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Sample records for alpha1-proteinase inhibitor human

  1. Production of alpha 1-proteinase inhibitor (human).

    Science.gov (United States)

    Hein, R H; Van Beveren, S M; Shearer, M A; Coan, M H; Brockway, W J

    1990-03-01

    A method for large scale isolation of alpha 1-proteinase inhibitor (alpha 1-PI) is described. This method employs waste Cohn Fraction IV-1 as the starting material and involves fractional precipitation with polyethylene glycol followed by ion exchange chromatography on diethylaminoethanol (DEAE)-Sepharose. The process also incorporates a ten hour, at 60 degrees C, heat-treatment step to reduce or eliminate the risk of transmission of viral disease. The final product, having a purity of approximately 60%, is freeze-dried. This preparation behaves almost identically to the alpha 1-PI in plasma and is suitable for replacement therapy in hereditary emphysema.

  2. Neutrophils degrade subendothelial matrices in the presence of alpha-1-proteinase inhibitor. Cooperative use of lysosomal proteinases and oxygen metabolites.

    OpenAIRE

    Weiss, S J; Regiani, S

    1984-01-01

    Triggered neutrophils rapidly degraded labeled matrices secreted by cultured, venous endothelial cells via a process dependent on elastase but not oxygen metabolites. In the presence of high concentrations of alpha-1-proteinase inhibitor, the ability of the stimulated neutrophil to solubilize the matrix was impaired. However, at lower concentrations of alpha-1-proteinase inhibitor the neutrophil could enhance the degradative potential of its released elastase by a H2O2-dependent process. Coin...

  3. Pulmonary penetration of alpha 1-proteinase inhibitor administered parenterally to dogs

    International Nuclear Information System (INIS)

    Smith, R.M.; Spragg, R.G.; Moser, K.M.; Cochrane, C.G.; McCarren, J.P.

    1987-01-01

    To study the penetration of alpha 1-proteinase inhibitor (A1Pl) into the lungs of healthy dogs, 83 mg/kg of active A1Pl was administered intravenously over 30 min followed by a bolus of 131 I-A1Pl. Animals were lavaged 2 to 72 h after infusion, sequential gamma camera scans were acquired, and urine was analyzed for the excretion of desmosine. After a distribution phase, infused A1Pl left the bloodstream with a half-life of 103 +/- 24 h. Analysis of plasma antiprotease activity demonstrated preservation of function of the infused A1Pl. Lavage fluid A1Pl concentration and activity were significantly increased 24 h after infusion. Gamma camera scans demonstrated that lung, liver, and spleen acquired 131 I-A1Pl similarly; radioactivities per gram of tissue of these organs were similar at autopsy. Excretion of desmosine did not decrease from a baseline of 157 +/- 59 nmol/24 h after A1Pl infusion, indicating no effect of A1Pl infusion on background elastolysis. These data suggest that intravenous administration of A1Pl can raise lung antiproteinase levels within 24 h despite the absence of preferential uptake by the lung of the infused protein

  4. Phage display of the serpin alpha-1 proteinase inhibitor randomized at consecutive residues in the reactive centre loop and biopanned with or without thrombin.

    Directory of Open Access Journals (Sweden)

    Benjamin M Scott

    Full Text Available In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2-P1 yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352-356 (P7-P3 was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7-P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1 as a serpin amenable to phage display and suggest the utility of this approach for the selection

  5. Bioequivalence of a Liquid Formulation of Alpha1-Proteinase Inhibitor Compared with Prolastin®-C (Lyophilized Alpha1-PI) in Alpha1-Antitrypsin Deficiency.

    Science.gov (United States)

    Barker, Alan F; Campos, Michael A; Brantly, Mark L; Stocks, James M; Sandhaus, Robert A; Lee, Douglas; Steinmann, Kimberly; Lin, Jiang; Sorrells, Susan

