-stress and proteotoxicity inflicted by the proteasome inhibitors on the leukemic cells since characteristic ER stress responses, such as ATF4 and CHOP synthesis, XBP1 splicing, activation of MAP kinases and eventually apoptosis were efficiently abrogated by the translational inhibitor cycloheximide.Although PP1

  9. Crystal Structure of the Extended-Spectrum β-Lactamase PER-2 and Insights into the Role of Specific Residues in the Interaction with β-Lactams and β-Lactamase Inhibitors

    Science.gov (United States)

    Ruggiero, Melina; Kerff, Frédéric; Herman, Raphaël; Sapunaric, Frédéric; Galleni, Moreno; Gutkind, Gabriel; Charlier, Paulette; Sauvage, Eric

    2014-01-01

    PER-2 belongs to a small (7 members to date) group of extended-spectrum β-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most β-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward β-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singular trans bond between residues 166 to 167, which generates an inverted Ω loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A β-lactamases. PER β-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A (“A” indicates an insertion according to Ambler's scheme for residue numbering in PER β-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different β-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior. PMID:25070104

  10. JAK inhibitors in autoinflammation.

    Science.gov (United States)

    Hoffman, Hal M; Broderick, Lori

    2018-06-11

    Interferonopathies are a subset of autoinflammatory disorders with a prominent type I IFN gene signature. Treatment of these patients has been challenging, given the lack of response to common autoinflammatory therapeutics including IL-1 and TNF blockade. JAK inhibitors (Jakinibs) are a family of small-molecule inhibitors that target the JAK/STAT signaling pathway and have shown clinical efficacy, with FDA and European Medicines Agency (EMA) approval for arthritic and myeloproliferative syndromes. Sanchez and colleagues repurposed baricitinib to establish a significant role for JAK inhibition as a novel therapy for patients with interferonopathies, demonstrating the power of translational rare disease research with lifesaving effects.

  11. Cathepsin D inhibitors

    Directory of Open Access Journals (Sweden)

    M. Gacko

    2007-11-01

    Full Text Available Inhibitors of cathepsin D belong to chemical compounds that estrify carboxyl groups of the Asp33 and Asp231residues of its catalytic site, penta-peptides containing statin, i.e. the amino acid similar in structure to the tetraedric indirectproduct, and polypeptides found in the spare organs of many plants and forming permanent noncovalent complexes withcathepsin. Cathepsin D activity is also inhibited by alpha2-macroglobulin and antibodies directed against this enzyme.Methods used to determine the activity and concentration of these inhibitors and their analytical, preparative and therapeuticapplications are discussed.

  12. Sildenafil Decreases BACE1 and Cathepsin B Levels and Reduces APP Amyloidogenic Processing in the SAMP8 Mouse.

    Science.gov (United States)

    Orejana, Lourdes; Barros-Miñones, Lucía; Jordan, Joaquin; Cedazo-Minguez, Angel; Tordera, Rosa M; Aguirre, Norberto; Puerta, Elena

    2015-06-01

    The senescence-accelerated mouse-prone 8 (SAMP8), used as a model of aging, displays many established pathological features of Alzheimer's disease. Cognitive impairments and increased levels of hyperphosphorylated tau are found in the hippocampus of SAMP8 mice along with an increased β-secretase activity and amyloid-β (Aβ) depositions that increase in number and extent with age. Based on a previous study from our laboratory showing an amelioration of cognitive impairments and tau pathology by sildenafil, in this study we tested whether this drug could also modulate the amyloid precursor protein amyloidogenic processing in this mouse model. Our results show that the protein levels of the β-secretases β-site amyloid precursor protein cleaving enzyme 1 and cathepsin B are higher in the hippocampus of 9-month-old SAMP8 mice than those of age-matched senescence-resistant-1. Sildenafil (7.5mg/kg for 4 weeks) attenuated learning and memory impairments shown by SAMP8 mice in the passive avoidance test. The increased expression of β-site amyloid precursor protein cleaving enzyme 1 was also reduced by sildenafil, an effect paralleled to decreases in the activities of two β-site amyloid precursor protein cleaving enzyme 1 modulators, calpain and cyclin-dependent kinase 5 protein. Interestingly, sildenafil enhanced both Akt and glycogen synthase kinase-3β (ser9) phosphorylation, which could be mediating the reduction in cathepsin B levels found in the hippocampus of sildenafil-treated SAMP8 mice. Sildenafil-induced reduction in β-site amyloid precursor protein cleaving enzyme 1 and cathepsin B expression in SAMP8 mice was associated with a decrease in hippocampal Aβ42 levels which, in turn, could mediate the parallel decline in glial fibrillary acidic protein expression observed in these animals. These findings highlight the therapeutic potential of sildenafil in Alzheimer's disease pathogenesis. © The Author 2014. Published by Oxford University Press on behalf of the Gerontological Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  13. 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.

  14. A cyclic peptidic serine protease inhibitor

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Xu, Peng; Jiang, Longguang

    2014-01-01

    Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...... pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending......, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity...

  15. Transglutaminase inhibitor from milk

    NARCIS (Netherlands)

    Jong, G.A.H. de; Wijngaards, G.; Koppelman, S.J.

    2003-01-01

    Cross-linking experiments of skimmed bovine milk with bacterial transglutaminase isolated from Streptoverticillium mobaraense showed only some degree of formation of high-molecular-weight casein polymers. Studies on the nature of this phenomenon revealed that bovine milk contains an inhibitor of

  16. Inhibitors of histone demethylases

    DEFF Research Database (Denmark)

    Lohse, Brian; Kristensen, Jesper L; Kristensen, Line H

    2011-01-01

    Methylated lysines are important epigenetic marks. The enzymes involved in demethylation have recently been discovered and found to be involved in cancer development and progression. Despite the relative recent discovery of these enzymes a number of inhibitors have already appeared. Most of the i...

  17. Aromatase inhibitors and breast cancer prevention.

    Science.gov (United States)

    Litton, Jennifer Keating; Arun, Banu K; Brown, Powel H; Hortobagyi, Gabriel N

    2012-02-01

    Endocrine therapy with selective estrogen receptor modulators (SERMs) has been the mainstay of breast cancer prevention trials to date. The aromatase inhibitors, which inhibit the final chemical conversion of androgens to estrogens, have shown increased disease-free survival benefit over tamoxifen in patients with primary hormone receptor-positive breast cancer, as well as reducing the risk of developing contralateral breast cancers. The aromatase inhibitors are being actively evaluated as prevention agents for women with a history of ductal carcinoma in situ as well as for women who are considered to be at high risk for developing primary invasive breast cancer. This review evaluates the available prevention data, as evidenced by the decrease in contralateral breast cancers, when aromatase inhibitors are used in the adjuvant setting, as well as the emerging data of the aromatase inhibitors specifically tested in the prevention setting for women at high risk. Exemestane is a viable option for breast cancer prevention. We continue to await further follow-up on exemestane as well as other aromatase inhibitors in the prevention setting for women at high risk of developing breast cancer or with a history of ductal carcinoma in situ.

  18. [Insect cholinesterases and irreversible inhibitors. Statistical treatment of the data].

    Science.gov (United States)

    Moralev, S N

    2010-01-01

    The data on sensitivity of cholinesterases (ChE) of different insects to reversible inhibitors, as well as the data on physico-chemical parameters of amino acids constituting their active centers, were treated by factor analysis and juxtaposed. It is shown that both these characteristics are related to taxonomical belonging of insects. It is revealed the "material substrate" of the factors determining inhibitor action specificity, which are specific sites in ChE active center.

  19. Invertase proteinaceous inhibitor of Cyphomandra betacea Sendt fruits.

    Science.gov (United States)

    Ordóñez, R M; Isla, M I; Vattuone, M A; Sampietro, A R

    2000-01-01

    This work describes a new invertase proteinaceous inhibitor from Cyphomandra betacea Sendt. (tomate de arbol) fruits. The proteinaceous inhibitor was isolated and purified from a cell wall preparation. The pH stability, kinetics of the inhibition of the C. betacea invertase, inhibition of several higher plant invertases and lectin nature of the inhibitor were studied. The inhibitor structure involves a single polypeptide (Mr = 19000), as shown by gel filtration and SDS-PAGE determinations. N-terminal aminoacid sequence was determined. The properties and some structural features of the inhibitor are compared with the proteinaceous inhibitors from several plant species (Beta vulgaris L., Ipomoea batatas L. and Lycopersicon esculentum Mill.). All these inhibitors share lectinic properties, some common epitopes, some aminoacid sequences and a certain lack of specificity towards invertases of different species, genera and even plant family. In consequence, the inhibitors appear to belong to the same lectin family. It is now known that some lectins are part of the defence mechanism of higher plants against fungi and bacteria and this is a probable role of the proteinaceous inhibitors.

  20. Acid corrosion inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Chen, N G

    1964-04-28

    An acid corrosion inhibitor is prepared by a 2-stage vacuum evaporation of effluents obtained from the ammonia columns of the coking oven plant. The effluent, leaving a scrubber in which the phenols are removed at a temperature of 98$C, passes through a quartz filter and flows into a heated chamber in which it is used for preheating a solution circulating through a vacuum unit, maintaining the temperature of the solution at 55$ to 60$C. The effluent enters a large tank in which it is boiled at 55$ to 60$C under 635 to 640 mm Hg pressure. Double evaporation of this solution yields a very effective acid corrosion inhibitor. Its corrosion-preventing effect is 97.9% compared with 90.1% for thiourea and 88.5% for urotropin under identical conditions.

  1. Benzoylurea Chitin Synthesis Inhibitors.

    Science.gov (United States)

    Sun, Ranfeng; Liu, Chunjuan; Zhang, Hao; Wang, Qingmin

    2015-08-12

    Benzoylurea chitin synthesis inhibitors are widely used in integrated pest management (IPM) and insecticide resistance management (IRM) programs due to their low toxicity to mammals and predatory insects. In the past decades, a large number of benzoylurea derivatives have been synthesized, and 15 benzoylurea chitin synthesis inhibitors have been commercialized. This review focuses on the history of commercial benzolyphenylureas (BPUs), synthetic methods, structure-activity relationships (SAR), action mechanism research, environmental behaviors, and ecotoxicology. Furthermore, their disadvantages of high risk to aquatic invertebrates and crustaceans are pointed out. Finally, we propose that the para-substituents at anilide of benzoylphenylureas should be the functional groups, and bipartite model BPU analogues are discussed in an attempt to provide new insight for future development of BPUs.

  2. Blocking the proliferation of human tumor cell lines by peptidase inhibitors from Bauhinia seeds.

    Science.gov (United States)

    Nakahata, Adriana Miti; Mayer, Barbara; Neth, Peter; Hansen, Daiane; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela

    2013-03-01

    In cancer tumors, growth, invasion, and formation of metastasis at a secondary site play a pivotal role, participating in diverse processes in the development of the pathology, such as degradation of extracellular matrix. Bauhinia seeds contain relatively large quantities of peptidase inhibitors, and two Bauhinia inhibitors were obtained in a recombinant form from the Bauhinia bauhinioides species, B. bauhinoides cruzipain inhibitor, which is a cysteine and serine peptidase inhibitor, and B. bauhinioides kallikrein inhibitor, which is a serine peptidase inhibitor. While recombinant B. bauhinoides cruzipain inhibitor inhibits human neutrophil elastase cathepsin G and the cysteine proteinase cathepsin L, recombinant B. bauhinioides kallikrein inhibitor inhibits plasma kallikrein and plasmin. The effects of recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor on the viability of tumor cell lines with a distinct potential of growth from the same tissue were compared to those of the clinical cytotoxic drug 5-fluorouracil. At 12.5 µM concentration, recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor were more efficient than 5-fluorouracil in inhibiting MKN-28 and Hs746T (gastric), HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), and THP-1 and K562 (leukemia) cell lines. Additionally, recombinant B. bauhinoides cruzipain inhibitor inhibited 40 % of the migration of Hs746T, the most invasive gastric cell line, while recombinant B. bauhinioides kallikrein inhibitor did not affect cell migration. Recombinant B. bauhinioides kallikrein inhibitor and recombinant B. bauhinoides cruzipain inhibitor, even at high doses, did not affect hMSC proliferation while 5-fluorouracil greatly reduced the proliferation rates of hMSCs. Therefore, both recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor might be considered for further studies

  3. Mutants of common bean alpha-amylase inhibitor-2 as an approach to investigate binding specificity to alpha-amylases Mutantes do inibidor-2 de alfa-amilase do feijão-comum para investigação da especificidade de ligação a alfa-amilases

    Directory of Open Access Journals (Sweden)

    Maria Cristina Mattar da Silva

    2004-03-01

    Full Text Available Despite the presence of a family of defense proteins, Phaseolus vulgaris can be attacked by bruchid insects resulting in serious damage to stored grains. The two distinct active forms of a-amylase inhibitors, a-AI1 and a-AI2, in P. vulgaris show different specificity toward a-amylases. Zabrotes subfasciatus a-amylase is inhibited by a-AI2 but not by a-AI1. In contrast, porcine a-amylase is inhibited by a-AI1 but not by a-AI2. The objective of this work was to understand the molecular basis of the specificity of two inhibitors in P. vulgaris (a-AI1 and a-AI2 in relation to a-amylases. Mutants of a-AI2 were made and expressed in tobacco plants. The results showed that all the a-AI2 mutant inhibitors lost their activity against the insect a-amylases but none exhibited activity toward the mammalian a-amylase. The replacement of His33 of a-AI2 with the a-AI1-like sequence Ser-Tyr-Asn abolished inhibition of Z. subfasciatus a-amylase. From structural modeling, the conclusion is that the size and complexity of the amylase-inhibitor interface explain why mutation of the N-terminal loop and resultant abolition of Z. subfasciatus a-amylase inhibition are not accompanied by gain of inhibitory activity against porcine a-amylase.Apesar de possuir uma família de proteínas de defesa, o feijão-comum (Phaseolus vulgaris L. pode ser atacado por insetos bruquídeos causando sérios danos aos grãos armazenados. O P. vulgaris possui duas formas ativas de inibidores de a-amilases, denominadas a-AI1 e a-AI2, que apresentam diferentes especificidades em relação às a-amilases. A a-amilase de Zabrotes subfasciatus é inibida por a-AI2 mas não por a-AI1. Em contraste, a a-amilase pancreática de porco é inibida por a-AI1 mas não é por a-AI2. O objetivo deste trabalho foi entender as bases moleculares da especificidade desses inibidores em relação às a-amilases. Para tanto, foram construídos mutantes do a-AI2, os quais foram expressados em plantas de fumo

  4. Pre-therapeutic dosimetry of normal organs and tissues of {sup 177}Lu-PSMA-617 prostate-specific membrane antigen (PSMA) inhibitor in patients with castration-resistant prostate cancer

    Energy Technology Data Exchange (ETDEWEB)

    Kabasakal, Levent; AbuQbeitah, Mohammad; Ayguen, Aslan; Yeyin, Nami [Istanbul University, Department of Nuclear Medicine, Cerrahpasa Medical Faculty, Istanbul (Turkey); Ocak, Meltem [Istanbul University, Department of Pharmaceutical Technology, Pharmacy Faculty, Istanbul (Turkey); Demirci, Emre [Sisli Etfal Training and Research Hospital, Department of Nuclear Medicine, Istanbul (Turkey); Toklu, Turkay [Yeditepe University Medical Faculty, Department of Nuclear Medicine, Istanbul (Turkey)

    2015-12-15

    {sup 177}Lu-617-prostate-specific membrane antigen (PSMA) ligand seems to be a promising tracer for radionuclide therapy of progressive prostate cancer. However, there are no published data regarding the radiation dose given to the normal tissues. The aim of the present study was to estimate the pretreatment radiation doses in patients who will undergo radiometabolic therapy using a tracer amount of {sup 177}Lu-labeled PSMA ligand. The study included seven patients with progressive prostate cancer with a mean age of 63.9 ± 3.9 years. All patients had prior PSMA positron emission tomography (PET) imaging and had intense tracer uptake at the lesions. The injected {sup 177}Lu-PSMA-617 activity ranged from 185 to 210 MBq with a mean of 192.6 ± 11.0 MBq. To evaluate bone marrow absorbed dose 2-cc blood samples were withdrawn in short variable times (3, 15, 30, 60, and 180 min and 24, 48, and 120 h) after injection. Whole-body images were obtained at 4, 24, 48, and 120 h post-injection (p.i.). The geometric mean of anterior and posterior counts was determined through region of interest (ROI) analysis. Attenuation correction was applied using PSMA PET/CT images. The OLINDA/EXM dosimetry program was used for curve fitting, residence time calculation, and absorbed dose calculations. The calculated radiation-absorbed doses for each organ showed substantial variation. The highest radiation estimated doses were calculated for parotid glands and kidneys. Calculated radiation-absorbed doses per megabecquerel were 1.17 ± 0.31 mGy for parotid glands and 0.88 ± 0.40 mGy for kidneys. The radiation dose given to the bone marrow was significantly lower than those of kidney and parotid glands (p < 0.05). The calculated radiation dose to bone marrow was 0.03 ± 0.01 mGy/MBq. Our first results suggested that {sup 177}Lu-PSMA-617 therapy seems to be a safe method. The dose-limiting organ seems to be the parotid glands rather than kidneys and bone marrow. The lesion radiation doses are

  5. DGAT inhibitors for obesity.

    Science.gov (United States)

    Matsuda, Daisuke; Tomoda, Hiroshi

    2007-10-01

    Obesity is characterized by the accumulation of triacylglycerol in adipocytes. Diacylglycerol acyltransferase (DGAT) catalyzes the final reaction of triacylgycerol synthesis. Two isozymes of DGAT, DGAT1 and DGAT2, have been reported. Increased DGAT2 activity has a role in steatosis, while DGAT1 plays a role in very (V)LDL synthesis; increased plasma VLDL concentrations may promote obesity and thus DGAT1 is considered a potential therapeutic target of inhibition for obesity control. Several DGAT inhibitors of natural and synthetic origin have been reported, and their future prospect as anti-obesity drugs is discussed in this review.

  6. The binding mechanism of a peptidic cyclic serine protease inhibitor

    DEFF Research Database (Denmark)

    Jiang, Longguang; Svane, Anna Sigrid P.; Sørensen, Hans Peter

    2011-01-01

    Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries......, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical...... inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding...

  7. Structure based design of 11β-HSD1 inhibitors.

    Science.gov (United States)

    Singh, Suresh; Tice, Colin

    2010-11-01

    Controlling elevated tissue-specific levels of cortisol may provide a novel therapeutic approach for treating metabolic syndrome. This concept has spurred large scale medicinal chemistry efforts in the pharmaceutical industry for the design of 11β-HSD1 inhibitors. High resolution X-ray crystal structures of inhibitors in complex with the enzyme have facilitated the structure-based design of diverse classes of molecules. A summary of binding modes, trends in structure-activity relationships, and the pharmacodynamic data of inhibitors from each class is presented.

  8. Pulmonary Toxicity of Cholinesterase Inhibitors

    National Research Council Canada - National Science Library

    Hilmas, Corey; Adler, Michael; Baskin, Steven I; Gupta, Ramesh C

    2006-01-01

    .... Whereas nerve agents were produced primarily for military deployment, other cholinesterase inhibitors were used for treating conditions such as myasthenia gravis and as pretreaunents for nerve agent exposure...

  9. RENAL SAFETY OF PROTON PUMP INHIBITORS

    Directory of Open Access Journals (Sweden)

    A. I. Dyadyk

    2017-01-01

    Full Text Available Proton pump inhibitors are a widely used in clinical practice, and are taken by millions of patients around the world for a long time. While proton pump inhibitors are well-tolerated class of drugs, the number of publications has been raised about adverse renal effects, specially their association with acute tubulointerstitial nephritis. It is one of the leading causes of acute renal injury and have catastrophic long-term consequences called chronic kidney disease. In this review, we consider epidemiology, pathogenesis, diagnostic criteria (including biopsy and morphological pattern, clinical manifestations and treatment of proton pump inhibitors-induced acute tubulointerstitial nephritis. A subclinical course without classical manifestations of a cell-mediated hypersensitivity reaction (fever, skin rash, eosinophilia, arthralgia is characteristic of acute tubulointerstitial nephritis. Increased serum creatinine, decreased glomerular filtration rate, electrolyte disorders, pathological changes in urine tests are not highly specific indicators, but allow to suspect the development of acute tubulointerstitial nephritis. The “gold” standard of diagnosis is the intravital morphological examination of the kidney tissue. Timely diagnosis and immediate discontinuation of the potentially causative drug is the mainstay of therapy and the first necessary step in the early management of suspected or biopsy-proven drug-induced acute tubulointerstitial nephritis. The usage of proton pump inhibitors should be performed only on strict indications with optimal duration of treatment and careful monitoring of kidney function. Multiple comorbidities (older age, heart failure, diabetes, cirrhosis, chronic kidney disease, hypovolemia increase potential nephrotoxicity. Awareness of this iatrogenic complication will improve diagnosis of proton pump inhibitors-induced acute tubulointerstitial nephritis by multidisciplinary specialists and increase the possibility

  10. Cost of care of haemophilia with inhibitors.

    Science.gov (United States)

    Di Minno, M N D; Di Minno, G; Di Capua, M; Cerbone, A M; Coppola, A

    2010-01-01

    In Western countries, the treatment of patients with inhibitors is presently the most challenging and serious issue in haemophilia management, direct costs of clotting factor concentrates accounting for >98% of the highest economic burden absorbed for the healthcare of patients in this setting. Being designed to address questions of resource allocation and effectiveness, decision models are the golden standard to reliably assess the overall economic implications of haemophilia with inhibitors in terms of mortality, bleeding-related morbidity, and severity of arthropathy. However, presently, most data analyses stem from retrospective short-term evaluations, that only allow for the analysis of direct health costs. In the setting of chronic diseases, the cost-utility analysis, that takes into account the beneficial effects of a given treatment/healthcare intervention in terms of health-related quality of life, is likely to be the most appropriate approach. To calculate net benefits, the quality adjusted life year, that significantly reflects such health gain, has to be compared with specific economic impacts. Differences in data sources, in medical practice and/or in healthcare systems and costs, imply that most current pharmacoeconomic analyses are confined to a narrow healthcare payer perspective. Long-term/lifetime prospective or observational studies, devoted to a careful definition of when to start a treatment; of regimens (dose and type of product) to employ, and of inhibitor population (children/adults, low-responding/high responding inhibitors) to study, are thus urgently needed to allow for newer insights, based on reliable data sources into resource allocation, effectiveness and cost-utility analysis in the treatment of haemophiliacs with inhibitors.

  11. Potential non-oncological applications of histone deacetylase inhibitors.

    Science.gov (United States)

    Ververis, Katherine; Karagiannis, Tom C

    2011-01-01

    Histone deacetylase inhibitors have emerged as a new class of anticancer therapeutic drugs. Their clinical utility in oncology stems from their intrinsic cytotoxic properties and combinatorial effects with other conventional cancer therapies. To date, the histone deacetylase inhibitors suberoylanilide hydroxamic acid (Vorinostat, Zolinza®) and depsipeptide (Romidepsin, Istodax®) have been approved by the US Food and Drug Administration for the treatment of refractory cutaneous T-cell lymphoma. Further, there are currently over 100 clinical trials involving the use of histone deacetylase inhibitors in a wide range of solid and hematological malignancies. The therapeutic potential of histone deacetylase inhibitors has also been investigated for numerous other diseases. For example, the cytotoxic properties of histone deacetylase inhibitors are currently being harnessed as a potential treatment for malaria, whereas the efficacy of these compounds for HIV relies on de-silencing latent virus. The anti-inflammatory properties of histone deacetylase inhibitors are the predominant mechanisms for other diseases, such as hepatitis, systemic lupus erythematosus and a wide range of neurodegenerative conditions. Additionally, histone deacetylase inhibitors have been shown to be efficacious in animal models of cardiac hypertrophy and asthma. Broad-spectrum histone deacetylase inhibitors are clinically available and have been used almost exclusively in preclinical systems to date. However, it is emerging that class- or isoform-specific compounds, which are becoming more readily available, may be more efficacious particularly for non-oncological applications. The aim of this review is to provide an overview of the effects and clinical potential of histone deacetylase inhibitors in various diseases. Apart from applications in oncology, the discussion is focused on the potential efficacy of histone deacetylase inhibitors for the treatment of neurodegenerative diseases, cardiac

  12. Structure-Based Design of CSDK4-Specific Inhibitors

    National Research Council Canada - National Science Library

    Marmorstein, Ronen

    2003-01-01

    ...) are implicated in more than 80% of human neoplasias (Ortega et al., 2002). For example, the gene encoding the CDK4/6 inhibitory protein, p16INK4, is deleted or mutated in the majority of leukemias, bladder cancers and familial melanomas (Roussel, 1999...

  13. Challenges in using allylthiourea and chlorate as specific nitrification inhibitors

    DEFF Research Database (Denmark)

    Tatari, Karolina; Gülay, Arda; Thamdrup, B.

    2017-01-01

    Allylthiourea (ATU) and chlorate (ClO3-) are often used to selectively inhibit nitritation and nitratation. In this work we identified challenges with use of these compounds in inhibitory assays with filter material from a biological rapid sand filter for groundwater treatment. Inhibition...... was stronger at higher ATU levels and resulted in no NH4+ detection at 0.5 mM ATU. ClO3- at typical concentrations for inhibition assays (1-10 mM) inhibited nitratation by less than 6%, while nitritation was instead inhibited by 91% when NH4+ was supplied. On the other hand, nitratation was inhibited by 67......-71% at 10-20 mM ClO3- when NO2- was supplied, suggesting significant nitratation inhibition at higher NO2- concentrations. No chlorite (ClO2-) was detected in the effluent, and thus we could not confirm that nitritation inhibition was caused by ClO3- reduction to ClO2-. In conclusion, ATU and ClO3- should...

  14. Biological abatement of cellulase inhibitors

    Science.gov (United States)

    Bio-abatement uses a fungus to metabolize and remove fermentation inhibitors. To determine whether bio-abatement could alleviate enzyme inhibitor effects observed in biomass liquors after pretreatment, corn stover at 10% (w/v) solids was pretreated with either dilute acid or liquid hot water. The ...

  15. Proteinaceous alpha-araylase inhibitors

    DEFF Research Database (Denmark)

    Svensson, Birte; Fukuda, Kenji; Nielsen, P.K.

    2004-01-01

    -amylase inhibitors belong to seven different protein structural families, most of which also contain evolutionary related proteins without inhibitory activity. Two families include bifunctional inhibitors acting both on alpha-amylases and proteases. High-resolution structures are available of target alpha...

  16. Corrosion inhibitors. Manufacture and technology

    International Nuclear Information System (INIS)

    Ranney, M.W.

    1976-01-01

    Detailed information is presented relating to corrosion inhibitors. Areas covered include: cooling water, boilers and water supply plants; oil well and refinery operations; fuel and lubricant additives for automotive use; hydraulic fluids and machine tool lubes; grease compositions; metal surface treatments and coatings; and general processes for corrosion inhibitors

  17. A novel method for screening the glutathione transferase inhibitors

    Directory of Open Access Journals (Sweden)

    Węgrzyn Grzegorz

    2009-03-01

    Full Text Available Abstract Background Glutathione transferases (GSTs belong to the family of Phase II detoxification enzymes. GSTs catalyze the conjugation of glutathione to different endogenous and exogenous electrophilic compounds. Over-expression of GSTs was demonstrated in a number of different human cancer cells. It has been found that the resistance to many anticancer chemotherapeutics is directly correlated with the over-expression of GSTs. Therefore, it appears to be important to find new GST inhibitors to prevent the resistance of cells to anticancer drugs. In order to search for glutathione transferase (GST inhibitors, a novel method was designed. Results Our results showed that two fragments of GST, named F1 peptide (GYWKIKGLV and F2 peptide (KWRNKKFELGLEFPNL, can significantly inhibit the GST activity. When these two fragments were compared with several known potent GST inhibitors, the order of inhibition efficiency (measured in reactions with 2,4-dinitrochlorobenzene (CDNB and glutathione as substrates was determined as follows: tannic acid > cibacron blue > F2 peptide > hematin > F1 peptide > ethacrynic acid. Moreover, the F1 peptide appeared to be a noncompetitive inhibitor of the GST-catalyzed reaction, while the F2 peptide was determined as a competitive inhibitor of this reaction. Conclusion It appears that the F2 peptide can be used as a new potent specific GST inhibitor. It is proposed that the novel method, described in this report, might be useful for screening the inhibitors of not only GST but also other enzymes.

  18. F8 haplotype and inhibitor risk: results from the Hemophilia Inhibitor Genetics Study (HIGS) Combined Cohort

    Science.gov (United States)

    Schwarz, John; Astermark, Jan; Menius, Erika D.; Carrington, Mary; Donfield, Sharyne M.; Gomperts, Edward D.; Nelson, George W.; Oldenburg, Johannes; Pavlova, Anna; Shapiro, Amy D.; Winkler, Cheryl A.; Berntorp, Erik

    2012-01-01

    Background Ancestral background, specifically African descent, confers higher risk for development of inhibitory antibodies to factor VIII (FVIII) in hemophilia A. It has been suggested that differences in the distribution of factor VIII gene (F8) haplotypes, and mismatch between endogenous F8 haplotypes and those comprising products used for treatment could contribute to risk. Design and Methods Data from the HIGS Combined Cohort were used to determine the association between F8 haplotype 3 (H3) vs. haplotypes 1 and 2 (H1+H2) and inhibitor risk among individuals of genetically-determined African descent. Other variables known to affect inhibitor risk including type of F8 mutation and HLA were included in the analysis. A second research question regarding risk related to mismatch in endogenous F8 haplotype and recombinant FVIII products used for treatment was addressed. Results H3 was associated with higher inhibitor risk among those genetically-identified (N=49) as of African ancestry, but the association did not remain significant after adjustment for F8 mutation type and the HLA variables. Among subjects of all racial ancestries enrolled in HIGS who reported early use of recombinant products (N=223), mismatch in endogenous haplotype and the FVIII proteins constituting the products used did not confer greater risk for inhibitor development. Conclusion H3 was not an independent predictor of inhibitor risk. Further, our findings did not support a higher risk of inhibitors in the presence of a haplotype mismatch between the FVIII molecule infused and that of the individual. PMID:22958194

  19. Reverse transcriptase inhibitors as microbicides.

    Science.gov (United States)

    Lewi, Paul; Heeres, Jan; Ariën, Kevin; Venkatraj, Muthusamy; Joossens, Jurgen; Van der Veken, Pieter; Augustyns, Koen; Vanham, Guido

    2012-01-01

    The CAPRISA 004 study in South Africa has accelerated the development of vaginal and rectal microbicides containing antiretrovirals that target specific enzymes in the reproduction cycle of HIV, especially reverse transcriptase inhibitors (RTI). In this review we discuss the potential relevance of HIV-1 RTIs as microbicides, focusing in the nucleotide RTI tenofovir and six classes of nonnucleoside RTIs (including dapivirine, UC781, urea and thiourea PETTs, DABOs and a pyrimidinedione). Although tenofovir and dapivirine appear to be most advanced in clinical trials as potential microbicides, several issues remain unresolved, e.g., the importance of nonhuman primates as a "gatekeeper" for clinical trials, the emergence and spread of drug-resistant mutants, the combination of microbicides that target different phases of viral reproduction and the accessibility to microbicides in low-income countries. Thus, here we discuss the latest research on RTI as microbicides in the light of the continuing spread of the HIV pandemic from the point of view of medicinal chemistry, virological, and pharmaceutical studies.

  20. AZT as a telomerase inhibitor

    International Nuclear Information System (INIS)

    Gomez, Daniel E.; Armando, Romina G.; Alonso, Daniel F.

    2012-01-01

    Telomerase is a highly specialized reverse transcriptase (RT) and the maintenance of telomeric length is determined by this specific enzyme. The human holoenzyme telomerase is a ribonucleoprotein composed by a catalytic subunit, hTERT, an RNA component, hTR, and a group of associated proteins. Telomerase is normally expressed in embryonic cells and is repressed during adulthood. The enzyme is reexpressed in around 85% of solid tumors. This observation makes it a potential target for developing drugs that could be developed for therapeutic purposes. The identification of the hTERT as a functional catalytic RT prompted studies of inhibiting telomerase with the HIV RT inhibitor azidothymidine (AZT). Previously, we have demonstrated that AZT binds preferentially to telomeres, inhibits telomerase and enhances tumor cell senescence, and apoptosis after AZT treatment in breast mammary adenocarcinoma cells. Since then, several studies have considered AZT for telomerase inhibition and have led to potential clinical strategies for anticancer therapy. This review covers present thinking of the inhibition of telomerase by AZT and future treatment protocols using the drug.

  1. Discovery of novel selenium derivatives as Pin1 inhibitors by high-throughput screening

    International Nuclear Information System (INIS)

    Subedi, Amit; Shimizu, Takeshi; Ryo, Akihide; Sanada, Emiko; Watanabe, Nobumoto; Osada, Hiroyuki

    2016-01-01

    Peptidyl prolyl cis/trans isomerization by Pin1 regulates various oncogenic signals during cancer progression, and its inhibition through multiple approaches has established Pin1 as a therapeutic target. However, lack of simplified screening systems has limited the discovery of potent Pin1 inhibitors. We utilized phosphorylation-dependent binding of Pin1 to its specific substrate to develop a screening system for Pin1 inhibitors. Using this system, we screened a chemical library, and identified a novel selenium derivative as Pin1 inhibitor. Based on structure-activity guided chemical synthesis, we developed more potent Pin1 inhibitors that inhibited cancer cell proliferation. -- Highlights: •Novel screening for Pin1 inhibitors based on Pin1 binding is developed. •A novel selenium compound is discovered as Pin1 inhibitor. •Activity guided chemical synthesis of selenium derivatives resulted potent Pin1 inhibitors.

  2. A novel small molecule inhibitor of hepatitis C virus entry.

    Directory of Open Access Journals (Sweden)

    Carl J Baldick

    Full Text Available Small molecule inhibitors of hepatitis C virus (HCV are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc, blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.

  3. A porphodimethene chemical inhibitor of uroporphyrinogen decarboxylase.

    Directory of Open Access Journals (Sweden)

    Kenneth W Yip

    Full Text Available Uroporphyrinogen decarboxylase (UROD catalyzes the conversion of uroporphyrinogen to coproporphyrinogen during heme biosynthesis. This enzyme was recently identified as a potential anticancer target; its inhibition leads to an increase in reactive oxygen species, likely mediated by the Fenton reaction, thereby decreasing cancer cell viability and working in cooperation with radiation and/or cisplatin. Because there is no known chemical UROD inhibitor suitable for use in translational studies, we aimed to design, synthesize, and characterize such a compound. Initial in silico-based design and docking analyses identified a potential porphyrin analogue that was subsequently synthesized. This species, a porphodimethene (named PI-16, was found to inhibit UROD in an enzymatic assay (IC50 = 9.9 µM, but did not affect porphobilinogen deaminase (at 62.5 µM, thereby exhibiting specificity. In cellular assays, PI-16 reduced the viability of FaDu and ME-180 cancer cells with half maximal effective concentrations of 22.7 µM and 26.9 µM, respectively, and only minimally affected normal oral epithelial (NOE cells. PI-16 also combined effectively with radiation and cisplatin, with potent synergy being observed in the case of cisplatin in FaDu cells (Chou-Talalay combination index <1. This work presents the first known synthetic UROD inhibitor, and sets the foundation for the design, synthesis, and characterization of higher affinity and more effective UROD inhibitors.

  4. Structural Principles in the Development of Cyclic Peptidic Enzyme Inhibitors

    Science.gov (United States)

    Xu, Peng; Andreasen, Peter A.; Huang, Mingdong

    2017-01-01

    This review summarizes our studies in the development of small cyclic peptides for specifically modulating enzyme activity. Serine proteases share highly similar active sites but perform diverse physiological and pathological functions. From a phage-display peptide library, we isolated two mono-cyclic peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), which inhibit the activity of human and murine urokinase-type plasminogen activators (huPA and muPA) with Ki values in the micromolar or sub-micromolar range, respectively. The following affinity maturations significantly enhanced the potencies of the two peptides, 10-fold and >250-fold for upain-1 and mupain-1, respectively. The most potent muPA inhibitor has a potency (Ki = 2 nM) and specificity comparable to mono-clonal antibodies. Furthermore, we also found an unusual feature of mupain-1 that its inhibitory potency can be enhanced by increasing the flexibility, which challenges the traditional viewpoint that higher rigidity leading to higher affinity. Moreover, by changing a few key residues, we converted mupain-1 from a uPA inhibitor to inhibitors of other serine proteases, including plasma kallikrein (PK) and coagulation factor XIa (fXIa). PK and fXIa inhibitors showed Ki values in the low nanomolar range and high specificity. Our studies demonstrate the versatility of small cyclic peptides to engineer inhibitory potency against serine proteases and to provide a new strategy for generating peptide inhibitors of serine proteases. PMID:29104489

  5. [ACE inhibitors and the kidney].

    Science.gov (United States)

    Hörl, W H

    1996-01-01

    Treatment with ACE inhibitors results in kidney protection due to reduction of systemic blood pressure, intraglomerular pressure, an antiproliferative effect, reduction of proteinuria and a lipid-lowering effect in proteinuric patients (secondary due to reduction of protein excretion). Elderly patients with diabetes melitus, coronary heart disease or peripheral vascular occlusion are at risk for deterioration of kidney function due to a high frequency of renal artery stenosis in these patients. In patients with renal insufficiency dose reduction of ACE inhibitors is necessary (exception: fosinopril) but more important is the risk for development of hyperkalemia. Patients at risk for renal artery stenosis and patients pretreated with diuretics should receive a low ACE inhibitor dosage initially ("start low - go slow"). For compliance reasons once daily ACE inhibitor dosage is recommended.

  6. Universal, class-specific and drug-specific reversal agents for the new oral anticoagulants.

    Science.gov (United States)

    Ansell, Jack E

    2016-02-01

    Although there is controversy about the absolute need for a reversal agent for the new direct oral anticoagulants (DOACs), the absence of such an agent is a barrier to more widespread use of these agents. For the management of major life-threatening bleeding with the DOACs, most authorities recommend the use of four factor prothrombin complex concentrates, although the evidence to support their use in terms of improving outcomes is meager. At the present time, there are three antidotes in development and poised to enter the market. Idarucizumab is a drug-specific antidote targeted to reverse the direct thrombin inhibitor, dabigatran. Andexanet alfa is a class-specific antidote targeted to reverse the oral direct factor Xa inhibitors as well as the indirect inhibitor, enoxaparin. Ciraparantag is a universal antidote targeted to reverse the direct thrombin and factor Xa inhibitors as well as the indirect inhibitor, enoxaparin.

  7. Insect P450 inhibitors and insecticides: challenges and opportunities.

    Science.gov (United States)

    Feyereisen, René

    2015-06-01

    P450 enzymes are encoded by a large number of genes in insects, often over a hundred. They play important roles in insecticide metabolism and resistance, and growing numbers of P450 enzymes are now known to catalyse important physiological reactions, such as hormone metabolism or cuticular hydrocarbon synthesis. Ways to inhibit P450 enzymes specifically or less specifically are well understood, as P450 inhibitors are found as drugs, as fungicides, as plant growth regulators and as insecticide synergists. Yet there are no P450 inhibitors as insecticides on the market. As new modes of action are constantly needed to support insecticide resistance management, P450 inhibitors should be considered because of their high potential for insect selectivity, their well-known mechanisms of action and the increasing ease of rational design and testing. © 2014 Society of Chemical Industry.

  8. [Isomeric derivatives of lupinine and epilupinine--organophosphorus inhibitors of cholinesterases].

    Science.gov (United States)

    Basova, N E; Kormilitsyn, B N; Perchenok, A Iu; Rosengart, E V; Saakov, V S; Suvorov, A A

    2012-01-01

    The isomeric-structure analysis data of anticholinesterase action of organophosphorous inhibitors with similar structure help in the search of specific effectors and detection of differences in reactivity of various animals' enzymes. This study compared the data of efficacy in respect of 4 mammal and 5 arthropoda cholinesterase preparations for 26 quinolizidine inhibitors, which molecules contain both the isomeric unbranched and branched alkoxyl radicals in the phosphoryl group, and the epimeric lupinine and epilupinine derivatives in the leaving group. The changes in the alkoxyl radical structure of inhibitor molecules act on their efficacy only with respect to the mammal enzymes ("group" inhibitor specificity). The differences between lupinine and epilupinine derivatives were revealed. Highly specific inhibitors of different enzymes were detected among the tested compounds.

  9. Phospholipid Binding Protein C Inhibitor (PCI) Is Present on Microparticles Generated In Vitro and In Vivo

    Science.gov (United States)

    Einfinger, Katrin; Badrnya, Sigrun; Furtmüller, Margareta; Handschuh, Daniela; Lindner, Herbert; Geiger, Margarethe

    2015-01-01

    Protein C inhibitor is a secreted, non-specific serine protease inhibitor with broad protease reactivity. It binds glycosaminoglycans and anionic phospholipids, which can modulate its activity. Anionic phospholipids, such as phosphatidylserine are normally localized to the inner leaflet of the plasma membrane, but are exposed on activated and apoptotic cells and on plasma membrane-derived microparticles. In this report we show by flow cytometry that microparticles derived from cultured cells and activated platelets incorporated protein C inhibitor during membrane blebbing. Moreover, protein C inhibitor is present in/on microparticles circulating in normal human plasma as judged from Western blots, ELISAs, flow cytometry, and mass spectrometry. These plasma microparticles are mainly derived from megakaryocytes. They seem to be saturated with protein C inhibitor, since they do not bind added fluorescence-labeled protein C inhibitor. Heparin partially removed microparticle-bound protein C inhibitor, supporting our assumption that protein C inhibitor is bound via phospholipids. To assess the biological role of microparticle-bound protein C inhibitor we performed protease inhibition assays and co-precipitated putative binding partners on microparticles with anti-protein C inhibitor IgG. As judged from amidolytic assays microparticle-bound protein C inhibitor did not inhibit activated protein C or thrombin, nor did microparticles modulate the activity of exogenous protein C inhibitor. Among the proteins co-precipitating with protein C inhibitor, complement factors, especially complement factor 3, were most striking. Taken together, our data do not support a major role of microparticle-associated protein C inhibitor in coagulation, but rather suggest an interaction with proteins of the complement system present on these phospholipid vesicles. PMID:26580551

  10. Molecular Dynamics simulations of Inhibitor of Apoptosis Proteins and identification of potential small molecule inhibitors.