    2017-12-01

    This study evaluated the bioequivalence, safety, and immunogenicity of a new liquid formulation of human plasma-derived alpha 1 -proteinase inhibitor, Liquid Alpha 1 -PI, compared with the Lyophilized Alpha 1 -PI formulation (Prolastin®-C), for augmentation therapy in patients with alpha 1 -antitrypsin deficiency (AATD). In this double-blind, randomized, 20-week crossover study, 32 subjects with AATD were randomized to receive 8 weekly infusions of 60 mg/kg of Liquid Alpha 1 -PI or Lyophilized Alpha 1 -PI. Serial blood samples were drawn for 7 days after the last dose followed by 8 weeks of the alternative treatment. The primary endpoint was bioequivalence at steady state, as measured by area under the concentration versus time curve from 0 to 7 days (AUC 0-7 days ) postdose using an antigenic content assay. Bioequivalence was defined as 90% confidence interval (CI) for the ratio of the geometric least squares (LS) mean of AUC 0-7 days for both products within the limits of 0.80 and 1.25. Safety and immunogenicity were assessed. Mean alpha 1 -PI concentration versus time curves for both formulations were superimposable. Mean AUC 0-7 days was 20 320 versus 19 838 mg × h/dl for Liquid Alpha 1 -PI and Lyophilized Alpha 1 -PI, respectively. The LS mean ratio of AUC 0-7 days (90% CI) for Liquid Alpha 1 -PI versus Lyophilized Alpha 1 -PI was 1.05 (1.03-1.08), indicating bioequivalence. Liquid Alpha 1 -PI was well tolerated and adverse events were consistent with Lyophilized Alpha 1 -PI. Immunogenicity to either product was not detected. In conclusion, Liquid Alpha 1 -PI is bioequivalent to Lyophilized Alpha 1 -PI, with a similar safety profile. The liquid formulation would eliminate the need for reconstitution and shorten preparation time for patients receiving augmentation therapy for AATD.

  6. Clinical utility of alpha-1 proteinase inhibitor in the management of adult patients with severe alpha-1 antitrypsin deficiency: a review of the current literature

    Directory of Open Access Journals (Sweden)

    Parr DG

    2017-07-01

    Full Text Available David G Parr, Beatriz Lara Department of Respiratory Medicine, Cardio-Respiratory Division, University Hospital Coventry, Coventry, UK Abstract: Alpha-1 antitrypsin (AAT functions primarily to inhibit neutrophil elastase, and its deficiency predisposes individuals to the development of chronic obstructive pulmonary disease (COPD. The putative protective serum concentration is generally considered to be above a threshold of 11 µM/L, and therapeutic augmentation of AAT above this value is believed to retard the progression of emphysema. Several AAT preparations, all derived from human donor plasma, have been commercialized since approval by the US Food and Drug Administration (FDA in 1987. Biochemical efficacy has been demonstrated by augmentation of pulmonary antiprotease activity, but demonstration of clinical efficacy in randomized, placebo-controlled trials has been hampered by the practical difficulties of performing conventional studies in a rare disease with a relatively long natural history. Computed tomography has been applied to measure lung density as a more specific and sensitive surrogate outcome measure of emphysema than physiologic indices, such as forced expiratory volume in 1 second, and studies consistently show a therapeutic reduction in the rate of lung density decline. However, convincing evidence of benefit using traditional clinical measures remains elusive. Intravenous administration of AAT at a dose of 60 mg/kg/week is the commonest regime in use and has well-documented safety and tolerability. International and national guidelines on the management of AAT deficiency recommend intravenous augmentation therapy to supplement optimized usual COPD treatment in patients with severe deficiency and evidence of lung function impairment. Keywords: alpha-1 antitrypsin deficiency, augmentation or replacement therapy, computed tomography, emphysema, COPD

  7. Comparison of concentrations of two proteinase inhibitors, porcine pancreatic elastase inhibitory capacity, and cell profiles in sequential bronchoalveolar lavage samples.

    Science.gov (United States)

    Morrison, H M; Kramps, J A; Dijkman, J H; Stockley, R A

    1986-01-01

    Bronchoalveolar lavage is used to obtain cells and proteins from the lower respiratory tract for diagnosis and research. Uncertainity exists about which site in the lung is sampled by the lavage fluid and what effect different lavage volumes have on recovery of the constituents of lavage fluid. Dilution of alveolar lining fluid by lavage fluid is variable and results are usually expressed as protein ratios to surmount this problem. We have compared cell profiles and the concentrations of two proteinase inhibitors--the low molecular weight bronchial protease inhibitor antileucoprotease and alpha 1 proteinase inhibitor, together with alpha 1 proteinase inhibitor function and its relationship to the cell profile in sequential bronchoalveolar lavage fluid samples from patients undergoing bronchoscopy. There was no difference in total or differential cell counts or albumin or alpha 1 proteinase inhibitor concentrations between the first and second halves of the lavage. Both the concentration of antileucoprotease and the ratio of antileucoprotease to albumin were, however, lower in the second half of the lavage (2p less than 0.01 and 2p less than 0.05 respectively). There was no difference in the function of alpha 1 proteinase inhibitor (assessed by inhibition of porcine pancreatic elastase--PPE) between aliquots (0.28 mole PPE inhibited/mol alpha 1 proteinase inhibitor; range 0-1.19 for the first half and 0.37 mol PPE inhibited/mol alpha 1 proteinase inhibitor; range 0.10-0.80 for the second half). About 60-70% of alpha 1 proteinase inhibitor in each half of the lavage fluid was inactive as an inhibitor. The function of alpha 1 proteinase inhibitor did not differ between bronchitic smokers and ex-smokers. Alpha 1 proteinase inhibitor function was not related to the number of total white cells, macrophages, or neutrophils in the lavage fluid. Contamination of lavage by red blood cells was found to alter the concentration of alpha 1 proteinase inhibitor but not its

  8. Human tyrosinase inhibitor in rum distillate wastewater.

    Science.gov (United States)

    Takara, Kensaku; Iwasaki, Hironori; Ujihara, Kunihiro; Wada, Koji

    2008-01-01

    An inhibitor of human tyrosinase activity in rum distillate wastewater was isolated and identified as (S)-(+)-imperanene (1). (S)-(+)-Imperanene significantly inhibited tyrosinase isolated from HMV-II cells (IC(50) 1.85 mM). Inhibition kinetics studies revealed that imperanene is a competitive inhibitor of tyrosinase when L-3,4-dihydroxyphenylalanine is used as the substrate. The inhibitory activities of 1, O-beta-D-glucopyranosyl imperanene (2) and O-beta-D-glucopyranosyl-3-methoxyl imperanene (3) were 1>2>3.

  9. A Selective Cyclic Peptidic Human SIRT5 Inhibitor

    Directory of Open Access Journals (Sweden)

    Jiajia Liu

    2016-09-01

    Full Text Available In the current study, we discovered that a side chain-to-side chain cyclic pentapeptide harboring a central Nε-carboxyethyl-thiocarbamoyl-lysine residue behaved as a strong and selective (versus human SIRT1/2/3/6 inhibitor against human SIRT5-catalyzed deacylation reaction. This compound was also found to be proteolytically much more stable than its linear counterpart. This compound could be a valuable lead for developing stronger, selective, metabolically stable, and cell permeable human SIRT5 inhibitors.

  10. Expression of functional human C1 inhibitor in COS cells

    NARCIS (Netherlands)

    Eldering, E.; Nuijens, J. H.; Hack, C. E.

    1988-01-01

    Full length human C1 inhibitor cDNA was cloned into a vector suitable for transient expression in COS-1 cells. Transfected COS cells secreted an immunoreactive protein of Mr approximately 110,000 that appeared to be functionally equivalent to the plasma-derived protein as established by the

  11. Determination of activities of human carbonic anhydrase II inhibitors ...

    African Journals Online (AJOL)

    intensive search for novel drugs is ongoing, through synthesis ... previously synthesized using microwave irradiation [10], were evaluated for their potential as inhibitors of human CA-II. The basic structure of all 44 curcumin analogs is depicted in Figure. 1. Figure 1: .... algorithm was used for exploring ligand poses inside the ...

  12. Molecular pharmacokinetic determinants of anticancer kinase inhibitors in humans

    Directory of Open Access Journals (Sweden)

    Julie Scholler

    2011-06-01

    Full Text Available This review presents the published data regarding the molecular determinants (drug metabolizing enzymes, drug transporters and orphan nuclear receptors of approved anticancer kinase inhibitors pharmacokinetics in humans. The clinical impact of these determinants (drug disposition and drug–drug interactions is also discussed.