    Science.gov (United States)

    Jayakumar, Jayanthi; Anishetty, Sharmila

    2014-05-01

    Chemotherapeutic resistance due to over expression of Inhibitor of Apoptosis Proteins (IAPs) XIAP, survivin and livin has been observed in various cancers. In the current study, Molecular Dynamics (MD) simulations were carried out for all three IAPs and a common ligand binding scaffold was identified. Further, a novel sequence based motif specific to these IAPs was designed. SMAC is an endogenous inhibitor of IAPs. Screening of ChemBank for compounds similar to lead SMAC-non-peptidomimetics yielded a cemadotin related compound NCIMech_000654. Cemadotin is a derivative of natural anti-tumor peptide dolastatin-15; hence these compounds were docked against all three IAPs. Based on our analysis, we propose that NCIMech_000654/dolastatin-15/cemadotin derivatives may be investigated for their potential in inhibiting XIAP, survivin and livin. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. 5 alpha-reductase inhibitors and prostatic disease.

    Science.gov (United States)

    Schröder, F H

    1994-08-01

    5 alpha-Reductase inhibitors are a new class of substances with very specific effects on type I and type II 5 alpha R which may be of use in the treatment of skin disease, such as male pattern baldness, male acne and hirsutism, as well as prostatic hyperplasia and prostate cancer. At least two types of 5 alpha R inhibitors with a different pH optimum have been described. cDNA encoding for both the type I and the type II enzyme has been cloned. Most of the orally effective 5 alpha R inhibitors belong to the class of 4-azasteroids. The radical substituted in the 17 position of the steroid ring seems to be related to species specific variations and to the types of 5 alpha R enzymes in different species and organ systems. 5 alpha R inhibitors lead to a decrease of plasma DHT by about 65% while there is a slight rise in plasma testosterone. The decrease of tissue DHT in the ventral prostate of the intact rat, the dog and in humans is more pronounced and amounts to about 85%. There is a reciprocal rise of tissue T in these systems. The application of an inhibitor of 5 alpha R type II leads to a shrinkage of BPH in men by about 30%. In the rat a similar shrinkage accompanied by a significant decrease of total organ DNA occurs. This decrease, however, is not as pronounced as can be achieved with castration.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. A Coupled CFD/FEM Structural Analysis to Determine Deformed Shapes of the RSRM Inhibitors

    Science.gov (United States)

    Dill, Richard A.; Whitesides, R. Harold

    1996-01-01

    Recent trends towards an increase in the stiffness of the acrylonitrile butadiene rubber (NBR) insulation material used in the construction of the redesigned solid rocket motor (RSRM) propellant inhibitors prompted questions about possible effects on RSRM performance. The specific objectives of the computational fluid dynamics (CFD) task included: (1) the definition of pressure loads to calculate the deformed shape of stiffer inhibitors, (2) the calculation of higher port velocities over the inhibitors to determine shifts in the vortex shedding or edge tone frequencies, and (3) the quantification of higher slag impingement and collection rates on the inhibitors and in the submerged nose nozzle cavity.

  13. Metal corrosion inhibitors and ecology

    International Nuclear Information System (INIS)

    Krasts, H.; Svarce, J.; Berge, B.

    1999-01-01

    The use of metal corrosion inhibitors in water is one of the cheapest method to protect metals against corrosion. However, the used inhibitors can come to surface water in the course of time and can become as source of environmental pollution. It is important to co-ordinate amount of substances in the elaborated inhibitors not only with demands for metal protection, but also with demands for quality of surface water and drinking water according to normative statements: 3.5 mg/l (as PO 4 ) for hexametaphosphate, tripolyphosphate and phosphonate; 40 mg/l (as SiO 2 for silicate, up to 1 mg/l for CU 2+ ; up to 5 mg/l for Zn 2+ ; up to 1 mg/l for B; up to 0.5 mg/l for Mo 2+ . The examples of the elaborated inhibitors are given. Many organic substances can be used as corrosion inhibitors, but there is shortage of standard methods for their analysis in water in Latvia. Removing of salt's deposits from boilers needs elaboration of a separate normative statement for dispersing waste water which content chloride at high concentration and heavy metals. (authors)

  14. Treatment with a JNK inhibitor increases, whereas treatment with a p38 inhibitor decreases, H2O2-induced calf pulmonary arterial endothelial cell death.

    Science.gov (United States)

    Park, Woo Hyun

    2017-08-01

    Oxidative stress induces apoptosis in endothelial cells (ECs). Reactive oxygen species (ROS) promote cell death by regulating the activity of various mitogen-activated protein kinases (MAPKs) in ECs. The present study investigated the effects of MAPK inhibitors on cell survival and glutathione (GSH) levels upon H 2 O 2 treatment in calf pulmonary artery ECs (CPAECs). H 2 O 2 treatment inhibited the growth and induced the death of CPAECs, as well as causing GSH depletion and the loss of mitochondrial membrane potential (MMP). While treatment with the MEK or JNK inhibitor impaired the growth of H 2 O 2 -treated CPAECs, treatment with the p38 inhibitor attenuated this inhibition of growth. Additionally, JNK inhibitor treatment increased the proportion of sub-G 1 phase cells in H 2 O 2 -treated CPAECs and further decreased the MMP. However, treatment with a p38 inhibitor reversed the effects of H 2 O 2 treatment on cell growth and the MMP. Similarly, JNK inhibitor treatment further increased, whereas p38 inhibitor treatment decreased, the proportion of GSH-depleted cells in H 2 O 2 -treated CPAECs. Each of the MAPK inhibitors affected cell survival, and ROS or GSH levels differently in H 2 O 2 -untreated, control CPAECs. The data suggest that the exposure of CPAECs to H 2 O 2 caused the cell growth inhibition and cell death through GSH depletion. Furthermore, JNK inhibitor treatment further enhanced, whereas p38 inhibitors attenuated, these effects. Thus, the results of the present study suggest a specific protective role for the p38 inhibitor, and not the JNK inhibitor, against H 2 O 2 -induced cell growth inhibition and cell death.

  15. Inhibitors of Angiogenesis.

    Science.gov (United States)

    Büning, H; Hacker, U T

    Angiogenesis plays a pivotal role in malignant, ischemic, inflammatory, infectious and immune disorders. The increasing molecular understanding of angiogenic processes fostered the development of strategies to induce or inhibit angiogenesis for therapeutic purposes. Here, we focus on anti-angiogenic therapies, which represent a standard of care in the treatment of different cancer types and in neovascular age-related macular degeneration. Specifically, strategies related to the blockade of angiogenic proteins and receptors will be outlined covering both preclinical and clinical aspects. Finally, examples of gene therapy based anti-angiogenic approaches are presented.

  16. Positron emitter labeled enzyme inhibitors

    International Nuclear Information System (INIS)

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

    1990-01-01

    This invention involves a new strategy for imagining and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography

  17. ROCK inhibitors in ocular disease

    Directory of Open Access Journals (Sweden)

    Eva Halasz

    2016-12-01

    Full Text Available Rho kinases (ROCKs have a crucial role in actin-cytoskeletal reorganization and thus are involved in broad aspects of cell motility, from smooth muscle contraction to neurite outgrowth. The first marketed ROCK inhibitor, called fasudil, has been used safely for treatment of cerebral vasospasm since 1995 in Japan. During the succeeding decades ROCK inhibitors have been applied in many pathological conditions from central nervous system disorders to cardiovascular disease as potential therapeutic agents or experimental tools to help understand the underlying (pathomechanisms. In 2014, a fasudil derivate named ripasudil was accepted for clinical use in glaucoma and ocular hypertension. Since ROCK kinases are widely expressed in ocular tissues, they have been implicated in the pathology of many ocular conditions such as corneal dysfunction, glaucoma, cataract, diabetic retinopathy, age-related macular degeneration, and retinal detachment. This paper aims to provide an overview of the most recent status/application of ROCK inhibitors in the field of eye disease.

  18. Inhibition of β-Secretase Activity by Monoterpenes, Sesquiterpenes, and C13 Norisoprenoids.

    Science.gov (United States)

    Marumoto, Shinsuke; Okuno, Yoshiharu; Miyazawa, Mitsuo

    2017-08-01

    Inhibition of β-secretase (BACE1) is currently regarded as the leading treatment strategy for Alzheimer's disease. In the present study, we aimed to screen the in vitro inhibitory activity of 80 types of aroma compounds (monoterpenes, sesquiterpenes, and C 13 norisoprenoids), including plant-based types, at a 200-μM concentration against a recombinant human BACE1. The results showed that the most potent inhibitor of BACE1 was geranyl acetone followed by (+)-camphor, (-)-fenchone, (+)-fenchone, and (-)-camphor with the half-maximal inhibitory concentration (IC 50 ) values of 51.9 ± 3.9, 95.9 ± 11.0, 106.3 ± 14.9, 117.0 ± 18.6, and 134.1 ± 16.4 μM, respectively. Furthermore, the mechanism of inhibition of BACE1 by geranyl acetone was analyzed using Dixon kinetics plus Cornish-Bowden plots, which revealed mixed-type mode. Therefore aroma compounds may be used as potential lead molecules for designing anti-BACE1 agents.

  19. Developmental defects in zebrafish for classification of EGF pathway inhibitors

    International Nuclear Information System (INIS)

    Pruvot, Benoist; Curé, Yoann; Djiotsa, Joachim; Voncken, Audrey; Muller, Marc

    2014-01-01

    One of the major challenges when testing drug candidates targeted at a specific pathway in whole animals is the discrimination between specific effects and unwanted, off-target effects. Here we used the zebrafish to define several developmental defects caused by impairment of Egf signaling, a major pathway of interest in tumor biology. We inactivated Egf signaling by genetically blocking Egf expression or using specific inhibitors of the Egf receptor function. We show that the combined occurrence of defects in cartilage formation, disturbance of blood flow in the trunk and a decrease of myelin basic protein expression represent good indicators for impairment of Egf signaling. Finally, we present a classification of known tyrosine kinase inhibitors according to their specificity for the Egf pathway. In conclusion, we show that developmental indicators can help to discriminate between specific effects on the target pathway from off-target effects in molecularly targeted drug screening experiments in whole animal systems. - Highlights: • We analyze the functions of Egf signaling on zebrafish development. • Genetic blocking of Egf expression causes cartilage, myelin and circulatory defects. • Chemical inhibition of Egf receptor function causes similar defects. • Developmental defects can reveal the specificity of Egf pathway inhibitors

  20. Developmental defects in zebrafish for classification of EGF pathway inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Pruvot, Benoist; Curé, Yoann; Djiotsa, Joachim; Voncken, Audrey; Muller, Marc, E-mail: m.muller@ulg.ac.be

    2014-01-15

    One of the major challenges when testing drug candidates targeted at a specific pathway in whole animals is the discrimination between specific effects and unwanted, off-target effects. Here we used the zebrafish to define several developmental defects caused by impairment of Egf signaling, a major pathway of interest in tumor biology. We inactivated Egf signaling by genetically blocking Egf expression or using specific inhibitors of the Egf receptor function. We show that the combined occurrence of defects in cartilage formation, disturbance of blood flow in the trunk and a decrease of myelin basic protein expression represent good indicators for impairment of Egf signaling. Finally, we present a classification of known tyrosine kinase inhibitors according to their specificity for the Egf pathway. In conclusion, we show that developmental indicators can help to discriminate between specific effects on the target pathway from off-target effects in molecularly targeted drug screening experiments in whole animal systems. - Highlights: • We analyze the functions of Egf signaling on zebrafish development. • Genetic blocking of Egf expression causes cartilage, myelin and circulatory defects. • Chemical inhibition of Egf receptor function causes similar defects. • Developmental defects can reveal the specificity of Egf pathway inhibitors.

  1. Inhibition of hydrogenase synthesis by DNA gyrase inhibitors in Bradyrhizobium japonicum

    International Nuclear Information System (INIS)

    Novak, P.D.; Maier, R.J.

    1987-01-01

    Derepression of an uptake hydrogenase in Bradyrhizobium japonicum is dependent on a microaerophilic environment. Addition of DNA gyrase inhibitors during derepression of hydrogenase specifically prevented expression of the hydrogenase enzyme. Antibodies to individual hydrogenase subunits failed to detect the protein after derepression in the presence of inhibitors, although there was no general inhibition of protein synthesis. The general pattern of proteins synthesized from 14 C-labeled amino acids during derepression was no significantly different whether proteins were labeled in the presence or in the absence of gyrase inhibitors. In contrast, if transcription or translation was inhibited by addition of inhibitors of those functions, virtually no proteins were labeled during derepression. This indicated that most of the 14 C-labeled proteins were synthesized de novo during derepression, synthesis of most proteins was unaffected by gyrase inhibitors, and the dependence of hydrogenase synthesis on gyrase activity was a specific one

  2. Congenital ventricular septal defects and prenatal exposure to cyclooxygenase inhibitors

    Directory of Open Access Journals (Sweden)

    F. Burdan

    2006-07-01

    Full Text Available Ventricular septal defects (VSDs are common congenital abnormalities which have been reported to be associated with maternal fever and various environmental factors. The aim of the present study was to evaluate the effect of prenatal exposure to cyclooxygenase (COX inhibitors on heart defects. A retrospective statistical analysis was performed using data collected in our laboratory during various teratological studies carried out on albino CRL:(WIWUBR Wistar strain rats from 1997 to 2004. The observations were compared with concurrent and historic control data, as well as findings from other developmental toxicological studies with selective and nonselective COX-2 inhibitors. Despite the lack of significant differences in the frequency of VSDs between drug-exposed and control groups, statistical analysis by the two-sided Mantel-Haenszel test and historical control data showed a higher incidence of heart defects in offspring exposed to nonselective COX inhibitors (30.06/10,000. Unlike other specific inhibitors, aspirin (46.26/10,000 and ibuprofen (106.95/10,000 significantly increased the incidence of the VSD when compared with various control groups (5.38-19.72/10,000. No significant differences in length or weight were detected between fetuses exposed to COX inhibitors and born with VSD and non-malformed offsprings. However, a statistically significant increase of fetal body length and decrease of body mass index were found in fetuses exposed to COX inhibitors when compared with untreated control. We conclude that prenatal exposure to COX inhibitors, especially aspirin and ibuprofen, increased the incidence of VSDs in rat offspring but was not related to fetal growth retardation.

  3. Characterization of inhibitor(s) of β-glucuronidase enzyme activity in GUS-transgenic wheat

    KAUST Repository

    Ramadan, Ahmed M Ali; Eissa, Hala F.; El-Domyati, Fotouh M.; Saleh, Osama Mesilhy; Ibrahim, Nasser E.; Salama, M. I.; Mahfouz, Magdy M.; Bahieldin, Ahmed M.

    2011-01-01

    The uidA gene, encoding for β-glucuronidase (GUS), is the most frequently used reporter gene in plants. As a reporter enzyme, GUS can be assayed both qualitatively and quantitatively. In wheat, there are numerous reports of failure in detecting GUS enzyme activity in tissues of transgenic plants, while other reports have suggested presence of β-glucuronidase inhibitor(s) in wheat tissues. In the present study, we show that the β-glucuronidase enzyme activity is not only tissue-specific but also genotype-dependent. Our data demonstrate that the glucuronic acid could be the candidate inhibitor for β-glucuronidase enzyme activity in wheat leaves and roots. It should be noted that the assays to detect β-glucuronidase enzyme activity in wheat should be interpreted carefully. Based on the data of our present study, we recommend studying the chemical pathways, the unintended effects and the possible loss-of-function of any candidate transgene prior to transformation experiments. © 2011 Springer Science+Business Media B.V.

  4. Characterization of inhibitor(s) of β-glucuronidase enzyme activity in GUS-transgenic wheat

    KAUST Repository

    Ramadan, Ahmed M Ali

    2011-06-26

    The uidA gene, encoding for β-glucuronidase (GUS), is the most frequently used reporter gene in plants. As a reporter enzyme, GUS can be assayed both qualitatively and quantitatively. In wheat, there are numerous reports of failure in detecting GUS enzyme activity in tissues of transgenic plants, while other reports have suggested presence of β-glucuronidase inhibitor(s) in wheat tissues. In the present study, we show that the β-glucuronidase enzyme activity is not only tissue-specific but also genotype-dependent. Our data demonstrate that the glucuronic acid could be the candidate inhibitor for β-glucuronidase enzyme activity in wheat leaves and roots. It should be noted that the assays to detect β-glucuronidase enzyme activity in wheat should be interpreted carefully. Based on the data of our present study, we recommend studying the chemical pathways, the unintended effects and the possible loss-of-function of any candidate transgene prior to transformation experiments. © 2011 Springer Science+Business Media B.V.

  5. Azidoblebbistatin, a photoreactive myosin inhibitor

    Science.gov (United States)

    Képiró, Miklós; Várkuti, Boglárka H.; Bodor, Andrea; Hegyi, György; Drahos, László; Kovács, Mihály; Málnási-Csizmadia, András

    2012-01-01

    Photoreactive compounds are important tools in life sciences that allow precisely timed covalent crosslinking of ligands and targets. Using a unique technique we have synthesized azidoblebbistatin, which is a derivative of blebbistatin, the most widely used myosin inhibitor. Without UV irradiation azidoblebbistatin exhibits identical inhibitory properties to those of blebbistatin. Using UV irradiation, azidoblebbistatin can be covalently crosslinked to myosin, which greatly enhances its in vitro and in vivo effectiveness. Photo-crosslinking also eliminates limitations associated with the relatively low myosin affinity and water solubility of blebbistatin. The wavelength used for photo-crosslinking is not toxic for cells and tissues, which confers a great advantage in in vivo tests. Because the crosslink results in an irreversible association of the inhibitor to myosin and the irradiation eliminates the residual activity of unbound inhibitor molecules, azidoblebbistatin has a great potential to become a highly effective tool in both structural studies of actomyosin contractility and the investigation of cellular and physiological functions of myosin II. We used azidoblebbistatin to identify previously unknown low-affinity targets of the inhibitor (EC50 ≥ 50 μM) in Dictyostelium discoideum, while the strongest interactant was found to be myosin II (EC50 = 5 μM). Our results demonstrate that azidoblebbistatin, and potentially other azidated drugs, can become highly useful tools for the identification of strong- and weak-binding cellular targets and the determination of the apparent binding affinities in in vivo conditions. PMID:22647605

  6. Biological abatement of cellulase inhibitors.

    Science.gov (United States)

    Cao, Guangli; Ximenes, Eduardo; Nichols, Nancy N; Zhang, Leyu; Ladisch, Michael

    2013-10-01

    Removal of enzyme inhibitors released during lignocellulose pretreatment is essential for economically feasible biofuel production. We tested bio-abatement to mitigate enzyme inhibitor effects observed in corn stover liquors after pretreatment with either dilute acid or liquid hot water at 10% (w/v) solids. Bio-abatement of liquors was followed by enzymatic hydrolysis of cellulose. To distinguish between inhibitor effects on enzymes and recalcitrance of the substrate, pretreated corn stover solids were removed and replaced with 1% (w/v) Solka Floc. Cellulose conversion in the presence of bio-abated liquors from dilute acid pretreatment was 8.6% (0.1x enzyme) and 16% (1x enzyme) higher than control (non-abated) samples. In the presence of bio-abated liquor from liquid hot water pretreated corn stover, 10% (0.1x enzyme) and 13% (1x enzyme) higher cellulose conversion was obtained compared to control. Bio-abatement yielded improved enzyme hydrolysis in the same range as that obtained using a chemical (overliming) method for mitigating inhibitors. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Phosphodiesterase inhibitors in clinical urology.

    Science.gov (United States)

    Ückert, Stefan; Kuczyk, Markus A; Oelke, Matthias

    2013-05-01

    To date, benign diseases of the male and female lower urinary and genital tract, such as erectile dysfunction, bladder overactivity, lower urinary tract symptomatology secondary to benign prostatic hyperplasia and symptoms of female sexual dysfunction (including arousal and orgasmic disorders), can be therapeutically approached by influencing the function of the smooth musculature of the respective tissues. The use of isoenzyme-selective phosphodiesterase (PDE) inhibitors is considered a great opportunity to treat various diseases of the human urogenital tract. PDE inhibitors, in particular the PDE5 (cyclic GMP PDE) inhibitors avanafil, lodenafil, sildenafil, tadalafil, udenafil and vardenafil, are regarded as efficacious, having a fast onset of drug action and an improved effect-to-adverse event ratio, combining a high response rate with the advantage of an on-demand intake. The purpose of this review is to summarize recent as well as potential future indications, namely, erectile dysfunction, Peyronie's disease, overactive bladder, urinary stone disease, lower urinary tract symptomatology secondary to benign prostatic hyperplasia and premature ejaculation, for the use of PDE inhibitors in clinical urology.

  8. Inhibitors of mTOR

    NARCIS (Netherlands)

    Klümpen, Heinz-Josef; Beijnen, Jos H.; Gurney, Howard; Schellens, Jan H. M.

    2010-01-01

    Inhibitors of mammalian target of rapamycin (mTOR) have been approved for the treatment of renal cell carcinoma and appear to have a role in the treatment of other malignancies. The primary objective of this drug review is to provide pharmacokinetic and dynamic properties of the commonly used drugs

  9. Retroviral proteinases and their inhibitors

    Czech Academy of Sciences Publication Activity Database

    Sedláček, Juraj

    2000-01-01

    Roč. 3, 3,4 (2000), s. 23-24 [ Proteolytic enzymes and their inhibitors in physiology and pathogenesis. 14.09.2000, Plzen] Institutional research plan: CEZ:AV0Z5052915 Subject RIV: EB - Genetics ; Molecular Biology

  10. Monoamine depletion by reuptake inhibitors

    Directory of Open Access Journals (Sweden)

    Hinz M

    2011-10-01

    Full Text Available Marty Hinz1, Alvin Stein2, Thomas Uncini31Clinical Research, NeuroResearch Clinics Inc, Cape Coral, FL; 2Stein Orthopedic Associates, Plantation, FL; 3DBS Labs Inc, Duluth, MN, USABackground: Disagreement exists regarding the etiology of cessation of the observed clinical results with administration of reuptake inhibitors. Traditionally, when drug effects wane, it is known as tachyphylaxis. With reuptake inhibitors, the placebo effect is significantly greater than the drug effect in the treatment of depression and attention deficit hyperactivity disorder, leading some to assert that waning of drug effects is placebo relapse, not tachyphylaxis.Methods: Two groups were retrospectively evaluated. Group 1 was composed of subjects with depression and Group 2 was composed of bariatric subjects treated with reuptake inhibitors for appetite suppression.Results: In Group 1, 200 subjects with depression were treated with citalopram 20 mg per day. A total of 46.5% (n = 93 achieved relief of symptoms (Hamilton-D rating score ≤ 7, of whom 37 (39.8% of whom experienced recurrence of depression symptoms, at which point an amino acid precursor formula was started. Within 1–5 days, 97.3% (n = 36 experienced relief of depression symptoms. In Group 2, 220 subjects were treated with phentermine 30 mg in the morning and citalopram 20 mg at 4 pm. In this group, 90.0% (n = 198 achieved adequate appetite suppression. The appetite suppression ceased in all 198 subjects within 4–48 days. Administration of an amino acid precursor formula restored appetite suppression in 98.5% (n = 195 of subjects within 1–5 days.Conclusion: Reuptake inhibitors do not increase the total number of monoamine molecules in the central nervous system. Their mechanism of action facilitates redistribution of monoamines from one place to another. In the process, conditions are induced that facilitate depletion of monoamines. The "reuptake inhibitor monoamine depletion theory" of this paper

  11. HDAC inhibitors: modulating leukocyte differentiation, survival, proliferation and inflammation.

    Science.gov (United States)

    Sweet, Matthew J; Shakespear, Melanie R; Kamal, Nabilah A; Fairlie, David P

    2012-01-01

    Therapeutic effects of histone deacetylase (HDAC) inhibitors in cancer models were first linked to their ability to cause growth arrest and apoptosis of tumor cells. It is now clear that these agents also have pleiotropic effects on angiogenesis and the immune system, and some of these properties are likely to contribute to their anti-cancer activities. It is also emerging that inhibitors of specific HDACs affect the differentiation, survival and/or proliferation of distinct immune cell populations. This is true for innate immune cells such as macrophages, as well as cells of the acquired immune system, for example, T-regulatory cells. These effects may contribute to therapeutic profiles in some autoimmune and chronic inflammatory disease models. Here, we review our current understanding of how classical HDACs (HDACs 1-11) and their inhibitors impact on differentiation, survival and proliferation of distinct leukocyte populations, as well as the likely relevance of these effects to autoimmune and inflammatory disease processes. The ability of HDAC inhibitors to modulate leukocyte survival may have implications for the rationale of developing selective inhibitors as anti-inflammatory drugs.

  12. Antitumour agents as inhibitors of tryptophan 2,3-dioxygenase

    Energy Technology Data Exchange (ETDEWEB)

    Pantouris, Georgios; Mowat, Christopher G., E-mail: C.G.Mowat@ed.ac.uk

    2014-01-03

    Highlights: •∼2800 National Cancer Institute USA compounds have been screened as potential inhibitors of TDO and/or IDO. •Seven compounds with anti-tumour properties have been identified as potent inhibitors. •NSC 36398 (taxifolin, dihydroquercetin) is selective for TDO with a K{sub i} of 16 M. •This may help further our understanding of the role of TDO in cancer. -- Abstract: The involvement of tryptophan 2,3-dioxygenase (TDO) in cancer biology has recently been described, with the enzyme playing an immunomodulatory role, suppressing antitumour immune responses and promoting tumour cell survival and proliferation. This finding reinforces the need for specific inhibitors of TDO that may potentially be developed for therapeutic use. In this work we have screened ∼2800 compounds from the library of the National Cancer Institute USA and identified seven potent inhibitors of TDO with inhibition constants in the nanomolar or low micromolar range. All seven have antitumour properties, killing various cancer cell lines. For comparison, the inhibition potencies of these compounds were tested against IDO and their inhibition constants are reported. Interestingly, this work reveals that NSC 36398 (dihydroquercetin, taxifolin), with an in vitro inhibition constant of ∼16 μM, is the first TDO-selective inhibitor reported.

  13. Kinetic characterization of ebselen, chelerythrine and apomorphine as glutaminase inhibitors.

    Science.gov (United States)

    Thomas, Ajit G; Rojas, Camilo; Tanega, Cordelle; Shen, Min; Simeonov, Anton; Boxer, Matthew B; Auld, Douglas S; Ferraris, Dana V; Tsukamoto, Takashi; Slusher, Barbara S

    2013-08-23

    Glutaminase catalyzes the hydrolysis of glutamine to glutamate and plays a central role in the proliferation of neoplastic cells via glutaminolysis, as well as in the generation of excitotoxic glutamate in central nervous system disorders such as HIV-associated dementia (HAD) and multiple sclerosis. Both glutaminase siRNA and glutaminase inhibition have been shown to be effective in in vitro models of cancer and HAD, suggesting a potential role for small molecule glutaminase inhibitors. However, there are no potent, selective inhibitors of glutaminase currently available. The two prototypical glutaminase inhibitors, BPTES and DON, are either insoluble or non-specific. In a search for more drug-like glutaminase inhibitors, we conducted a screen of 1280 in vivo active drugs (Library of Pharmacologically Active Compounds (LOPAC(1280))) and identified ebselen, chelerythrine and (R)-apomorphine. The newly identified inhibitors exhibited 10 to 1500-fold greater affinities than DON and BPTES and over 100-fold increased efficiency of inhibition. Although non-selective, it is noteworthy that the affinity of ebselen for glutaminase is more potent than any other activity yet described. It is possible that the previously reported biological activity seen with these compounds is due, in part, to glutaminase inhibition. Ebselen, chelerythrine and apomorphine complement the armamentarium of compounds to explore the role of glutaminase in disease. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Lonafarnib is a potential inhibitor for neovascularization.

    Directory of Open Access Journals (Sweden)

    Linlin Sun

    Full Text Available Atherosclerosis is a common cardiovascular disease that involves the build-up of plaque on the inner walls of the arteries. Intraplaque neovacularization has been shown to be essential in the pathogenesis of atherosclerosis. Previous studies showed that small-molecule compounds targeting farnesyl transferase have the ability to prevent atherosclerosis in apolipoprotein E-deficient mice, but the underlying mechanism remains to be elucidated. In this study, we found that lonafarnib, a specific inhibitor of farnesyl transferase, elicits inhibitory effect on vascular endothelial capillary assembly in vitro in a dose-dependent manner. In addition, we showed that lonafarnib treatment led to a dose-dependent decrease in scratch wound closure in vitro, whereas it had little effect on endothelial cell proliferation. These data indicate that lonafarnib inhibits neovascularization via directly targeting endothelial cells and disturbing their motility. Moreover, we demonstrated that pharmacological inhibition of farnesyl transferase by lonafarnib significantly impaired centrosome reorientation toward the leading edge of endothelial cells. Mechanistically, we found that the catalytic β subunit of farnesyl transferase associated with a cytoskeletal protein important for the establishment and maintenance of cell polarity. Additionally, we showed that lonafarnib remarkably inhibited the expression of the cytoskeletal protein and interrupted its interaction with farnesyl transferase. Our findings thus offer novel mechanistic insight into the protective effect of farnesyl transferase inhibitors on atherosclerosis and provide encouraging evidence for the potential use of this group of agents in inhibiting plaque neovascularization.

  15. Matrix Metalloproteinase Responsive Delivery of Myostatin Inhibitors.

    Science.gov (United States)

    Braun, Alexandra C; Gutmann, Marcus; Ebert, Regina; Jakob, Franz; Gieseler, Henning; Lühmann, Tessa; Meinel, Lorenz

    2017-01-01

    The inhibition of myostatin - a member of the transforming growth factor (TGF-β) family - drives regeneration of functional skeletal muscle tissue. We developed a bioresponsive drug delivery system (DDS) linking release of a myostatin inhibitor (MI) to inflammatory flares of myositis to provide self-regulated MI concentration gradients within tissues of need. A protease cleavable linker (PCL) - responding to MMP upregulation - is attached to the MI and site-specifically immobilized on microparticle surfaces. The PCL disintegrated in a matrix metalloproteinase (MMP) 1, 8, and particularly MMP-9 concentration dependent manner, with MMP-9 being an effective surrogate biomarker correlating with the activity of myositis. The bioactivity of particle-surface bound as well as released MI was confirmed by luciferase suppression in stably transfected HEK293 cells responding to myostatin induced SMAD phosphorylation. We developed a MMP-responsive DDS for MI delivery responding to inflammatory flare of a diseased muscle matching the kinetics of MMP-9 upregulation, with MMP-9 kinetics matching (patho-) physiological myostatin levels. ᅟ: Graphical Abstract Schematic illustration of the matrix metalloproteinase responsive delivery system responding to inflammatory flares of muscle disease. The protease cleavable linker readily disintegrates upon entry into the diseased tissue, therby releasing the mystatin inhibitor.

  16. Serotonin and Norepinephrine Reuptake Inhibitors (SNRIs)

    Science.gov (United States)

    Serotonin and norepinephrine reuptake inhibitors (SNRIs) Antidepressant SNRIs help relieve depression symptoms, such as irritability and sadness, ... effects they may cause. By Mayo Clinic Staff Serotonin and norepinephrine reuptake inhibitors (SNRIs) are a class ...

  17. Contemporary protease inhibitors and cardiovascular risk

    DEFF Research Database (Denmark)

    Lundgren, Jens; Mocroft, Amanda; Ryom, Lene

    2018-01-01

    PURPOSE OF REVIEW: To review the evidence linking use of HIV protease inhibitors with excess risk of cardiovascular disease (CVD) in HIV+ populations. RECENT FINDINGS: For the two contemporary most frequently used protease inhibitors, darunavir and atazanavir [both pharmacologically boosted...

  18. Reduction rules for reset/inhibitor nets

    NARCIS (Netherlands)

    Verbeek, H.M.W.; Wynn, M.T.; Aalst, van der W.M.P.; Hofstede, ter A.H.M.

    2010-01-01

    Reset/inhibitor nets are Petri nets extended with reset arcs and inhibitor arcs. These extensions can be used to model cancellation and blocking. A reset arc allows a transition to remove all tokens from a certain place when the transition fires. An inhibitor arc can stop a transition from being

  19. 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...

  20. Structure of a retro-binding peptide inhibitor complexed with human alpha-thrombin.

    Science.gov (United States)

    Tabernero, L; Chang, C Y; Ohringer, S L; Lau, W F; Iwanowicz, E J; Han, W C; Wang, T C; Seiler, S M; Roberts, D G; Sack, J S

    1995-02-10

    The crystallographic structure of the ternary complex between human alpha-thrombin, hirugen and the peptidyl inhibitor Phe-alloThr-Phe-O-CH3, which is acylated at its N terminus with 4-guanidino butanoic acid (BMS-183507), has been determined at 2.6 A resolution. The structure reveals a unique "retro-binding" mode for this tripeptide active site inhibitor. The inhibitor binds with its alkyl-guanidine moiety in the primary specificity pocket and its two phenyl rings occupying the hydrophobic proximal and distal pockets of the thrombin active site. In this arrangement the backbone of the tripeptide forms a parallel beta-strand to the thrombin main-chain at the binding site. This is opposite to the orientation of the natural substrate, fibrinogen, and all the small active site-directed thrombin inhibitors whose bound structures have been previously reported. BMS-183507 is the first synthetic inhibitor proved to bind in a retro-binding fashion to thrombin, in a fashion similar to that of the N-terminal residues of the natural inhibitor hirudin. Furthermore, this new potent thrombin inhibitor (Ki = 17.2 nM) is selective for thrombin over other serine proteases tested and may be a template to be considered in designing hirudin-based thrombin inhibitors with interactions at the specificity pocket.

  1. Analgésicos inibidores específicos da ciclooxigenase-2: avanços terapêuticos Analgésicos inhibidores específicos de la ciclooxigenase-2: avanzos terapéuticos Specific cyclooxygenase-2 inhibitor analgesics: therapeutic advances

    Directory of Open Access Journals (Sweden)

    Wilson Andrade Carvalho

    2004-06-01

    of more selective drugs to decrease inflammation without affecting COX-1 that protects stomach and kidneys and giving origin to a new generation of anti-inflammatory compounds called specific COX-2 inhibitors. CONCLUSIONS: Although there is significantly lower gastrointestinal toxicity in patients treated with selective COX-2 inhibitors, other severe adverse effects have been observed, including renal failure and cardiovascular effects, such as myocardial infarction acute and thrombosis. Despite these potential side effects, these new drugs are being tested in different clinical conditions, especially in cancer prevention and Alzheimer's disease.

  2. Molecular modeling of auxin transport inhibitors

    International Nuclear Information System (INIS)

    Gardner, G.; Black-Schaefer, C.; Bures, M.G.

    1990-01-01

    Molecular modeling techniques have been used to study the chemical and steric properties of auxin transport inhibitors. These bind to a specific site on the plant plasma membrane characterized by its affinity for N-1-naphthylphthalamic acid (NPA). A three-dimensional model was derived from critical features of ligands for the NPA receptor, and a suggested binding conformation is proposed. This model, along with three-dimensional structural searching techniques, was then used to search the Abbott corporate database of chemical structures. Of the 467 compounds that satisfied the search criteria, 77 representative molecules were evaluated for their ability to compete for [ 3 H]NPA binding to corn microsomal membranes. Nineteen showed activity that ranged from 16 to 85% of the maximum NPA binding. Four of the most active of these, from chemical classes not included in the original compound set, also inhibited polar auxin transport through corn coleoptile sections

  3. Stabilization versus inhibition of TAFIa by competitive inhibitors in vitro

    NARCIS (Netherlands)

    Walker, J.B.; Hughes, B.; James, I.; Haddock, P.; Kluft, C.; Bajzar, L.

    2003-01-01

    Two competitive inhibitors of TAFIa (activated thrombin-activable fibrinolysis inhibitor), 2-guanidinoethyl-mercaptosuccinic acid and potato tuber carboxypeptidase inhibitor, variably affect fibrinolysis of clotted human plasma. Depending on their concentration, the inhibitors shortened, prolonged,

  4. Specifying Specification.

    Science.gov (United States)

    Paulo, Norbert

    2016-03-01

    This paper tackles the accusation that applied ethics is no serious academic enterprise because it lacks theoretical bracing. It does so in two steps. In the first step I introduce and discuss a highly acclaimed method to guarantee stability in ethical theories: Henry Richardson's specification. The discussion shows how seriously ethicists take the stability of the connection between the foundational parts of their theories and their further development as well as their "application" to particular problems or cases. A detailed scrutiny of specification leads to the second step, where I use insights from legal theory to inform the debate around stability from that point of view. This view reveals some of specification's limitations. I suggest that, once specification is sufficiently specified, it appears astonishingly similar to deduction as used in legal theory. Legal theory also provides valuable insight into the functional range of deduction and its relation to other forms of reasoning. This leads to a richer understanding of stability in normative theories and to a smart division of labor between deduction and other forms of reasoning. The comparison to legal theory thereby provides a framework for how different methods such as specification, deduction, balancing, and analogy relate to one another.

  5. Kinetic characterization of ebselen, chelerythrine and apomorphine as glutaminase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, Ajit G.; Rojas, Camilo [Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Tanega, Cordelle; Shen, Min; Simeonov, Anton; Boxer, Matthew B.; Auld, Douglas S. [National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850 (United States); Ferraris, Dana V. [Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Tsukamoto, Takashi [Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Slusher, Barbara S., E-mail: bslusher@jhmi.edu [Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States)

    2013-08-23

    Highlights: •Ebselen, chelerythrine and apomorphine were identified as glutaminase inhibitors. •These had greater affinities and efficiency of inhibition than known prototypes. •Their previously reported biological activity could be due to glutaminase inhibition. -- Abstract: Glutaminase catalyzes the hydrolysis of glutamine to glutamate and plays a central role in the proliferation of neoplastic cells via glutaminolysis, as well as in the generation of excitotoxic glutamate in central nervous system disorders such as HIV-associated dementia (HAD) and multiple sclerosis. Both glutaminase siRNA and glutaminase inhibition have been shown to be effective in in vitro models of cancer and HAD, suggesting a potential role for small molecule glutaminase inhibitors. However, there are no potent, selective inhibitors of glutaminase currently available. The two prototypical glutaminase inhibitors, BPTES and DON, are either insoluble or non-specific. In a search for more drug-like glutaminase inhibitors, we conducted a screen of 1280 in vivo active drugs (Library of Pharmacologically Active Compounds (LOPAC{sup 1280})) and identified ebselen, chelerythrine and (R)-apomorphine. The newly identified inhibitors exhibited 10 to 1500-fold greater affinities than DON and BPTES and over 100-fold increased efficiency of inhibition. Although non-selective, it is noteworthy that the affinity of ebselen for glutaminase is more potent than any other activity yet described. It is possible that the previously reported biological activity seen with these compounds is due, in part, to glutaminase inhibition. Ebselen, chelerythrine and apomorphine complement the armamentarium of compounds to explore the role of glutaminase in disease.

  6. Kinetic characterization of ebselen, chelerythrine and apomorphine as glutaminase inhibitors

    International Nuclear Information System (INIS)

    Thomas, Ajit G.; Rojas, Camilo; Tanega, Cordelle; Shen, Min; Simeonov, Anton; Boxer, Matthew B.; Auld, Douglas S.; Ferraris, Dana V.; Tsukamoto, Takashi; Slusher, Barbara S.

    2013-01-01

    Highlights: •Ebselen, chelerythrine and apomorphine were identified as glutaminase inhibitors. •These had greater affinities and efficiency of inhibition than known prototypes. •Their previously reported biological activity could be due to glutaminase inhibition. -- Abstract: Glutaminase catalyzes the hydrolysis of glutamine to glutamate and plays a central role in the proliferation of neoplastic cells via glutaminolysis, as well as in the generation of excitotoxic glutamate in central nervous system disorders such as HIV-associated dementia (HAD) and multiple sclerosis. Both glutaminase siRNA and glutaminase inhibition have been shown to be effective in in vitro models of cancer and HAD, suggesting a potential role for small molecule glutaminase inhibitors. However, there are no potent, selective inhibitors of glutaminase currently available. The two prototypical glutaminase inhibitors, BPTES and DON, are either insoluble or non-specific. In a search for more drug-like glutaminase inhibitors, we conducted a screen of 1280 in vivo active drugs (Library of Pharmacologically Active Compounds (LOPAC 1280 )) and identified ebselen, chelerythrine and (R)-apomorphine. The newly identified inhibitors exhibited 10 to 1500-fold greater affinities than DON and BPTES and over 100-fold increased efficiency of inhibition. Although non-selective, it is noteworthy that the affinity of ebselen for glutaminase is more potent than any other activity yet described. It is possible that the previously reported biological activity seen with these compounds is due, in part, to glutaminase inhibition. Ebselen, chelerythrine and apomorphine complement the armamentarium of compounds to explore the role of glutaminase in disease

  7. Probe specificity

    International Nuclear Information System (INIS)

    Laget, J.M.