  13. Discovery of novel human acrosin inhibitors by virtual screening

    Science.gov (United States)

    Liu, Xuefei; Dong, Guoqiang; Zhang, Jue; Qi, Jingjing; Zheng, Canhui; Zhou, Youjun; Zhu, Ju; Sheng, Chunquan; Lü, Jiaguo

    2011-10-01

    Human acrosin is an attractive target for the discovery of male contraceptive drugs. For the first time, structure-based drug design was applied to discover structurally diverse human acrosin inhibitors. A parallel virtual screening strategy in combination with pharmacophore-based and docking-based techniques was used to screen the SPECS database. From 16 compounds selected by virtual screening, a total of 10 compounds were found to be human acrosin inhibitors. Compound 2 was found to be the most potent hit (IC50 = 14 μM) and its binding mode was investigated by molecular dynamics simulations. The hit interacted with human acrosin mainly through hydrophobic and hydrogen-bonding interactions, which provided a good starting structure for further optimization studies.

  14. ROCK inhibitor prevents the dedifferentiation of human articular chondrocytes

    International Nuclear Information System (INIS)

    Matsumoto, Emi; Furumatsu, Takayuki; Kanazawa, Tomoko; Tamura, Masanori; Ozaki, Toshifumi

    2012-01-01

    Highlights: ► ROCK inhibitor stimulates chondrogenic gene expression of articular chondrocytes. ► ROCK inhibitor prevents the dedifferentiation of monolayer-cultured chondrocytes. ► ROCK inhibitor enhances the redifferentiation of cultured chondrocytes. ► ROCK inhibitor is useful for preparation of un-dedifferentiated chondrocytes. ► ROCK inhibitor may be a useful reagent for chondrocyte-based regeneration therapy. -- Abstract: Chondrocytes lose their chondrocytic phenotypes in vitro. The Rho family GTPase ROCK, involved in organizing the actin cytoskeleton, modulates the differentiation status of chondrocytic cells. However, the optimum method to prepare a large number of un-dedifferentiated chondrocytes is still unclear. In this study, we investigated the effect of ROCK inhibitor (ROCKi) on the chondrogenic property of monolayer-cultured articular chondrocytes. Human articular chondrocytes were subcultured in the presence or absence of ROCKi (Y-27632). The expression of chondrocytic marker genes such as SOX9 and COL2A1 was assessed by quantitative real-time PCR analysis. Cellular morphology and viability were evaluated. Chondrogenic redifferentiation potential was examined by a pellet culture procedure. The expression level of SOX9 and COL2A1 was higher in ROCKi-treated chondrocytes than in untreated cells. Chondrocyte morphology varied from a spreading form to a round shape in a ROCKi-dependent manner. In addition, ROCKi treatment stimulated the proliferation of chondrocytes. The deposition of safranin O-stained proteoglycans and type II collagen was highly detected in chondrogenic pellets derived from ROCKi-pretreated chondrocytes. Our results suggest that ROCKi prevents the dedifferentiation of monolayer-cultured chondrocytes, and may be a useful reagent to maintain chondrocytic phenotypes in vitro for chondrocyte-based regeneration therapy.

  15. Inhibitors

    Science.gov (United States)

    ... Icon View public health webinars on blood disorders Inhibitors Language: English (US) Español (Spanish) Recommend on Facebook ... because treatment of bleeds becomes less effective. About Inhibitors People with hemophilia, and many with VWD type ...

  16. Expression of human α1-proteinase inhibitor in Aspergillus niger

    Directory of Open Access Journals (Sweden)

    Punt Peter J

    2007-10-01

    Full Text Available Abstract Background Human α1-proteinase inhibitor (α1-PI, also known as antitrypsin, is the most abundant serine protease inhibitor (serpin in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, α1-PI is available for replacement therapy as an FDA licensed plasma-derived (pd product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant α1-PI (r-α1-PI could provide an attractive alternative. Although r-α1-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation. Results We have explored the possibility of expressing the gene for human α1-PI in the filamentous fungus Aspergillus niger (A. niger, a system reported to be capable of providing more "mammalian-like" glycosylation patterns to secretable proteins than commonly used yeast hosts. Our expression strategy was based on fusion of α1-PI with a strongly expressed, secreted leader protein (glucoamylase G2, separated by dibasic processing site (N-V-I-S-K-R that provides in vivo cleavage. SDS-PAGE, Western blot, ELISA, and α1-PI activity assays enabled us to select the transformant(s secreting a biologically active glycosylated r-α1-PI with yields of up to 12 mg/L. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS analysis further confirmed that molecular mass of the r-α1-PI was similar to that of the pd-α1-PI. In vitro stability of the r-α1-PI from A. niger was tested in comparison with pd-α1-PI reference and non-glycosylated human r-α1-PI from E. coli. Conclusion We examined the suitability of the filamentous fungus A. niger for the expression of the human gene for α1-PI, a medium size