    1986-11-01

    Specificity and complementarity of hadron and electron probes must be systematically developed to answer three questions currently asked in intermediate energy nuclear physics: what is nucleus structure at short distances, what is nature of short range correlations, what is three body force nature [fr

  8. The evolution of renin-angiotensin blockade: angiotensin-converting enzyme inhibitors as the starting point.

    Science.gov (United States)

    Sica, Domenic A

    2010-04-01

    The renin-angiotensin system has been a target in the treatment of hypertension for close to three decades. Several medication classes that block specific aspects of this system have emerged as useful therapies, including angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and, most recently, direct renin inhibitors. There has been a natural history to the development of each of these three drug classes, starting with their use as antihypertensive agents; thereafter, in each case they have been employed as end-organ protective agents. To date, there has been scant evidence to favor angiotensin receptor blockers or direct renin inhibitors over angiotensin-converting enzyme inhibitors in treating hypertension or in affording end-organ protection; thus, angiotensin-converting enzyme inhibitors remain the standard of care when renin-angiotensin system blockade is warranted.

  9. Crystal structures of HIV-1 nonnucleoside reverse transcriptase inhibitors: N-benzyl-4-methyl-benzimidazoles

    Science.gov (United States)

    Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

    2009-07-01

    HIV-1 nonnucleoside reverse transcriptase inhibitors are potentially specific and effective drugs in AIDS therapy. The presence of two aromatic systems with an angled orientation in the molecule of the inhibitor is crucial for interactions with HIV-1 RT. The inhibitor drives like a wedge into the cluster of aromatic residues of RT HIV-1 and restrains the enzyme in a conformation that blocks the chemical step of nucleotide incorporation. Structural studies provide useful information for designing new, more active inhibitors. The crystal structures of four NNRTIs are presented here. The investigated compounds are derivatives of N-benzyl-4-methyl-benzimidazole with various aliphatic and aromatic substituents at carbon 2 positions and a 2,6-dihalogeno-substituted N-benzyl moiety. Structural data reported here show that the conformation of the investigated compounds is relatively rigid. Such feature is important for the nonnucleoside inhibitor binding to HIV-1 reverse transcriptase.

  10. Co-evolution of insect proteases and plant protease inhibitors.

    Science.gov (United States)

    Jongsma, Maarten A; Beekwilder, Jules

    2011-08-01

    Plants are at the basis of the food chain, but there is no such thing as a "free lunch" for herbivores. To promote reproductive success, plants evolved multi-layered defensive tactics to avoid or discourage herbivory. To the detriment of plants, herbivores, in turn, evolved intricate strategies to find, eat, and successfully digest essential plant parts to raise their own offspring. In this battle the digestive tract is the arena determining final victory or defeat as measured by growth or starvation of the herbivore. Earlier, specific molecular opponents were identified as proteases and inhibitors: digestive proteases of herbivores evolved structural motifs to occlude plant protease inhibitors, or alternatively, the insects evolved proteases capable of specifically degrading the host plant inhibitors. In response plant inhibitors evolved hyper-variable and novel protein folds to remain active against potential herbivores. At the level of protease regulation in herbivorous insects, it was shown that inhibition-insensitive digestive proteases are up-regulated when sensitive proteases are inhibited. The way this regulation operates in mammals is known as negative feedback by gut-luminal factors, so-called 'monitor peptides' that are sensitive to the concentration of active enzymes. We propose that regulation of gut enzymes by endogenous luminal factors has been an open invitation to plants to "hijack" this regulation by evolving receptor antagonists, although yet these plant factors have not been identified. In future research the question of the co-evolution of insect proteases and plant inhibitors should, therefore, be better approached from a systems level keeping in mind that evolution is fundamentally opportunistic and that the plant's fitness is primarily improved by lowering the availability of essential amino acids to an herbivore by any available mechanism.

  11. Developing selective histone deacetylases (HDACs) inhibitors through ebselen and analogs.

    Science.gov (United States)

    Wang, Yuren; Wallach, Jason; Duane, Stephanie; Wang, Yuan; Wu, Jianghong; Wang, Jeffrey; Adejare, Adeboye; Ma, Haiching

    2017-01-01

    Histone deacetylases (HDACs) are key regulators of gene expression in cells and have been investigated as important therapeutic targets for cancer and other diseases. Different subtypes of HDACs appear to play disparate roles in the cells and are associated with specific diseases. Therefore, substantial effort has been made to develop subtype-selective HDAC inhibitors. In an effort to discover existing scaffolds with HDAC inhibitory activity, we screened a drug library approved by the US Food and Drug Administration and a National Institutes of Health Clinical Collection compound library in HDAC enzymatic assays. Ebselen, a clinical safe compound, was identified as a weak inhibitor of several HDACs, including HDAC1, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, and HDAC9 with half maximal inhibitory concentrations approximately single digit of µM. Two ebselen analogs, ebselen oxide and ebsulfur (a diselenide analog of ebselen), also inhibited these HDACs, however with improved potencies on HDAC8. Benzisothiazol, the core structure of ebsulfur, specifically inhibited HDAC6 at a single digit of µM but had no inhibition on other HDACs. Further efforts on structure-activity relationship based on the core structure of ebsulfur led to the discovery of a novel class of potent and selective HDAC6 inhibitors with RBC-2008 as the lead compound with single-digit nM potency. This class of histone deacetylase inhibitor features a novel pharmacophore with an ebsulfur scaffold selectively targeting HDAC6. Consistent with its inhibition on HDAC6, RBC-2008 significantly increased the acetylation levels of α-tubulin in PC-3 cells. Furthermore, treatment with these compounds led to cell death of multiple tumor cell lines in a dose-dependent manner. These results demonstrated that ebselen and ebsulfur analogs are inhibitors of HDACs, supporting further preclinical development of this class of compounds for potential therapeutic applications.

  12. 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.

  13. Rhomboid protease inhibitors: Emerging tools and future therapeutics

    Czech Academy of Sciences Publication Activity Database

    Stříšovský, Kvido

    2016-01-01

    Roč. 60, Dec (2016), s. 52-62 ISSN 1084-9521 R&D Projects: GA MŠk(CZ) LK11206; GA MŠk LO1302 EU Projects: European Commission(XE) 304154 - Rhomboid substrates Institutional support: RVO:61388963 Keywords : rhomboid protease * inhibitor * disease * mechanism * substrate specificity Subject RIV: CE - Biochemistry Impact factor: 6.614, year: 2016 http://www.sciencedirect.com/science/article/pii/S1084952116302592

  14. 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.

  15. Calcineurin-inhibitor pain syndrome.

    Science.gov (United States)

    Prommer, Eric

    2012-07-01

    There has been increased recognition of calcineurin, a phosphoprotein serine/threonine phosphatase enzyme, in the regulation of many physiologic systems. Calcineurin mediates activation of lymphocytes, which play a role in immune response. Widely distributed in the central nervous system, calcinuerin also plays an important role in sensory neural function, via its role in the regulation of newly discovered 2-pore potassium channels, which greatly influence neuronal resting membrane potentials. Calcinuerin inhibition is the mechanism of action of immunomodulatory drugs such as cyclosporine and tacrolimus, which are widely used in transplantation medicine to prevent rejection. While important for immunosuppression, the use of calcineurin inhibitors has been associated with the development of a new pain syndrome called the calcineurin pain syndrome, which appears to be an untoward complication of the interruption of the physiologic function of calcineurin. This is a narrative review focusing on the epidemiology, pathophysiology, characterization of a newly recognized pain syndrome associated with the use of calcineurin inhibitors. The use of immunosuppressants however is associated with several well-known toxicities to which the calcineurin pain syndrome can be added. The development of this syndrome most likely involves altered nociceptive processing due to the effect of calcineurin inhibition on neuronal firing, as well as effects of calcineurin on vascular tone. The most striking aspect of the treatment of this syndrome is the response to calcium channel blockers, which suggest that the effects of calcineurin inhibition on vascular tone play an important role in the development of the calcineurin pain syndrome. The calcineurin syndrome is a newly recognized complication associated with the use of calcineurin inhibitors. There is no standard therapy at this time but anecdotal reports suggest the effectiveness of calcium channel blockers.

  16. Crystallization inhibitors for amorphous oxides

    International Nuclear Information System (INIS)

    Reznitskij, L.A.; Filippova, S.E.

    1993-01-01

    Data for the last 10 years, in which experimental results of studying the temperature stabilization of x-ray amorphous oxides (including R 3 Fe 5 O 12 R-rare earths, ZrO 2 , In 2 O 3 , Sc 2 O 3 ) and their solid solution are presented, are generalized. Processes of amorphous oxide crystallization with the production of simple oxides, solid solutions and chemical compounds with different polyhedral structure, are investigated. Energy and crystallochemical criteria for selecting the doping inhibitor-components stabilizing the amorphous state are ascertained, temperatures and enthalpies of amorpous oxide crystallization are determined, examination of certain provisions of iso,orphous miscibility theory is conducted

  17. Inhibitors of plant hormone transport

    Czech Academy of Sciences Publication Activity Database

    Klíma, Petr; Laňková, Martina; Zažímalová, Eva

    2016-01-01

    Roč. 253, č. 6 (2016), s. 1391-1404 ISSN 0033-183X R&D Projects: GA MŠk(CZ) LD15088 Institutional support: RVO:61389030 Keywords : polar auxin transport * acid-binding protein * gnom arf-gef * equilibrative nucleoside transporter * efflux carrier polarity * plasma-membrane-protein * cultured tobacco cells * arabidopsis-thaliana * gravitropic response * brefeldin-a * Plant hormones * Transport * Inhibitors * Auxin * Cytokinins * Strigolactones * Abscisic acid * Cell biology Subject RIV: ED - Physiology Impact factor: 2.870, year: 2016

  18. Synthesis and evaluation of iodide uptake inhibitors in thyroid gland

    International Nuclear Information System (INIS)

    Lacotte, Pierre

    2012-01-01

    This work was intended to discover small organic molecules acting as iodide uptake inhibitors in thyroid cells. These compounds can indeed be derivatized into biochemical probes for further characterization of proteins involved in iodide transport mechanisms. On the long term, these inhibitors also appear as attractive drug candidates for treatment of thyroid pathologies or radioprotection against iodine isotopes. A similar strategy was adopted for both of the two inhibitor families. First, we synthesized a chemical library of around 100 analogues; we measured their IC50 against iodide uptake in FRTL-5 cells to get structure-activity relationships. Absolute configuration of stereo-genic centers was also investigated, and a preferential stereochemistry was found to be responsible for activity. From this basis, around twenty 'second-generation' analogues were synthesized by combining fragments contributing to biological activity. Biological evaluation indicated that nine were very potent inhibitors, with IC50 ≤ 6 nM and satisfying physicochemical properties required for drug candidates. Finally, one photoactivatable biotinylated probe was developed in each family and used for photoaffinity labeling. Several specifically labeled proteins are still under identification and constitute new potential therapeutic targets. (author)

  19. Novel endogenous angiogenesis inhibitors and their therapeutic potential.

    Science.gov (United States)

    Rao, Nithya; Lee, Yu Fei; Ge, Ruowen

    2015-10-01

    Angiogenesis, the formation of new blood vessels from the pre-existing vasculature is essential for embryonic development and tissue homeostasis. It also plays critical roles in diseases such as cancer and retinopathy. A delicate balance between pro- and anti-angiogenic factors ensures normal physiological homeostasis. Endogenous angiogenesis inhibitors are proteins or protein fragments that are formed in the body and have the ability to limit angiogenesis. Many endogenous angiogenesis inhibitors have been discovered, and the list continues to grow. Endogenous protein/peptide inhibitors are relatively less toxic, better tolerated and have a lower risk of drug resistance, which makes them attractive as drug candidates. In this review, we highlight ten novel endogenous protein angiogenesis inhibitors discovered within the last five years, including ISM1, FKBPL, CHIP, ARHGAP18, MMRN2, SOCS3, TAp73, ZNF24, GPR56 and JWA. Although some of these proteins have been well characterized for other biological functions, we focus on their new and specific roles in angiogenesis inhibition and discuss their potential for therapeutic application.

  20. The design strategy of selective PTP1B inhibitors over TCPTP.

    Science.gov (United States)

    Li, XiangQian; Wang, LiJun; Shi, DaYong

    2016-08-15

    Protein tyrosine phosphatase 1B (PTP1B) has already been well studied as a highly validated therapeutic target for diabetes and obesity. However, the lack of selectivity limited further studies and clinical applications of PTP1B inhibitors, especially over T-cell protein tyrosine phosphatase (TCPTP). In this review, we enumerate the published specific inhibitors of PTP1B, discuss the structure-activity relationships by analysis of their X-ray structures or docking results, and summarize the characteristic of selectivity related residues and groups. Furthermore, the design strategy of selective PTP1B inhibitors over TCPTP is also proposed. We hope our work could provide an effective way to gain specific PTP1B inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Tubulin Inhibitor-Based Antibody-Drug Conjugates for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Hao Chen

    2017-08-01

    Full Text Available Antibody-drug conjugates (ADCs are a class of highly potent biopharmaceutical drugs generated by conjugating cytotoxic drugs with specific monoclonal antibodies through appropriate linkers. Specific antibodies used to guide potent warheads to tumor tissues can effectively reduce undesired side effects of the cytotoxic drugs. An in-depth understanding of antibodies, linkers, conjugation strategies, cytotoxic drugs, and their molecular targets has led to the successful development of several approved ADCs. These ADCs are powerful therapeutics for cancer treatment, enabling wider therapeutic windows, improved pharmacokinetic/pharmacodynamic properties, and enhanced efficacy. Since tubulin inhibitors are one of the most successful cytotoxic drugs in the ADC armamentarium, this review focuses on the progress in tubulin inhibitor-based ADCs, as well as lessons learned from the unsuccessful ADCs containing tubulin inhibitors. This review should be helpful to facilitate future development of new generations of tubulin inhibitor-based ADCs for cancer therapy.

  2. Laura: Soybean variety lacking Kunitz trypsin inhibitor

    Directory of Open Access Journals (Sweden)

    Srebrić Mirjana

    2010-01-01

    Full Text Available Grain of conventional soybean varieties requires heat processing to break down trypsin inhibitor's activity before using as food or animal feed. At the same time, protein denaturation and other qualitative changes occur in soybean grain, especially if the temperature of heating is not controlled. Two types of trypsin inhibitor were found in soybean grain the Kunitz trypsin inhibitor and the Bowman-Birk inhibitor. Mature grain of soybean Laura is lacking Kunitz trypsin inhibitor. Grain yield of variety Laura is equal to high yielding varieties from the maturity group I, where it belongs. Lacking of Kunitz-trypsin inhibitor makes soybean grain suitable for direct feeding in adult non ruminant animals without previous thermal processing. Grain of variety Laura can be processed for a shorter period of time than conventional soybeans. This way we save energy, and preserve valuable nutritional composition of soybean grain, which is of interest in industrial processing.

  3. Vanadium Compounds as PTP Inhibitors

    Directory of Open Access Journals (Sweden)

    Elsa Irving

    2017-12-01

    Full Text Available Phosphotyrosine signaling is regulated by the opposing actions of protein tyrosine kinases (PTKs and protein tyrosine phosphatases (PTPs. Here we discuss the potential of vanadium derivatives as PTP enzyme inhibitors and metallotherapeutics. We describe how vanadate in the V oxidized state is thought to inhibit PTPs, thus acting as a pan-inhibitor of this enzyme superfamily. We discuss recent developments in the biological and biochemical actions of more complex vanadium derivatives, including decavanadate and in particular the growing number of oxidovanadium compounds with organic ligands. Pre-clinical studies involving these compounds are discussed in the anti-diabetic and anti-cancer contexts. Although in many cases PTP inhibition has been implicated, it is also clear that many such compounds have further biochemical effects in cells. There also remain concerns surrounding off-target toxicities and long-term use of vanadium compounds in vivo in humans, hindering their progress through clinical trials. Despite these current misgivings, interest in these chemicals continues and many believe they could still have therapeutic potential. If so, we argue that this field would benefit from greater focus on improving the delivery and tissue targeting of vanadium compounds in order to minimize off-target toxicities. This may then harness their full therapeutic potential.

  4. Proton pump inhibitors and osteoporosis

    DEFF Research Database (Denmark)

    Andersen, Bjarne Nesgaard; Johansen, Per Birger; Abrahamsen, Bo

    2016-01-01

    PURPOSE OF REVIEW: The purpose of the review is to provide an update on recent advances in the evidence based on proton pump inhibitors (PPI) as a possible cause of osteoporosis and osteoporotic fractures. This review focuses, in particular, on new studies published in the last 18 months and a di......PURPOSE OF REVIEW: The purpose of the review is to provide an update on recent advances in the evidence based on proton pump inhibitors (PPI) as a possible cause of osteoporosis and osteoporotic fractures. This review focuses, in particular, on new studies published in the last 18 months...... and a discussion of these findings and how this has influenced our understanding of this association, the clinical impact and the underlying pathophysiology. RECENT FINDINGS: New studies have further strengthened existing evidence linking use of PPIs to osteoporosis. Short-term use does not appear to pose a lower...... risk than long-term use. There is a continued lack of conclusive studies identifying the pathogenesis. Direct effects on calcium absorption or on osteoblast or osteoclast action cannot at present plausibly explain the mechanism. SUMMARY: The use of PPIs is a risk factor for development of osteoporosis...

  5. Structures of human Golgi-resident glutaminyl cyclase and its complexes with inhibitors reveal a large loop movement upon inhibitor binding.

    Science.gov (United States)

    Huang, Kai-Fa; Liaw, Su-Sen; Huang, Wei-Lin; Chia, Cho-Yun; Lo, Yan-Chung; Chen, Yi-Ling; Wang, Andrew H-J

    2011-04-08

    Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD150, was shown to be able to reduce the deposition of pyroglutamate-modified amyloid-β peptides in brain of transgenic mouse models of Alzheimer disease, leading to a significant improvement of learning and memory in those transgenic animals. Here, we report the 1.05-1.40 Å resolution structures, solved by the sulfur single-wavelength anomalous dispersion phasing method, of the Golgi-luminal catalytic domain of the recently identified Golgi-resident QC (gQC) and its complex with PBD150. We also describe the high-resolution structures of secretory QC (sQC)-PBD150 complex and two other gQC-inhibitor complexes. gQC structure has a scaffold similar to that of sQC but with a relatively wider and negatively charged active site, suggesting a distinct substrate specificity from sQC. Upon binding to PBD150, a large loop movement in gQC allows the inhibitor to be tightly held in its active site primarily by hydrophobic interactions. Further comparisons of the inhibitor-bound structures revealed distinct interactions of the inhibitors with gQC and sQC, which are consistent with the results from our inhibitor assays reported here. Because gQC and sQC may play different biological roles in vivo, the different inhibitor binding modes allow the design of specific inhibitors toward gQC and sQC.

  6. HRT Specification

    CERN Document Server

    Möller, M

    1996-01-01

    In the context of the AIS Project (Advanced Informatics Systems for administration and management) a study has been conducted that resulted in the definition of a high level information systems model. Thirteen proposed systems were defined for detailed analysis. The Finance, Foundation, Human Resources, Logistics and Purchasing areas have been studied in detail. These studies have lead to the purchase and implementation of the ORIAC and SIRIAC packages, the Foundation database, the Oracle HR package, the Triton package and EDH and BHT. This specification describes the Human Resources Toolkit (HRT) intended to be used for accessing data in the HR and Foundation systems. This toolkit should help the divisions carry out their Human Resource management, planning and follow-up. It will have extensive report generation capabilities and offer a variety of standard graphs. It should have an easy-to-use graphical user interface and run on the CERN standard desktop platforms.

  7. Interdisciplinary management of EGFR-inhibitor-induced skin reactions: a German expert opinion.

    Science.gov (United States)

    Potthoff, K; Hofheinz, R; Hassel, J C; Volkenandt, M; Lordick, F; Hartmann, J T; Karthaus, M; Riess, H; Lipp, H P; Hauschild, A; Trarbach, T; Wollenberg, A

    2011-03-01

    Anti-epidermal growth factor receptor treatment strategies, i.e. monoclonal antibodies such as cetuximab and panitumumab, or epidermal growth factor receptor (EGFR) small molecule tyrosine kinase inhibitors, such as erlotinib and gefitinib, have expanded the treatment options for different tumor types. Dermatologic toxic effects are the most common side-effects of EGFR inhibitor therapy. They can profoundly affect the patient's quality of life. The aim of this study was to provide interdisciplinary expert recommendations on how to treat patients with skin reactions undergoing anti-EGFR treatment. An expert panel from Germany with expertise in medical oncology, dermatology or clinical pharmacology was convened to develop expert recommendations based on published peer-reviewed literature. The expert recommendations for the state-of-the-art treatment of skin reactions induced by EGFR inhibitor therapy include recommendations for diagnostics and grading as well as grade-specific and stage-adapted treatment approaches and preventive measures. It was concluded that EGFR-inhibitor-related dermatologic reactions should always be treated combining basic care of the skin and a specific therapy adapted to stage and grade of skin reaction. For grade 2 and above, specific treatment recommendations for early- and later-stage skin reactions induced by EGFR-inhibitor therapy were proposed. This paper presents a German national expert opinion for the treatment of skin reactions in patients receiving EGFR inhibitor therapy.

  8. Novel mechanisms to inhibit HIV reservoir seeding using Jak inhibitors.

    Directory of Open Access Journals (Sweden)

    Christina Gavegnano

    2017-12-01

    Full Text Available Despite advances in the treatment of HIV infection with ART, elucidating strategies to overcome HIV persistence, including blockade of viral reservoir establishment, maintenance, and expansion, remains a challenge. T cell homeostasis is a major driver of HIV persistence. Cytokines involved in regulating homeostasis of memory T cells, the major hub of the HIV reservoir, trigger the Jak-STAT pathway. We evaluated the ability of tofacitinib and ruxolitinib, two FDA-approved Jak inhibitors, to block seeding and maintenance of the HIV reservoir in vitro. We provide direct demonstration for involvement of the Jak-STAT pathway in HIV persistence in vivo, ex vivo, and in vitro; pSTAT5 strongly correlates with increased levels of integrated viral DNA in vivo, and in vitro Jak inhibitors reduce the frequency of CD4+ T cells harboring integrated HIV DNA. We show that Jak inhibitors block viral production from infected cells, inhibit γ-C receptor cytokine (IL-15-induced viral reactivation from latent stores thereby preventing transmission of infectious particles to bystander activated T cells. These results show that dysregulation of the Jak-STAT pathway is associated with viral persistence in vivo, and that Jak inhibitors target key events downstream of γ-C cytokine (IL-2, IL-7 and IL-15 ligation to their receptors, impacting the magnitude of the HIV reservoir in all memory CD4 T cell subsets in vitro and ex vivo. Jak inhibitors represent a therapeutic modality to prevent key events of T cell activation that regulate HIV persistence and together, specific, potent blockade of these events may be integrated to future curative strategies.

  9. HIV protease inhibitors in pregnancy : pharmacology and clinical use.

    Science.gov (United States)

    Andany, Nisha; Loutfy, Mona R

    2013-03-01

    The impact of antiretroviral therapy (ART) on the natural history of HIV-1 infection has resulted in dramatic reductions in disease-associated morbidity and mortality. Additionally, the epidemiology of HIV-1 infection worldwide is changing, as women now represent a substantial proportion of infected adults. As more highly effective and tolerable antiretroviral regimens become available, and as the prevention of mother-to-child transmission becomes an attainable goal in the management of HIV-infected individuals, more and more HIV-positive women are choosing to become pregnant and have children. Consequently, it is important to consider the efficacy and safety of antiretroviral agents in pregnancy. Protease inhibitors are a common class of medication used in the treatment of HIV-1 infection and are increasingly being used in pregnancy. However, several studies have raised concerns regarding pharmacokinetic alterations in pregnancy, particularly in the third trimester, which results in suboptimal drug concentrations and a theoretically higher risk of virologic failure and perinatal transmission. Drug level reductions have been observed with each individual protease inhibitor and dose adjustments in pregnancy are suggested for certain agents. Furthermore, studies have also raised concerns regarding the safety of protease inhibitors in pregnancy, particularly as they may increase the risk of pre-term birth and metabolic disturbances. Overall, protease inhibitors are safe and effective for the treatment of HIV-infected pregnant women. Specifically, ritonavir-boosted lopinavir- and atazanavir-based regimens are preferred in pregnancy, while ritonavir-boosted darunavir- and saquinavir-based therapies are reasonable alternatives. This paper reviews the use of protease inhibitors in pregnancy, focusing on pharmacokinetic and safety considerations, and outlines the recommendations for use of this class of medication in the HIV-1-infected pregnant woman.

  10. Medicinal Chemistry Projects Requiring Imaginative Structure-Based Drug Design Methods.

    Science.gov (United States)

    Moitessier, Nicolas; Pottel, Joshua; Therrien, Eric; Englebienne, Pablo; Liu, Zhaomin; Tomberg, Anna; Corbeil, Christopher R

    2016-09-20

    cytochrome P450 enzymes (CYPs)-for toxicology studies-the program Impacts was derived from Fitted and helped us to reveal a complex metabolism with unforeseen stereocenter isomerizations. These efforts, combined with those of other docking software developers, have strengthened our understanding of the complex drug-protein binding process while providing the medicinal chemistry community with useful tools that have led to drug discoveries. In this Account, we describe our contributions over the past 15 years-within their historical context-to the design of drug candidates, including BACE-1 inhibitors, POP covalent inhibitors, G-quadruplex binders, and aminoglycosides binding to nucleic acids. We also remark the necessary developments of docking programs, specifically Fitted, that enabled structure-based design to flourish and yielded multiple fruitful, rational medicinal chemistry campaigns.

  11. Transcriptional changes associated with resistance to inhibitors of epidermal growth factor receptor revealed using metaanalysis

    International Nuclear Information System (INIS)

    Younis, Sidra; Javed, Qamar; Blumenberg, Miroslav

    2015-01-01

    EGFR is important in maintaining metabolic homeostasis in healthy cells, but in tumors it activates downstream signaling pathways, causing proliferation, angiogenesis, invasion and metastasis. Consequently, EGFR is targeted in cancers using reversible, irreversible or antibody inhibitors. Unfortunately, tumors develop inhibitor resistance by mutations or overexpressing EGFR, or its ligand, or activating secondary, EGFR-independent pathways. Here we present a global metaanalysis comparing transcriptional profiles from matched pairs of EGFR inhibitor-sensitive vs. -resistant cell lines, using 15 datasets comprising 274 microarrays. We also analyzed separately pairs of cell lines derived using reversible, irreversible or antibody inhibitors. The metaanalysis identifies commonalities in cell lines resistant to EGFR inhibitors: in sensitive cell lines, the ontological categories involving the ErbB receptors pathways, cell adhesion and lipid metabolism are overexpressed; however, resistance to EGFR inhibitors is associated with overexpression of genes for ErbB receptors-independent oncogenic pathways, regulation of cell motility, energy metabolism, immunity especially inflammatory cytokines biosynthesis, cell cycle and responses to exogenous and endogenous stimuli. Specifically in Gefitinib-resistant cell lines, the immunity-associated genes are overexpressed, whereas in Erlotinib-resistant ones so are the mitochondrial genes and processes. Unexpectedly, lines selected using EGFR-targeting antibodies overexpress different gene ontologies from ones selected using kinase inhibitors. Specifically, they have reduced expression of genes for proliferation, chemotaxis, immunity and angiogenesis. This metaanalysis suggests that ‘combination therapies’ can improve cancer treatment outcomes. Potentially, use of mitochondrial blockers with Erlotinib, immunity blockers with Gefitinib, tyrosine kinase inhibitors with antibody inhibitors, may have better chance of avoiding

  12. Histone Deacetylase Inhibitors Prolong Cardiac Repolarization through Transcriptional Mechanisms.

    Science.gov (United States)

    Spence, Stan; Deurinck, Mark; Ju, Haisong; Traebert, Martin; McLean, LeeAnne; Marlowe, Jennifer; Emotte, Corinne; Tritto, Elaine; Tseng, Min; Shultz, Michael; Friedrichs, Gregory S

    2016-09-01

    Histone deacetylase (HDAC) inhibitors are an emerging class of anticancer agents that modify gene expression by altering the acetylation status of lysine residues of histone proteins, thereby inducing transcription, cell cycle arrest, differentiation, and cell death or apoptosis of cancer cells. In the clinical setting, treatment with HDAC inhibitors has been associated with delayed cardiac repolarization and in rare instances a lethal ventricular tachyarrhythmia known as torsades de pointes. The mechanism(s) of HDAC inhibitor-induced effects on cardiac repolarization is unknown. We demonstrate that administration of structurally diverse HDAC inhibitors to dogs causes delayed but persistent increases in the heart rate corrected QT interval (QTc), an in vivo measure of cardiac repolarization, at timepoints far removed from the Tmax for parent drug and metabolites. Transcriptional profiling of ventricular myocardium from dogs treated with various HDAC inhibitors demonstrated effects on genes involved in protein trafficking, scaffolding and insertion of various ion channels into the cell membrane as well as genes for specific ion channel subunits involved in cardiac repolarization. Extensive in vitro ion channel profiling of various structural classes of HDAC inhibitors (and their major metabolites) by binding and acute patch clamp assays failed to show any consistent correlations with direct ion channel blockade. Drug-induced rescue of an intracellular trafficking-deficient mutant potassium ion channel, hERG (G601S), and decreased maturation (glycosylation) of wild-type hERG expressed by CHO cells in vitro correlated with prolongation of QTc intervals observed in vivo The results suggest that HDAC inhibitor-induced prolongation of cardiac repolarization may be mediated in part by transcriptional changes of genes required for ion channel trafficking and localization to the sarcolemma. These data have broad implications for the development of these drug classes and

  13. Do non-nucleoside reverse transcriptase inhibitors contribute to lipodystrophy?

    Science.gov (United States)

    Nolan, David

    2005-01-01

    Lipodystrophy complications, including lipoatrophy (pathological fat loss) and metabolic complications, have emerged as important long-term toxicities associated with antiretroviral therapy in the current era. The wealth of data that has accumulated over the past 6 years has now clarified the contribution of specific antiretroviral drugs to the risk of these clinical endpoints, with evidence that lipoatrophy is strongly associated with the choice of nucleoside reverse transcriptase inhibitor therapy (specifically, stavudine and to a lesser extent zidovudine). The aetiological basis of metabolic complications of antiretroviral therapy has proven to be complex, in that the risk appears to be modulated by a number of lifestyle factors that have made the metabolic syndrome highly prevalent in the general population, with additional contributions from HIV disease status itself, as well as from individual drugs within the HIV protease inhibitor class. The currently licensed non-nucleoside reverse transcriptase inhibitor (NNRTI) drugs, efavirenz and nevirapine, have been proven to have a favourable safety profile in terms of lipodystrophy complications. However, it must be noted that NNRTI drugs also have individual toxicity profiles that must be accounted for when considering and/or monitoring their use in the treatment of HIV infection.

  14. Acquired resistance to EGFR inhibitors: mechanisms and prevention strategies

    International Nuclear Information System (INIS)

    Viloria-Petit, Alicia M.; Kerbel, Robert S.

    2004-01-01

    Potent and specific, or relatively specific, inhibitors of epidermal growth factor receptor (EGFR) signaling, including monoclonal antibodies and small molecular weight compounds, have been successfully developed. Both types of agent have been found to have significant antitumor activity, especially when used in combination with radio- hormone- and chemotherapy in preclinical studies. Because of the potentiation of the conventional drug activity in these combination settings, inhibitors of EGFR signaling have often been referred to as sensitizers for chemotherapy or radiation, as well as drug resistance reversal agents. Phase II clinical trials in head-and-neck as well as lung cancer suggested this concept of chemosensitization might translate into the clinic, but this remains to be definitively proven in randomized, double-blind Phase III trials. Given the extensive preclinical literature on EGFR blocking drugs and the advanced clinical development of such agents, it is surprising that the possibility of development of acquired resistance to the EGFR inhibitors themselves, a common clinical problem with virtually all other currently used anticancer drugs, remains a largely unexplored subject of investigation. Here we summarize some of the possible mechanisms that can result in acquired resistance to EGFR-targeting drugs. Alternative combination therapies to circumvent and delay this problem are suggested

  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. Isoform-specific inhibition of cyclophilins.

    Science.gov (United States)

    Daum, Sebastian; Schumann, Michael; Mathea, Sebastian; Aumüller, Tobias; Balsley, Molly A; Constant, Stephanie L; de Lacroix, Boris Féaux; Kruska, Fabian; Braun, Manfred; Schiene-Fischer, Cordelia

    2009-07-07

    Cyclophilins belong to the enzyme class of peptidyl prolyl cis-trans isomerases which catalyze the cis-trans isomerization of prolyl bonds in peptides and proteins in different folding states. Cyclophilins have been shown to be involved in a multitude of cellular functions like cell growth, proliferation, and motility. Among the 20 human cyclophilin isoenzymes, the two most abundant members of the cyclophilin family, CypA and CypB, exhibit specific cellular functions in several inflammatory diseases, cancer development, and HCV replication. A small-molecule inhibitor on the basis of aryl 1-indanylketones has now been shown to discriminate between CypA and CypB in vitro. CypA binding of this inhibitor has been characterized by fluorescence anisotropy- and isothermal titration calorimetry-based cyclosporin competition assays. Inhibition of CypA- but not CypB-mediated chemotaxis of mouse CD4(+) T cells by the inhibitor provided biological proof of discrimination in vivo.

  17. PARP Inhibitors in Ovarian Cancer.

    Science.gov (United States)

    Mittica, Gloria; Ghisoni, Eleonora; Giannone, Gaia; Genta, Sofia; Aglietta, Massimo; Sapino, Anna; Valabrega, Giorgio

    2018-03-05

    Treatment of Epithelial Ovarian Cancer (EOC), historically based on surgery and platinum doublet chemotherapy, is associated with high risk of relapse and poor prognosis for recurrent disease. In this landscape, the innovative treatment with PARP inhibitors (PARPis) demonstrated an outstanding activity in EOC, and is currently changing clinical practice in BRCA mutant patients. To highlight the mechanism of action, pharmacokinetics, clinical activity, indications and current strategies of development of Olaparib, Niraparib, Rucaparib, Talazoparib and Veliparib, the 5 most relevant PARPis. We performed a review on Pubmed using 'ovarian cancer' and the name of each PARPi (PARP inhibitor) discussed in the review as Medical Subject Headings (MeSH) keywords. The same search was performed on "clinicaltrial.gov" to identify ongoing clinical trials and on "google.com/patents" and "uspto.gov" for recent patents exploring PARPIs in ovarian cancer. Olaparib, Niraparib and Rucaparib are already approved for treatment of recurrent EOC and their indications are partially overlapping. Talazoparib and Veliparib are promising PARPis, but currently under investigation in early phase trials. Several studies are evaluating PARPis in monotherapy or in associations, in a wide range of settings (i.e. first line, neoadjuvant, platinum-sensitive and resistant disease). PARPis are valuable options in patients with recurrent ovarian cancer with promising activity in different stages of this disease. Further studies are required to better define optimal clinical settings, predictors of response beyond BRCA mutations and strategies to overcome secondary resistance of PARPis therapy in EOC. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. Inhibitors of Microglial Neurotoxicity: Focus on Natural Products

    Directory of Open Access Journals (Sweden)

    Kyoungho Suk

    2011-01-01

    Full Text Available Microglial cells play a dual role in the central nervous system as they have both neurotoxic and neuroprotective effects. Uncontrolled and excessive activation of microglia often contributes to inflammation-mediated neurodegeneration. Recently, much attention has been paid to therapeutic strategies aimed at inhibiting neurotoxic microglial activation. Pharmacological inhibitors of microglial activation are emerging as a result of such endeavors. In this review, natural products-based inhibitors of microglial activation will be reviewed. Potential neuroprotective activity of these compounds will also be discussed. Future works should focus on the discovery of novel drug targets that specifically mediate microglial neurotoxicity rather than neuroprotection. Development of new drugs based on these targets may require a better understanding of microglial biology and neuroinflammation at the molecular, cellular, and systems levels.

  19. Discovery of DNA repair inhibitors by combinatorial library profiling

    Science.gov (United States)

    Moeller, Benjamin J.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

    2011-01-01

    Small molecule inhibitors of DNA repair are emerging as potent and selective anti-cancer therapies, but the sheer magnitude of the protein networks involved in DNA repair processes poses obstacles to discovery of effective candidate drugs. To address this challenge, we used a subtractive combinatorial selection approach to identify a panel of peptide ligands that bind DNA repair complexes. Supporting the concept that these ligands have therapeutic potential, we show that one selected peptide specifically binds and non-competitively inactivates DNA-PKcs, a protein kinase critical in double-strand DNA break repair. In doing so, this ligand sensitizes BRCA-deficient tumor cells to genotoxic therapy. Our findings establish a platform for large-scale parallel screening for ligand-directed DNA repair inhibitors, with immediate applicability to cancer therapy. PMID:21343400

  20. Developing selective histone deacetylases (HDACs inhibitors through ebselen and analogs

    Directory of Open Access Journals (Sweden)

    Wang Y

    2017-05-01

    Full Text Available Yuren Wang,1 Jason Wallach,2 Stephanie Duane,1 Yuan Wang,1 Jianghong Wu,1 Jeffrey Wang,1 Adeboye Adejare,2 Haiching Ma1 1Reaction Biology Corp., Malvern, 2Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of the Sciences, Philadelphia, PA, USA Abstract: Histone deacetylases (HDACs are key regulators of gene expression in cells and have been investigated as important therapeutic targets for cancer and other diseases. Different subtypes of HDACs appear to play disparate roles in the cells and are associated with specific diseases. Therefore, substantial effort has been made to develop subtype-selective HDAC inhibitors. In an effort to discover existing scaffolds with HDAC inhibitory activity, we screened a drug library approved by the US Food and Drug Administration and a National Institutes of Health Clinical Collection compound library in HDAC enzymatic assays. Ebselen, a clinical safe compound, was identified as a weak inhibitor of several HDACs, including HDAC1, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, and HDAC9 with half maximal inhibitory concentrations approximately single digit of µM. Two ebselen analogs, ebselen oxide and ebsulfur (a diselenide analog of ebselen, also inhibited these HDACs, however with improved potencies on HDAC8. Benzisothiazol, the core structure of ebsulfur, specifically inhibited HDAC6 at a single digit of µM but had no inhibition on other HDACs. Further efforts on structure–activity relationship based on the core structure of ebsulfur led to the discovery of a novel class of potent and selective HDAC6 inhibitors with RBC-2008 as the lead compound with single-digit nM potency. This class of histone deacetylase inhibitor features a novel pharmacophore with an ebsulfur scaffold selectively targeting HDAC6. Consistent with its inhibition on HDAC6, RBC-2008 significantly increased the acetylation levels of α-tubulin in PC-3 cells. Furthermore, treatment with these compounds led to

  1. Discovery and SAR of hydantoin TACE inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Wensheng; Guo, Zhuyan; Orth, Peter; Madison, Vincent; Chen, Lei; Dai, Chaoyang; Feltz, Robert J.; Girijavallabhan, Vinay M.; Kim, Seong Heon; Kozlowski, Joseph A.; Lavey, Brian J.; Li, Dansu; Lundell, Daniel; Niu, Xiaoda; Piwinski, John J.; Popovici-Muller, Janeta; Rizvi, Razia; Rosner, Kristin E.; Shankar, Bandarpalle B.; Shih, Neng-Yang; Siddiqui, M.A.; Sun, J.; Tong, L.; Umland, S.; Wong, M.K.; Yang, D.Y.; Zhou, G. (Merck)

    2010-09-03

    We disclose inhibitors of TNF-{alpha} converting enzyme (TACE) designed around a hydantoin zinc binding moiety. Crystal structures of inhibitors bound to TACE revealed monodentate coordination of the hydantoin to the zinc. SAR, X-ray, and modeling designs are described. To our knowledge, these are the first reported X-ray structures of TACE with a hydantoin zinc ligand.

  2. Does plasminogen activator inhibitor-1 drive lymphangiogenesis?

    DEFF Research Database (Denmark)

    Bruyère, Françoise; Melen-Lamalle, Laurence; Blacher, Silvia

    2010-01-01

    The purpose of this study is to explore the function of plasminogen activator inhibitor-1 (PAI-1) during pathological lymphangiogenesis. PAI-1, the main physiological inhibitor of plasminogen activators is involved in pathological angiogenesis at least by controlling extracellular proteolysis and...

  3. Electrochemical Behaviour of Environmentally Friendly Inhibitor of ...

    African Journals Online (AJOL)

    Electrochemical Behaviour of Environmentally Friendly Inhibitor of Aloe Secundiflora Extract in Corrosion Control of Carbon Steel in Soft Water Media. ... The investigation was performed at different inhibitor concentrations under static and dynamic conditions using a Rotating Disk Electrode (RDE). The impedance and ...