  17. Recombinant human C1-inhibitor in the treatment of acute angioedema attacks

    NARCIS (Netherlands)

    Choi, Goda; Soeters, Maarten R.; Farkas, Henriette; Varga, Lilian; Obtulowicz, Krystyna; Bilo, Barbara; Porebski, Greg; Hack, C. Erik; Verdonk, Rene; Nuijens, Jan; Levi, Marcel

    2007-01-01

    BACKGROUND: Patients with hereditary C1-inhibitor deficiency have recurrent attacks of angioedema, preferably treated with C1-inhibitor concentrate. A recombinant human C1-inhibitor (rHuC1INH) was developed, derived from milk from transgenic rabbits. This study was undertaken to investigate the

  18. [Computational prediction of human immunodeficiency resistance to reverse transcriptase inhibitors].

    Science.gov (United States)

    Tarasova, O A; Filimonov, D A; Poroikov, V V

    2017-10-01

    Human immunodeficiency virus (HIV) causes acquired immunodeficiency syndrome (AIDS) and leads to over one million of deaths annually. Highly active antiretroviral treatment (HAART) is a gold standard in the HIV/AIDS therapy. Nucleoside and non-nucleoside inhibitors of HIV reverse transcriptase (RT) are important component of HAART, but their effect depends on the HIV susceptibility/resistance. HIV resistance mainly occurs due to mutations leading to conformational changes in the three-dimensional structure of HIV RT. The aim of our work was to develop and test a computational method for prediction of HIV resistance associated with the mutations in HIV RT. Earlier we have developed a method for prediction of HIV type 1 (HIV-1) resistance; it is based on the usage of position-specific descriptors. These descriptors are generated using the particular amino acid residue and its position; the position of certain residue is determined in a multiple alignment. The training set consisted of more than 1900 sequences of HIV RT from the Stanford HIV Drug Resistance database; for these HIV RT variants experimental data on their resistance to ten inhibitors are presented. Balanced accuracy of prediction varies from 80% to 99% depending on the method of classification (support vector machine, Naive Bayes, random forest, convolutional neural networks) and the drug, resistance to which is obtained. Maximal balanced accuracy was obtained for prediction of resistance to zidovudine, stavudine, didanosine and efavirenz by the random forest classifier. Average accuracy of prediction is 89%.

  19. The HSP90 Inhibitor Ganetespib Radiosensitizes Human Lung Adenocarcinoma Cells

    Energy Technology Data Exchange (ETDEWEB)

    Gomez-Casal, Roberto; Bhattacharya, Chitralekha [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Medicine, The University of Pittsburgh, Pittsburgh, PA 15213 (United States); Epperly, Michael W. [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Radiation Oncology, The University of Pittsburgh, Pittsburgh, PA 15213 (United States); Basse, Per H. [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Immunology, The University of Pittsburgh, Pittsburgh, PA 15213 (United States); Wang, Hong [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Biostatistics, The University of Pittsburgh, Pittsburgh, PA 15213 (United States); Wang, Xinhui [Harvard Medical School, Harvard University, 25 Shattuck Street, Boston, MA 02115 (United States); Proia, David A. [Synta Pharmaceuticals Corp., 45 Hartwell Avenue, Lexington, MA 02421 (United States); Greenberger, Joel S. [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Radiation Oncology, The University of Pittsburgh, Pittsburgh, PA 15213 (United States); Socinski, Mark A.; Levina, Vera, E-mail: levinav@upmc.edu [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Medicine, The University of Pittsburgh, Pittsburgh, PA 15213 (United States)