  4. Metabolic complications associated with HIV protease inhibitor therapy.

    Science.gov (United States)

    Nolan, David

    2003-01-01

    HIV protease inhibitors were introduced into clinical practice over 7 years ago as an important component of combination antiretroviral drug regimens which in many ways revolutionised the treatment of HIV infection. The significant improvements in prognosis that have resulted from the use of these regimens, combined with the need for lifelong treatment, have increasingly focused attention on the adverse effects of antiretroviral drugs and on the metabolic complications of HIV protease inhibitors in particular. In this review, the cluster of metabolic abnormalities characterised by triglyceride-rich dyslipidaemia and insulin resistance associated with HIV protease inhibitor therapy are considered, along with implications for cardiovascular risk in patients affected by these complications. Toxicity profiles of individual drugs within the HIV protease inhibitor class are examined, as there is an increased recognition of significant intra-class differences both in terms of absolute risk of metabolic complications as well as the particular metabolic phenotype associated with these drugs. Guidelines for clinical assessment and treatment are emphasised, along with pathophysiological mechanisms that may provide a rational basis for the treatment of metabolic complications. Finally, these drug-specific effects are considered within the context of HIV-specific effects on lipid metabolism as well as lifestyle factors that have contributed to a rapidly increasing incidence of similar metabolic syndromes in the general population. These data highlight the importance of individualising patient management in terms of choice of antiretroviral regimen, assessment of metabolic outcomes and use of therapeutic interventions, based on the assessment of baseline (pre-treatment) metabolic status as well as the presence of potentially modifiable cardiovascular risk factors.

  5. Potential physiological role of plant glycosidase inhibitors

    DEFF Research Database (Denmark)

    Bellincampi, D.; Carmadella, L.; Delcour, J.A.

    2004-01-01

    Carbohydrate-active enzymes including glycosidases, transglycosidases, glycosyltransferases, polysaccharide lyases and carbohydrate esterases are responsible for the enzymatic processing of carbohydrates in plants. A number of carbohydrate-active enzymes are produced by microbial pathogens...... and insects responsible of severe crop losses. Plants have evolved proteinaceous inhibitors to modulate the activity of several of these enzymes. The continuing discovery of new inhibitors indicates that this research area is still unexplored and may lead to new exciting developments. To date, the role...... of the inhibitors is not completely understood. Here we review recent results obtained on the best characterised inhibitors, pointing to their possible biological role in vivo. Results recently obtained with plant transformation technology indicate that this class of inhibitors has potential biotechnological...

  6. Discovery of HDAC inhibitors with potent activity against multiple malaria parasite life cycle stages.

    Science.gov (United States)

    Hansen, Finn K; Sumanadasa, Subathdrage D M; Stenzel, Katharina; Duffy, Sandra; Meister, Stephan; Marek, Linda; Schmetter, Rebekka; Kuna, Krystina; Hamacher, Alexandra; Mordmüller, Benjamin; Kassack, Matthias U; Winzeler, Elizabeth A; Avery, Vicky M; Andrews, Katherine T; Kurz, Thomas

    2014-07-23

    In this work we investigated the antiplasmodial activity of a series of HDAC inhibitors containing an alkoxyamide connecting-unit linker region. HDAC inhibitor 1a (LMK235), previously shown to be a novel and specific inhibitor of human HDAC4 and 5, was used as a starting point to rapidly construct a mini-library of HDAC inhibitors using a straightforward solid-phase supported synthesis. Several of these novel HDAC inhibitors were found to have potent in vitro activity against asexual stage Plasmodium falciparum malaria parasites. Representative compounds were shown to hyperacetylate P. falciparum histones and to inhibit deacetylase activity of recombinant PfHDAC1 and P. falciparum nuclear extracts. All compounds were also screened in vitro for activity against Plasmodium berghei exo-erythrocytic stages and selected compounds were further tested against late stage (IV and V) P. falciparum gametocytes. Of note, some compounds showed nanomolar activity against all three life cycle stages tested (asexual, exo-erythrocytic and gametocyte stages) and several compounds displayed significantly increased parasite selectivity compared to the reference HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). These data suggest that it may be possible to develop HDAC inhibitors that target multiple malaria parasite life cycle stages. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  7. Blockade of the ERK pathway markedly sensitizes tumor cells to HDAC inhibitor-induced cell death

    International Nuclear Information System (INIS)

    Ozaki, Kei-ichi; Minoda, Ai; Kishikawa, Futaba; Kohno, Michiaki

    2006-01-01

    Constitutive activation of the extracellular signal-regulated kinase (ERK) pathway is associated with the neoplastic phenotype of a large number of human tumor cells. Although specific blockade of the ERK pathway by treating such tumor cells with potent mitogen-activated protein kinase/ERK kinase (MEK) inhibitors completely suppresses their proliferation, it by itself shows only a modest effect on the induction of apoptotic cell death. However, these MEK inhibitors markedly enhance the efficacy of histone deacetylase (HDAC) inhibitors to induce apoptotic cell death: such an enhanced cell death is observed only in tumor cells in which the ERK pathway is constitutively activated. Co-administration of MEK inhibitor markedly sensitizes tumor cells to HDAC inhibitor-induced generation of reactive oxygen species, which appears to mediate the enhanced cell death induced by the combination of these agents. These results suggest that the combination of MEK inhibitors and HDAC inhibitors provides an efficient chemotherapeutic strategy for the treatment of tumor cells in which the ERK pathway is constitutively activated

  8. Structure-Based Design of Novel HIV-1 Protease Inhibitors to Combat Drug Resistance

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh,A.; Sridhar, P.; Leshchenko, S.; Hussain, A.; Li, J.; Kovalevsky, A.; Walters, D.; Wedelind, J.; Grum-Tokars, V.; et al.

    2006-01-01

    Structure-based design and synthesis of novel HIV protease inhibitors are described. The inhibitors are designed specifically to interact with the backbone of HIV protease active site to combat drug resistance. Inhibitor 3 has exhibited exceedingly potent enzyme inhibitory and antiviral potency. Furthermore, this inhibitor maintains impressive potency against a wide spectrum of HIV including a variety of multi-PI-resistant clinical strains. The inhibitors incorporated a stereochemically defined 5-hexahydrocyclopenta[b]furanyl urethane as the P2-ligand into the (R)-(hydroxyethylamino)sulfonamide isostere. Optically active (3aS,5R,6aR)-5-hydroxy-hexahydrocyclopenta[b]furan was prepared by an enzymatic asymmetrization of meso-diacetate with acetyl cholinesterase, radical cyclization, and Lewis acid-catalyzed anomeric reduction as the key steps. A protein-ligand X-ray crystal structure of inhibitor 3-bound HIV-1 protease (1.35 Angstroms resolution) revealed extensive interactions in the HIV protease active site including strong hydrogen bonding interactions with the backbone. This design strategy may lead to novel inhibitors that can combat drug resistance.

  9. Interaction of HIV-1 reverse transcriptase ribonuclease H with an acylhydrazone inhibitor.

    Science.gov (United States)

    Gong, Qingguo; Menon, Lakshmi; Ilina, Tatiana; Miller, Lena G; Ahn, Jinwoo; Parniak, Michael A; Ishima, Rieko

    2011-01-01

    HIV-1 reverse transcriptase is a bifunctional enzyme, having both DNA polymerase (RNA- and DNA-dependent) and ribonuclease H activities. HIV-1 reverse transcriptase has been an exceptionally important target for antiretroviral therapeutic development, and nearly half of the current clinically used antiretrovirals target reverse transcriptase DNA polymerase. However, no inhibitors of reverse transcriptase ribonuclease H are on the market or in preclinical development. Several drug-like small molecule inhibitors of reverse transcriptase ribonuclease H have been described, but little structural information is available about the interactions between reverse transcriptase ribonuclease H and inhibitors that exhibit antiviral activity. In this report, we describe NMR studies of the interaction of a new ribonuclease H inhibitor, BHMP07, with a catalytically active HIV-1 reverse transcriptase ribonuclease H domain fragment. We carried out solution NMR experiments to identify the interaction interface of BHMP07 with the ribonuclease H domain fragment. Chemical shift changes of backbone amide signals at different BHMP07 concentrations clearly demonstrate that BHMP07 mainly recognizes the substrate handle region in the ribonuclease H fragment. Using ribonuclease H inhibition assays and reverse transcriptase mutants, the binding specificity of BHMP07 was compared with another inhibitor, dihydroxy benzoyl naphthyl hydrazone. Our results provide a structural characterization of the ribonuclease H inhibitor interaction and are likely to be useful for further improvements of the inhibitors. © 2010 John Wiley & Sons A/S.

  10. Enzymes and Inhibitors in Neonicotinoid Insecticide Metabolism

    Science.gov (United States)

    Shi, Xueyan; Dick, Ryan A.; Ford, Kevin A.; Casida, John E.

    2009-01-01

    Neonicotinoid insecticide metabolism involves considerable substrate specificity and regioselectivity of the relevant CYP450, aldehyde oxidase, and phase II enzymes. Human CYP450 recombinant enzymes carry out the following conversions: CYP3A4, 2C19 and 2B6 for thiamethoxam (TMX) to clothianidin (CLO); 3A4, 2C19 and 2A6 for CLO to desmethyl-CLO; 2C19 for TMX to desmethyl-TMX. Human liver aldehyde oxidase reduces the nitro substituent of CLO to nitroso much more rapidly than that of TMX. Imidacloprid (IMI), CLO and several of their metabolites do not give detectable N-glucuronides but 5-hydroxy-IMI, 4,5-diol-IMI and 4-hydroxy-thiacloprid are converted to O-glucuronides in vitro with mouse liver microsomes and UDP-glucuronic acid or in vivo in mice. Mouse liver cytosol with S-adenosylmethionine converts desmethyl-CLO to CLO but not desmethyl-TMX to TMX. Two organophosphorus CYP450 inhibitors partially block IMI, thiacloprid and CLO metabolism in vivo in mice, elevating the brain and liver levels of the parent compounds while reducing amounts of the hydroxylated metabolites. PMID:19391582

  11. Suppression of Coronavirus Replication by Cyclophilin Inhibitors

    Directory of Open Access Journals (Sweden)

    Takashi Sasaki

    2013-05-01

    Full Text Available Coronaviruses infect a variety of mammalian and avian species and cause serious diseases in humans, cats, mice, and birds in the form of severe acute respiratory syndrome (SARS, feline infectious peritonitis (FIP, mouse hepatitis, and avian infectious bronchitis, respectively. No effective vaccine or treatment has been developed for SARS-coronavirus or FIP virus, both of which cause lethal diseases. It has been reported that a cyclophilin inhibitor, cyclosporin A (CsA, could inhibit the replication of coronaviruses. CsA is a well-known immunosuppressive drug that binds to cellular cyclophilins to inhibit calcineurin, a calcium-calmodulin-activated serine/threonine-specific phosphatase. The inhibition of calcineurin blocks the translocation of nuclear factor of activated T cells from the cytosol into the nucleus, thus preventing the transcription of genes encoding cytokines such as interleukin-2. Cyclophilins are peptidyl-prolyl isomerases with physiological functions that have been described for many years to include chaperone and foldase activities. Also, many viruses require cyclophilins for replication; these include human immunodeficiency virus, vesicular stomatitis virus, and hepatitis C virus. However, the molecular mechanisms leading to the suppression of viral replication differ for different viruses. This review describes the suppressive effects of CsA on coronavirus replication.

  12. Clinically Applicable Inhibitors Impacting Genome Stability.

    Science.gov (United States)

    Prakash, Anu; Garcia-Moreno, Juan F; Brown, James A L; Bourke, Emer

    2018-05-13

    Advances in technology have facilitated the molecular profiling (genomic and transcriptomic) of tumours, and has led to improved stratification of patients and the individualisation of treatment regimes. To fully realize the potential of truly personalised treatment options, we need targeted therapies that precisely disrupt the compensatory pathways identified by profiling which allow tumours to survive or gain resistance to treatments. Here, we discuss recent advances in novel therapies that impact the genome (chromosomes and chromatin), pathways targeted and the stage of the pathways targeted. The current state of research will be discussed, with a focus on compounds that have advanced into trials (clinical and pre-clinical). We will discuss inhibitors of specific DNA damage responses and other genome stability pathways, including those in development, which are likely to synergistically combine with current therapeutic options. Tumour profiling data, combined with the knowledge of new treatments that affect the regulation of essential tumour signalling pathways, is revealing fundamental insights into cancer progression and resistance mechanisms. This is the forefront of the next evolution of advanced oncology medicine that will ultimately lead to improved survival and may, one day, result in many cancers becoming chronic conditions, rather than fatal diseases.

  13. Interpreting clinical assays for histone deacetylase inhibitors

    International Nuclear Information System (INIS)

    Martinet, Nadine; Bertrand, Philippe

    2011-01-01

    As opposed to genetics, dealing with gene expressions by direct DNA sequence modifications, the term epigenetics applies to all the external influences that target the chromatin structure of cells with impact on gene expression unrelated to the sequence coding of DNA itself. In normal cells, epigenetics modulates gene expression through all development steps. When “imprinted” early by the environment, epigenetic changes influence the organism at an early stage and can be transmitted to the progeny. Together with DNA sequence alterations, DNA aberrant cytosine methylation and microRNA deregulation, epigenetic modifications participate in the malignant transformation of cells. Their reversible nature has led to the emergence of the promising field of epigenetic therapy. The efforts made to inhibit in particular the epigenetic enzyme family called histone deacetylases (HDACs) are described. HDAC inhibitors (HDACi) have been proposed as a viable clinical therapeutic approach for the treatment of leukemia and solid tumors, but also to a lesser degree for noncancerous diseases. Three epigenetic drugs are already arriving at the patient’s bedside, and more than 100 clinical assays for HDACi are registered on the National Cancer Institute website. They explore the eventual additive benefits of combined therapies. In the context of the pleiotropic effects of HDAC isoforms, more specific HDACi and more informative screening tests are being developed for the benefit of the patients

  14. Comparison of Effect of Brassinosteroid and Gibberellin Biosynthesis Inhibitors on Growth of Rice Seedlings

    Directory of Open Access Journals (Sweden)

    Tadashi Matusmoto

    2016-01-01

    Full Text Available Brassinosteroid (BR and gibberellin (GA are two predominant plant hormones that regulate plant cell elongation. Mutants disrupt the biosynthesis of these hormones and display different degrees of dwarf phenotypes in rice. Although the role of each plant hormone in promoting the longitudinal growth of plants has been extensively studied using genetic methods, their relationship is still poorly understood. In this study, we used two specific inhibitors targeting BR and GA biosynthesis to investigate the roles of BR and GA in growth of rice seedlings. Yucaizol, a specific inhibitor of BR biosynthesis, and Trinexapac-ethyl, a commercially available inhibitor of GA biosynthesis, were used. The effect of Yucaizol on rice seedlings indicated that Yucaizol significantly retarded stem elongation. The IC50 value was found to be approximately 0.8 μmol/L. Yucaizol also induced small leaf angle phenocopy in rice seedlings, similarly to BR-deficient rice, while Trinexapac-ethyl did not. When Yucaizol combined with Trinexapac-ethyl was applied to the rice plants, the mixture of these two inhibitors retarded stem elongation of rice at lower doses. Our results suggest that the use of a BR biosynthesis inhibitor combined with a GA biosynthesis inhibitor may be useful in the development of new technologies for controlling rice plant height.

  15. The Wonders of Phosphodiesterase‑5 Inhibitors: A Majestic History

    African Journals Online (AJOL)

    A milestone in drug discovery was the selective inhibitors of. PDE‑5 that ... the pharmacotherapeutics of PDE‑5 inhibitors and the majestic history that led to their discovery. ..... including HIV protease inhibitors, ketoconazole, itraconazole,.

  16. 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.

  17. Histone Deacetylase Inhibitors as Anticancer Drugs.

    Science.gov (United States)

    Eckschlager, Tomas; Plch, Johana; Stiborova, Marie; Hrabeta, Jan

    2017-07-01

    Carcinogenesis cannot be explained only by genetic alterations, but also involves epigenetic processes. Modification of histones by acetylation plays a key role in epigenetic regulation of gene expression and is controlled by the balance between histone deacetylases (HDAC) and histone acetyltransferases (HAT). HDAC inhibitors induce cancer cell cycle arrest, differentiation and cell death, reduce angiogenesis and modulate immune response. Mechanisms of anticancer effects of HDAC inhibitors are not uniform; they may be different and depend on the cancer type, HDAC inhibitors, doses, etc. HDAC inhibitors seem to be promising anti-cancer drugs particularly in the combination with other anti-cancer drugs and/or radiotherapy. HDAC inhibitors vorinostat, romidepsin and belinostat have been approved for some T-cell lymphoma and panobinostat for multiple myeloma. Other HDAC inhibitors are in clinical trials for the treatment of hematological and solid malignancies. The results of such studies are promising but further larger studies are needed. Because of the reversibility of epigenetic changes during cancer development, the potency of epigenetic therapies seems to be of great importance. Here, we summarize the data on different classes of HDAC inhibitors, mechanisms of their actions and discuss novel results of preclinical and clinical studies, including the combination with other therapeutic modalities.

  18. Histone Deacetylase Inhibitors as Anticancer Drugs

    Directory of Open Access Journals (Sweden)

    Tomas Eckschlager

    2017-07-01

    Full Text Available Carcinogenesis cannot be explained only by genetic alterations, but also involves epigenetic processes. Modification of histones by acetylation plays a key role in epigenetic regulation of gene expression and is controlled by the balance between histone deacetylases (HDAC and histone acetyltransferases (HAT. HDAC inhibitors induce cancer cell cycle arrest, differentiation and cell death, reduce angiogenesis and modulate immune response. Mechanisms of anticancer effects of HDAC inhibitors are not uniform; they may be different and depend on the cancer type, HDAC inhibitors, doses, etc. HDAC inhibitors seem to be promising anti-cancer drugs particularly in the combination with other anti-cancer drugs and/or radiotherapy. HDAC inhibitors vorinostat, romidepsin and belinostat have been approved for some T-cell lymphoma and panobinostat for multiple myeloma. Other HDAC inhibitors are in clinical trials for the treatment of hematological and solid malignancies. The results of such studies are promising but further larger studies are needed. Because of the reversibility of epigenetic changes during cancer development, the potency of epigenetic therapies seems to be of great importance. Here, we summarize the data on different classes of HDAC inhibitors, mechanisms of their actions and discuss novel results of preclinical and clinical studies, including the combination with other therapeutic modalities.

  19. Proton pump inhibitors and gastroenteritis

    International Nuclear Information System (INIS)

    Hassing, Robert-Jan; Verbon, Annelies; Visser, Herman de; Hofman, Albert; Stricker, Bruno H.

    2016-01-01

    An association between proton pump inhibitor (PPI) therapy and bacterial gastroenteritis has been suggested as well as contradicted. The aim of this study was to examine the association between the use of PPIs and occurrence of bacterial gastroenteritis in the prospective Rotterdam Study. The Rotterdam Study is a population-based cohort study among 14,926 subjects aged 45 years and older with up to 24 years of follow-up. Analyses were performed with a generalized estimating equations method in participants who handed-in a diagnostic stool sample. Furthermore, a nested case–control analysis was performed using the total cohort as a reference group. A bacterial microorganism was isolated in 125 samples, whereas 1174 samples were culture negative. In the generalized estimating equations analysis, we found that participants with a bacterial gastroenteritis were more likely than controls to be current users of PPIs (adjusted OR 1.94; 95 % CI 1.15–3.25). Different sensitivity analyses did not change this result. A considerably higher effect was observed (adjusted OR 6.14; 95 % CI 3.81–9.91), using the total cohort as a reference in a nested case–control analysis. Current PPI therapy is associated with an increased risk of bacterial gastroenteritis. However, by reducing the risk of selection and information bias in our study design, we demonstrated that the effect is lower than previously assumed.

  20. ALK inhibitors, a pharmaceutical perspective

    Directory of Open Access Journals (Sweden)

    Arturo eGalvani

    2012-02-01

    Full Text Available In 2007, the ALK tyrosine kinase, already known to be translocated and activated in Anaplastic Large Cell Lymphoma, and a few other rare cancers, was described as a potential therapeutic target for a subset of non small-cell lung cancer (NSCLC patients. Clinical proof of concept, culminating in the recent approval by the FDA of the Pfizer drug Xalkori (crizotinib, formerly known as PF-02341066 followed in record time. The drug was approved together with a companion diagnostic, the Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular, Inc. for detection of eligible patients. This remarkable example of the coming of age of personalized medicine in cancer therapy is hopefully only an auspice of things to come in this rapidly developing field. Perhaps unsurprisingly, however, the appearance of clinical acquired resistance to crizotinib has already been observed early on in clinical testing, with the identification of several ALK secondary point mutations which diminish drug efficacy, and which open the way for development of second-generation inhibitors. It is also emerging that acquired resistance to crizotinib may also occur through ALK-independent mechanisms, which still need to be elucidated in detail.