    2015-05-22

    The molecular chaperone HSP90 is involved in stabilization and function of multiple client proteins, many of which represent important oncogenic drivers in NSCLC. Utilization of HSP90 inhibitors as radiosensitizing agents is a promising approach. The antitumor activity of ganetespib, HSP90 inhibitor, was evaluated in human lung adenocarcinoma (AC) cells for its ability to potentiate the effects of IR treatment in both in vitro and in vivo. The cytotoxic effects of ganetespib included; G2/M cell cycle arrest, inhibition of DNA repair, apoptosis induction, and promotion of senescence. All of these antitumor effects were both concentration- and time-dependent. Both pretreatment and post-radiation treatment with ganetespib at low nanomolar concentrations induced radiosensitization in lung AC cells in vitro. Ganetespib may impart radiosensitization through multiple mechanisms: such as down regulation of the PI3K/Akt pathway; diminished DNA repair capacity and promotion of cellular senescence. In vivo, ganetespib reduced growth of T2821 tumor xenografts in mice and sensitized tumors to IR. Tumor irradiation led to dramatic upregulation of β-catenin expression in tumor tissues, an effect that was mitigated in T2821 xenografts when ganetespib was combined with IR treatments. These data highlight the promise of combining ganetespib with IR therapies in the treatment of AC lung tumors.

  20. Intrinsic thermodynamics of inhibitor binding to human carbonic anhydrase IX.

    Science.gov (United States)

    Linkuvienė, Vaida; Matulienė, Jurgita; Juozapaitienė, Vaida; Michailovienė, Vilma; Jachno, Jelena; Matulis, Daumantas

    2016-04-01

    Human carbonic anhydrase 9th isoform (CA IX) is an important marker of numerous cancers and is increasingly interesting as a potential anticancer drug target. Various synthetic aromatic sulfonamide-bearing compounds are being designed as potent inhibitors of CA IX. However, sulfonamide compound binding to CA IX is linked to several reactions, the deprotonation of the sulfonamide amino group and the protonation of the CA active site Zn(II)-bound hydroxide. These linked reactions significantly affect the affinities and other thermodynamic parameters such as enthalpies and entropies of binding. The observed and intrinsic affinities of compound binding to CA IX were determined by the fluorescent thermal shift assay. The enthalpies and entropies of binding were determined by the isothermal titration calorimetry. The pKa of CA IX was determined to be 6.8 and the enthalpy of CA IX-Zn(II)-bound hydroxide protonation was -24 kJ/mol. These values enabled the analysis of intrinsic thermodynamics of a library of compounds binding to CA IX. The most strongly binding compounds exhibited the intrinsic affinity of 0.01 nM and the observed affinity of 2 nM. The intrinsic thermodynamic parameters of compound binding to CA IX helped to draw the compound structure to thermodynamics relationship. It is important to distinguish the intrinsic from observed parameters of any disease target protein interaction with its inhibitors as drug candidates when drawing detailed compound structure to thermodynamics correlations. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Lansoprazole and carbonic anhydrase IX inhibitors sinergize against human melanoma cells.

    Science.gov (United States)

    Federici, Cristina; Lugini, Luana; Marino, Maria Lucia; Carta, Fabrizio; Iessi, Elisabetta; Azzarito, Tommaso; Supuran, Claudiu T; Fais, Stefano

    2016-01-01

    Proton Pump Inhibitors (PPIs) reduce tumor acidity and therefore resistance of tumors to drugs. Carbonic Anhydrase IX (CA IX) inhibitors have proven to be effective against tumors, while tumor acidity might impair their full effectiveness. To analyze the effect of PPI/CA IX inhibitors combined treatment against human melanoma cells. The combination of Lansoprazole (LAN) and CA IX inhibitors (FC9-399A and S4) has been investigated in terms of cell proliferation inhibition and cell death in human melanoma cells. The combination of these inhibitors was more effective than the single treatments in both inhibiting cell proliferation and in inducing cell death in human melanoma cells. These results represent the first successful attempt in combining two different proton exchanger inhibitors. This is the first evidence on the effectiveness of a new approach against tumors based on the combination of PPI and CA IX inhibitors, thus providing an alternative strategy against tumors.