  1. SGLT2 Inhibitors May Predispose to Ketoacidosis.

    Science.gov (United States)

    Taylor, Simeon I; Blau, Jenny E; Rother, Kristina I

    2015-08-01

    Sodium glucose cotransporter 2 (SGLT2) inhibitors are antidiabetic drugs that increase urinary excretion of glucose, thereby improving glycemic control and promoting weight loss. Since approval of the first-in-class drug in 2013, data have emerged suggesting that these drugs increase the risk of diabetic ketoacidosis. In May 2015, the Food and Drug Administration issued a warning that SGLT2 inhibitors may lead to ketoacidosis. Using PubMed and Google, we conducted Boolean searches including terms related to ketone bodies or ketoacidosis with terms for SGLT2 inhibitors or phlorizin. Priority was assigned to publications that shed light on molecular mechanisms whereby SGLT2 inhibitors could affect ketone body metabolism. SGLT2 inhibitors trigger multiple mechanisms that could predispose to diabetic ketoacidosis. When SGLT2 inhibitors are combined with insulin, it is often necessary to decrease the insulin dose to avoid hypoglycemia. The lower dose of insulin may be insufficient to suppress lipolysis and ketogenesis. Furthermore, SGLT2 is expressed in pancreatic α-cells, and SGLT2 inhibitors promote glucagon secretion. Finally, phlorizin, a nonselective inhibitor of SGLT family transporters decreases urinary excretion of ketone bodies. A decrease in the renal clearance of ketone bodies could also increase the plasma ketone body levels. Based on the physiology of SGLT2 and the pharmacology of SGLT2 inhibitors, there are several biologically plausible mechanisms whereby this class of drugs has the potential to increase the risk of developing diabetic ketoacidosis. Future research should be directed toward identifying which patients are at greatest risk for this side effect and also to optimizing pharmacotherapy to minimize the risk to patients.

  2. Enzyme structure and interaction with inhibitors

    International Nuclear Information System (INIS)

    London, R.E.

    1983-01-01

    This article reviews some of the results of studies on the 13 C-labeled enzyme dihydrofolate reductase (DHFR). Nuclear magnetic resonance (NMR) techniques are used in combination with isotopic labeling to learn about the structure and dynamics of this enzyme. 13 C-labeling is used for the purpose of studying enzyme/substrate and enzyme/inhibitor interactions. A second set of studies with DHFR was designed to investigate the basis for the high affinity between the inhibitor methotrexate and DHFR. The label was placed on the inhibitor, rather than the enzyme

  3. An Updated Review of Tyrosinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Te-Sheng Chang

    2009-05-01

    Full Text Available Tyrosinase is a multifunctional, glycosylated, and copper-containing oxidase, which catalyzes the first two steps in mammalian melanogenesis and is responsible for enzymatic browning reactions in damaged fruits during post-harvest handling and processing. Neither hyperpigmentation in human skin nor enzymatic browning in fruits are desirable. These phenomena have encouraged researchers to seek new potent tyrosinase inhibitors for use in foods and cosmetics. This article surveys tyrosinase inhibitors newly discovered from natural and synthetic sources. The inhibitory strength is compared with that of a standard inhibitor, kojic acid, and their inhibitory mechanisms are discussed.

  4. Monoamine oxidase A (MAO A) inhibitors decrease glioma progression

    Science.gov (United States)

    Vaikari, Vijaya Pooja; Kota, Rajesh; Chen, Kevin; Yeh, Tzu-Shao; Jhaveri, Niyati; Groshen, Susan L.; Olenyuk, Bogdan Z.; Chen, Thomas C.; Hofman, Florence M.; Shih, Jean C.

    2016-01-01

    Glioblastoma (GBM) is an aggressive brain tumor which is currently treated with temozolomide (TMZ). Tumors usually become resistant to TMZ and recur; no effective therapy is then available. Monoamine Oxidase A (MAO A) oxidizes monoamine neurotransmitters resulting in reactive oxygen species which cause cancer. This study shows that MAO A expression is increased in human glioma tissues and cell lines. MAO A inhibitors, clorgyline or the near-infrared-dye MHI-148 conjugated to clorgyline (NMI), were cytotoxic for glioma and decreased invasion in vitro. Using the intracranial TMZ-resistant glioma model, clorgyline or NMI alone or in combination with low-dose TMZ reduced tumor growth and increased animal survival. NMI was localized specifically to the tumor. Immunocytochemistry studies showed that the MAO A inhibitor reduced proliferation, microvessel density and invasion, and increased macrophage infiltration. In conclusion, we have identified MAO A inhibitors as potential novel stand-alone drugs or as combination therapy with low dose TMZ for drug-resistant gliomas. NMI can also be used as a non-invasive imaging tool. Thus has a dual function for both therapy and diagnosis. PMID:26871599

  5. Plasminogen activator inhibitor-2 in patients with monocytic leukemia.

    Science.gov (United States)

    Scherrer, A; Kruithof, E K; Grob, J P

    1991-06-01

    Plasma and tumor cells from 103 patients with leukemia or lymphoma at initial presentation were investigated for the presence of plasminogen activator inhibitor-2 (PAI-2) antigen, a potent inhibitor of urokinase. PAI-2 was detected in plasma and leukemic cells of the 21 patients with leukemia having a monocytic component [acute myelomonocytic (M4), acute monoblastic (M5), and chronic myelomonocytic leukemias], and in the three patients with acute undifferentiated myeloblastic leukemia (M0). In contrast, this serine protease inhibitor was undetectable in 79 patients with other subtypes of acute myeloid leukemia or other hematological malignancies. Serial serum PAI-2 determinations in 16 patients with acute leukemia at presentation, during therapy, remission, and relapse revealed that in the five patients with M4-M5, elevated PAI-2 levels rapidly normalized under therapy and during remission, but increased again in the patients with a relapse associated with an M4-M5 phenotype. Thus, PAI-2 seems to be a marker highly specific for the active stages of monocytic leukemia, i.e. presentation and relapse. The presence of PAI-2 in the plasma and cells of patients with M0 may give a clue to a monocytic origin of these cells.

  6. SGLT2 inhibitors in the management of type 2 diabetes.

    Science.gov (United States)

    Monica Reddy, R P; Inzucchi, Silvio E

    2016-08-01

    The glucose-lowering pharmacopeia continues to grow for patients with type 2 diabetes. The latest drug category, the SGLT2 inhibitors reduce glycated hemoglobin concentrations by increasing urinary excretion of glucose. They are used mainly in combination with metformin and other antihyperglycemic agents, including insulin. Their glucose-lowering potency is modest. Advantages include lack of hypoglycemia as a side effect, and mild reduction in blood pressure and body weight. Side effects include increased urinary frequency, owing to their mild diuretic action, symptoms of hypovolemia, genitourinary infections. There are also recent reports of rare cases of diabetic ketoacidosis occurring in insulin-treated patients. Recently, a large cardiovascular outcome trial reported that a specific SGLT2 inhibitor, empagliflozin, led to a reduction in the primary endpoint of major cardiovascular events. This effect was mainly the result of a surprising 38 % reduction in cardiovascular death, and the drug was also associated with nearly as large a reduction in heart failure hospitalization. These findings were notable because most drugs used in type 2 diabetes have not been shown to improve cardiovascular outcomes. Accordingly, there is growing interest in empagliflozin and the entire SGLT2 inhibitor class as drugs that could potentially change the manner in which we approach the management of hyperglycemia in patients with type 2 diabetes.

  7. Low molecular weight squash trypsin inhibitors from Sechium edule seeds.

    Science.gov (United States)

    Laure, Hélen J; Faça, Vítor M; Izumi, Clarice; Padovan, Júlio C; Greene, Lewis J

    2006-02-01

    Nine chromatographic components containing trypsin inhibitor activity were isolated from Sechium edule seeds by acetone fractionation, gel filtration, affinity chromatography and RP-HPLC in an overall yield of 46% of activity and 0.05% of protein. The components obtained with highest yield of total activity and highest specific activity were sequenced by Edman degradation and their molecular masses determined by mass spectrometry. The inhibitors contained 31, 32 and 27 residues per molecule and their sequences were: SETI-IIa, EDRKCPKILMRCKRDSDCLAKCTCQESGYCG; SETI-IIb, EEDRKCPKILMRCKRDSDCLAKCTCQESGYCG and SETI-V, CPRILMKCKLDTDCFPTCTCRPSGFCG. SETI-IIa and SETI-IIb, which differed by an amino-terminal E in the IIb form, were not separable under the conditions employed. The sequences are consistent with consensus sequences obtained from 37 other inhibitors: CPriI1meCk_DSDCla_C_C_G_CG, where capital letters are invariant amino acid residues and lower case letters are the most preserved in this position. SETI-II and SETI-V form complexes with trypsin with a 1:1 stoichiometry and have dissociation constants of 5.4x10(-11)M and 1.1x10(-9)M, respectively.

  8. Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target

    Science.gov (United States)

    Selzer, Paul M.; Pingel, Sabine; Hsieh, Ivy; Ugele, Bernhard; Chan, Victor J.; Engel, Juan C.; Bogyo, Matthew; Russell, David G.; Sakanari, Judy A.; McKerrow, James H.

    1999-09-01

    Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

  9. Selective Serotonin Reuptake Inhibitors (SSRIs)

    Science.gov (United States)

    ... and a rapid heart rate. Seek immediate medical attention if you have any of these signs or symptoms. Antidepressants and pregnancy. Talk to your doctor about the risks and benefits of using specific antidepressants. Some antidepressants may harm ...

  10. JAK inhibitors for the treatment of myeloproliferative neoplasms and other disorders [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    William Vainchenker

    2018-01-01

    Full Text Available JAK inhibitors have been developed following the discovery of the JAK2V617F in 2005 as the driver mutation of the majority of non-BCR-ABL1 myeloproliferative neoplasms (MPNs. Subsequently, the search for JAK2 inhibitors continued with the discovery that the other driver mutations (CALR and MPL also exhibited persistent JAK2 activation. Several type I ATP-competitive JAK inhibitors with different specificities were assessed in clinical trials and exhibited minimal hematologic toxicity. Interestingly, these JAK inhibitors display potent anti-inflammatory activity. Thus, JAK inhibitors targeting preferentially JAK1 and JAK3 have been developed to treat inflammation, autoimmune diseases, and graft-versus-host disease. Ten years after the beginning of clinical trials, only two drugs have been approved by the US Food and Drug Administration: one JAK2/JAK1 inhibitor (ruxolitinib in intermediate-2 and high-risk myelofibrosis and hydroxyurea-resistant or -intolerant polycythemia vera and one JAK1/JAK3 inhibitor (tofacitinib in methotrexate-resistant rheumatoid arthritis. The non-approved compounds exhibited many off-target effects leading to neurological and gastrointestinal toxicities, as seen in clinical trials for MPNs. Ruxolitinib is a well-tolerated drug with mostly anti-inflammatory properties. Despite a weak effect on the cause of the disease itself in MPNs, it improves the clinical state of patients and increases survival in myelofibrosis. This limited effect is related to the fact that ruxolitinib, like the other type I JAK2 inhibitors, inhibits equally mutated and wild-type JAK2 (JAK2WT and also the JAK2 oncogenic activation. Thus, other approaches need to be developed and could be based on either (1 the development of new inhibitors specifically targeting JAK2V617F or (2 the combination of the actual JAK2 inhibitors with other therapies, in particular with molecules targeting pathways downstream of JAK2 activation or the stability of JAK2

  11. Strategies for discontinuation of proton pump inhibitors

    DEFF Research Database (Denmark)

    Haastrup, Peter; Paulsen, Maja S; Begtrup, Luise M

    2014-01-01

    PURPOSE: Proton pump inhibitors (PPIs) are considered to be overprescribed. Consensus on how to attempt discontinuation is, however, lacking. We therefore conducted a systematic review of clinical studies on discontinuation of PPIs. METHODS: Systematic review based on clinical studies investigating...

  12. Predicting the Performance of Organic Corrosion Inhibitors

    Directory of Open Access Journals (Sweden)

    David A. Winkler

    2017-12-01

    Full Text Available The withdrawal of effective but toxic corrosion inhibitors has provided an impetus for the discovery of new, benign organic compounds to fill that role. Concurrently, developments in the high-throughput synthesis of organic compounds, the establishment of large libraries of available chemicals, accelerated corrosion inhibition testing technologies, and the increased capability of machine learning methods have made discovery of new corrosion inhibitors much faster and cheaper than it used to be. We summarize these technical developments in the corrosion inhibition field and describe how data-driven machine learning methods can generate models linking molecular properties to corrosion inhibition that can be used to predict the performance of materials not yet synthesized or tested. We briefly summarize the literature on quantitative structure–property relationships models of small organic molecule corrosion inhibitors. The success of these models provides a paradigm for rapid discovery of novel, effective corrosion inhibitors for a range of metals and alloys in diverse environments.

  13. Novel diamide-based inhibitors of IMPDH.

    Science.gov (United States)

    Gu, Henry H; Iwanowicz, Edwin J; Guo, Junqing; Watterson, Scott H; Shen, Zhongqi; Pitts, William J; Dhar, T G Murali; Fleener, Catherine A; Rouleau, Katherine; Sherbina, N Z; Witmer, Mark; Tredup, Jeffrey; Hollenbaugh, Diane

    2002-05-06

    A series of novel amide-based small molecule inhibitors of inosine monophosphate dehydrogenase is described. The synthesis and the structure-activity relationships (SARs) derived from in vitro studies are presented.

  14. The treatment of kleptomania with serotonin reuptake inhibitors.

    Science.gov (United States)

    Lepkifker, E; Dannon, P N; Ziv, R; Iancu, I; Horesh, N; Kotler, M

    1999-01-01

    Kleptomania is characterized by an irresistible impulse to steal objects not needed for personal use or for their monetary value. Several recent case reports have shown that Serotonin Specific Reuptake Inhibitors (SSRIs) could be effective in the treatment of kleptomania just as it is in other obsessive-compulsive spectrum disorders. We report five cases of kleptomania patients who were successfully treated with fluoxetine or paroxetine in combination with a psychotherapeutic intervention. In one case, the discontinuation of the medication repeatedly led to the resurgence of the kleptomanic behavior. Our case series illustrates the effectiveness of SSRIs in kleptomania. It thus supports the assumption that this syndrome involves a dysfunctional serotoninergic mechanism.

  15. Met receptor inhibitor SU11274 localizes in the endoplasmic reticulum.

    Science.gov (United States)

    Wiest, Edwin J; Smith, Heather Jensen; Hollingsworth, Michael A

    2018-07-02

    We discovered that SU11274, a class I c-Met inhibitor, fluoresces when excited by 488 nm laser light and showed rapid specific accumulation in distinct subcellular compartments. Given that SU11274 reduces cancer cell viability, we exploited these newly identified spectral properties to determine SU11274 intracellular distribution and accumulation in human pancreatic cancer cells. The aim of the studies reported here was to identify organelle(s) to which SU11274 is trafficked. We conclude that SU11274 rapidly and predominantly accumulates in the endoplasmic reticulum. Copyright © 2018. Published by Elsevier Inc.

  16. Monoamine Oxidase B Inhibitors in Parkinson's Disease.

    Science.gov (United States)

    Dezsi, Livia; Vecsei, Laszlo

    2017-01-01

    Parkinson's disease (PD) is a neurodegenerative disorder with a prevalence increasing with age. Oxidative stress and glutamate toxicity are involved in its pathomechanism. There are still many unmet needs of PD patients, including the alleviation of motor fluctuations and dyskinesias, and the development of therapies with neuroprotective potential. To give an overview of the pharmacological properties, the efficacy and safety of the monoamine oxidase B (MAO-B) inhibitors in the treatment of PD, with special focus on the results of randomized clinical trials. A literature search was conducted in PubMed for 'PD treatment', 'MAO-B inhibitors', 'selegiline', 'rasagiline', 'safinamide' and 'clinical trials' with 'MAO-B inhibitors' in 'Parkinson' disease'. MAO-B inhibitors have a favorable pharmacokinetic profile, improve the dopamine deficient state and may have neuroprotective properties. Safinamide exhibits an anti-glutamatergic effect as well. When applied as monotherapy, MAO-B inhibitors provide a modest, but significant improvement of motor function and delay the need for levodopa. Rasagiline and safinamide were proven safe and effective when added to a dopamine agonist in early PD. As add-on to levodopa, MAO-B inhibitors significantly reduced off-time and were comparable in efficacy to COMT inhibitors. Improvements were achieved as regards certain non-motor symptoms as well. Due to the efficacy shown in clinical trials and their favorable side-effect profile, MAO-B inhibitors are valuable drugs in the treatment of PD. They are recommended as monotherapy in the early stages of the disease and as add-on therapy to levodopa in advanced PD. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  17. The effect of chemical anti-inhibitors on fibrinolytic enzymes and inhibitors

    DEFF Research Database (Denmark)

    Sidelmann, Johannes Jakobsen; Jespersen, J; Kluft, C

    1997-01-01

    proteases. We studied the influence of chemical anti-inhibitors (chloramine T, flufenamate, sodium lauryl sulfate, and methylamine) on fibrinolytic serine proteases and fibrinolytic enzyme inhibitors using the physiological substrate fibrin as plasmin substrate. Low concentrations of chloramine T (0.01 mmol......%) and plasminogen activators (apparent recovery > 200%). Sodium lauryl sulfate eliminates the major fibrinolytic enzyme inhibitors, but increases the activity of plasmin (apparent recovery > 200%) and plasminogen activator, urokinase type (apparent recovery 130%). Methylamine affects only plasmin inhibition. We...

  18. Development of Radiosensitizer using farnesyltransferase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Jong Seok; Choe, Yong Kyung; Han, Mi Young; Kim, Kwang Dong [Korea Research Institute of Bioscience and Biotechnology, Taejon (Korea)

    1999-03-01

    We selected some compounds that were reported to have an activity of farneyltransferase inhibitor and tested the hypothesis that they might be used to radiosensitize cells transformed by ras oncogenes. The inhibition of ras processing using some, but not all, inhibitors resulted in higher levels of cell death after {gamma}-irradiation and increased radiosensitivity in H-ras-transformed NIH3T3 cells and MCF-10A human tumor cells. They did not induce additional cell death in control cells that doe not have ras mutation. Furthermore, the treatment of inhibitors alone induced a weak G0/G1 block, whereas inhibitors in combination with {gamma}-irradiation induced an additional enrichment in the G2/M phase of the cell cycle that typically represents irradiation-induced growth arrest. At present, the underling mechanism by which the farnesylltransferase inhibitors exert radiosensitizing effect is not known. In summary, our results suggest and lead to the possibility that some of farnesylation inhibitors may prove clinically useful not only as antitumor agents, but also radiosensitizers of tumors whose growth is dependent on ras function. (author). 15 refs., 10 figs., 4 tabs.

  19. Natural inhibitors of tumor-associated proteases

    International Nuclear Information System (INIS)

    Magdolen, U.; Krol, J.; Sato, S.; Schmitt, M.; Magdolen, V.; Krueger, A.; Mueller, M.M.; Sperl, S.

    2002-01-01

    The turnover and remodelling of extracellular matrix (ECM) is an essential part of many normal biological processes including development, morphogenesis, and wound healing. ECM turnover also occurs in severe pathological situations like artherosclerosis, fibrosis, tumor invasion and metastasis. The major proteases involved in this turnover are serine proteases (especially the urokinase-type plasminogen activator/plasmin system), matrix metalloproteases (a family of about 20 zinc-dependent endopeptidases including collagenases, gelatinases, stromelysins, and membrane-type metalloproteases), and cysteine proteases. In vivo, the activity of these proteases is tightly regulated in the extracellular space by zymogen activation and/or controlled inhibition. In the present review, we give an overview on the structure and biochemical properties of important tumor-associated protease inhibitors such as plasminogen activator inhibitor type 1 and type 2 (PAI-1, PAI-2), tissue inhibitors of metalloproteinases (TIMP-1, -2, -3, and -4), and the cysteine protease inhibitor cystatin C. Interestingly, some of these inhibitors of tumor-associated proteases display multiple functions which rather promote than inhibit tumor progression, when the presence of inhibitors in the tumor tissue is not balanced. (author)

  20. Emerging Corrosion Inhibitors for Interfacial Coating

    Directory of Open Access Journals (Sweden)

    Mona Taghavikish

    2017-12-01

    Full Text Available Corrosion is a deterioration of a metal due to reaction with environment. The use of corrosion inhibitors is one of the most effective ways of protecting metal surfaces against corrosion. Their effectiveness is related to the chemical composition, their molecular structures and affinities for adsorption on the metal surface. This review focuses on the potential of ionic liquid, polyionic liquid (PIL and graphene as promising corrosion inhibitors in emerging coatings due to their remarkable properties and various embedment or fabrication strategies. The review begins with a precise description of the synthesis, characterization and structure-property-performance relationship of such inhibitors for anti-corrosion coatings. It establishes a platform for the formation of new generation of PIL based coatings and shows that PIL corrosion inhibitors with various heteroatoms in different form can be employed for corrosion protection with higher barrier properties and protection of metal surface. However, such study is still in its infancy and there is significant scope to further develop new structures of PIL based corrosion inhibitors and coatings and study their behaviour in protection of metals. Besides, it is identified that the combination of ionic liquid, PIL and graphene could possibly contribute to the development of the ultimate corrosion inhibitor based coating.