  2. Characterization of Inhibitor of differentiation (Id) proteins in human cornea.

    Science.gov (United States)

    Mohan, Rajiv R; Morgan, Brandie R; Anumanthan, Govindaraj; Sharma, Ajay; Chaurasia, Shyam S; Rieger, Frank G

    2016-05-01

    Inhibitor of differentiation (Id) proteins are DNA-binding transcription factors involved in cellular proliferation, migration, inflammation, angiogenesis and fibrosis. However, their expression and role in the cornea is unknown. The present study was undertaken to characterize the expression of Id proteins and their interactions with the pro-fibrotic cytokine Transforming Growth Factor β1 (TGFβ1) and anti-fibrotic cytokine, bone morphogenic protein 7 (BMP7) in human cornea. Human donor corneas procured from Eye Bank were used. Id proteins were localized in human corneal sections using immunofluorescence. Primary cultures of human corneal fibroblasts (HCF) were established and treated with either TGFβ1 (5 ng/ml) or BMP7 (10 ng/ml) for 24 h in serum free medium. Expression of Id's in response to TGFβ1, BMP7 and TGFβ1 + BMP7 was analyzed by quantitative real time PCR (qRT-PCR) and western blot analysis. Id1 and Id2 proteins were ubiquitously expressed in the epithelial cells and stromal keratocytes in human cornea. The Id1 was localized to the basal epithelial cells as seen by immunohistochemistry. HCF expressed all known mammalian Id genes (Id1-Id4). In addition, Id1 and Id2 are selectively expressed in HCF. Treatment of human recombinant TGFβ1 (5 ng/ml) to serum-starved HCF showed a significant increase in Id genes (Id1, Id2 and Id4) at 2 h time point compared to BMP7 treatment, which showed time dependent increase in the expression of Id1-Id3 at 24-48 h. Combined treatment with TGFβ1 + BMP7 to HCF showed a significant increase in Id1 transcript and an increasing trend in Id3 and Id4 expression. The results of this study suggest that Id family of genes (Id1-Id4) are localized in the human cornea and expressed in the corneal fibroblasts. Also, Id's were differentially regulated with TGFβ1 and/or BMP7 in a time dependent manner and might serve as a therapeutic target in corneal fibrosis. Published by Elsevier Ltd.

  3. Human hematopoietic prostaglandin D synthase inhibitor complex structures.

    Science.gov (United States)

    Kado, Yuji; Aritake, Kosuke; Uodome, Nobuko; Okano, Yousuke; Okazaki, Nobuo; Matsumura, Hiroyoshi; Urade, Yoshihiro; Inoue, Tsuyoshi

    2012-04-01

    In mast and Th2 cells, hematopoietic prostaglandin (PG) D synthase (H-PGDS) catalyses the isomerization of PGH(2) in the presence of glutathione (GSH) to produce the allergic and inflammatory mediator PGD(2). We determined the X-ray structures of human H-PGDS inhibitor complexes with 1-amino-4-{4-[4-chloro-6-(2-sulpho-phenylamino)-[1,3,5]triazin-2-ylmethyl]-3-sulpho-phenylamino}-9,10-dioxo-9,10-dihydro-anthracene-2-sulphonic acid (Cibacron Blue) and 1-amino-4-(4-aminosulphonyl) phenyl-anthraquinone-2-sulphonic acid (APAS) at 2.0 Å resolution. When complexed with H-PGDS, Cibacron Blue had an IC(50) value of 40 nM and APAS 2.1 μM. The Cibacron Blue molecule was stabilized by four hydrogen bonds and π-π stacking between the anthraquinone ring and Trp104, the ceiling of the active site H-PGDS pocket. Among the four hydrogen bonds, the Cibacron Blue terminal sulphonic group directly interacted with conserved residues Lys112 and Lys198, which recognize the PGH(2) substrate α-chain. In contrast, the APAS anthraquinone ring was inverted to interact with Trp104, while its benzenesulphonic group penetrated the GSH-bound region at the bottom of the active site. Due to the lack of extended aromatic rings, APAS could not directly hydrogen bond with the two conserved lysine residues, thus decreasing the total number of hydrogen bond from four to one. These factors may contribute to the 50-fold difference in the IC(50) values obtained for the two inhibitors.

  4. Binding of Rasagiline-related Inhibitors to Human Monoamine Oxidases

    Science.gov (United States)

    Binda, Claudia; Hubálek, Frantisek; Li, Min; Herzig, Yaacov; Sterling, Jeffrey; Edmondson, Dale E.; Mattevi, Andrea

    2008-01-01

    Monoamine oxidases A and B (MAO A and B) catalyze neurotransmitters degradation and represent drug targets for the treatment of neurodegenerative disorders. Rasagiline is an irreversible, MAO B-selective inhibitor that has been approved as a novel anti-Parkinson’s drug. In this study we investigate the inhibition of recombinant human MAO A and MAO B by several rasagiline analogues. Different substituents added onto the rasagiline scaffold alter the binding affinity depending on the position on the aminoindan ring and on the size of the substituent. Compounds with a hydroxyl group on either the C4 or the C6 atom inhibit both isozymes, whereas a bulkier substituent such as a carbamate is tolerated only at the C4 position. The 1.7 Å crystal structure of MAO B in complex with 4-(N-methyl-N-ethyl-carbamoyloxy)-N-methyl-N-propargyl-1(R)-aminoindan shows that the binding mode is similar to that of rasagiline with the carbamate moiety occupying the entrance cavity space. 1(R)-aminoindan, the major metabolic product of rasagiline, and its analogues reversibly inhibit both MAO A and MAO B. The crystal structure of N-methyl-1(R)-aminoindan bound to MAO B shows that its aminoindan ring adopts a different orientation compared to that of rasagiline. PMID:16366596

  5. Structure–activity relationships of substituted oxyoxalamides as inhibitors of the human soluble epoxide hydrolase

    OpenAIRE

    Kim, In-Hae; Lee, In-Hee; Nishiwaki, Hisashi; Hammock, Bruce D.; Nishi, Kosuke

    2014-01-01

    We explored both structure–activity relationships among substituted oxyoxalamides used as the primary pharmacophore of inhibitors of the human sEH and as a secondary pharmacophore to improve water solubility of inhibitors. When the oxyoxalamide function was modified with a variety of alkyls or substituted alkyls, compound 6 with a 2-adamantyl group and a benzyl group was found to be a potent sEH inhibitor, suggesting that the substituted oxyoxalamide function is a promising primary pharmacoph...

  6. Purification and Characterization of Human Thrombin Activatable Fibrinolysis Inhibitor (TAFI)

    DEFF Research Database (Denmark)

    Christensen, Trine; Skottrup, Peter Durand; Valnickova, Zuzana

    Thrombin Activatable Fibrinolysis inhibitor (TAFI) is a basic carboxypeptidase, circulating in plasma as an enzymatic inactive precursor. TAFI shares ~40% overall sequence identity with pancreas Carboxypeptidase B (PCPB) with the activation peptide being less conserved. Following activation of TA...

  7. Inhibition of SRC-3 enhances sensitivity of human cancer cells to histone deacetylase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Zou, Zhengzhi, E-mail: zouzhengzhi@m.scnu.edu.cn [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510000 (China); Luo, Xiaoyong [Department of Oncology, The Affiliated Luoyang Central Hospital of Zhengzhou University, Luoyang 471000 (China); Nie, Peipei [KingMed Diagnostics and KingMed School of Laboratory Medicine, Guangzhou Medical University, Guangzhou 510000 (China); Wu, Baoyan; Zhang, Tao; Wei, Yanchun [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510000 (China); Wang, Wenyi [Xiamen Cancer Center, Department of Medical Oncology, The First Affiliated Hospital of Xiamen University, Xiamen 361000 (China); Geng, Guojun; Jiang, Jie [Xiamen Cancer Center, Department of Thoracic Surgery, The First Affiliated Hospital of Xiamen University, Xiamen 361000 (China); Mi, Yanjun, E-mail: myjgj_77@163.com [Xiamen Cancer Center, Department of Medical Oncology, The First Affiliated Hospital of Xiamen University, Xiamen 361000 (China)

    2016-09-09

    SRC-3 is widely expressed in multiple tumor types and involved in cancer cell proliferation and apoptosis. Histone deacetylase (HDAC) inhibitors are promising antitumor drugs. However, the poor efficacy of HDAC inhibitors in solid tumors has restricted its further clinical application. Here, we reported the novel finding that depletion of SRC-3 enhanced sensitivity of breast and lung cancer cells to HDAC inhibitors (SAHA and romidepsin). In contrast, overexpression of SRC-3 decreased SAHA-induced cancer cell apoptosis. Furthermore, we found that SRC-3 inhibitor bufalin increased cancer cell apoptosis induced by HDAC inhibitors. The combination of bufalin and SAHA was particular efficient in attenuating AKT activation and reducing Bcl-2 levels. Taken together, these accumulating data might guide development of new breast and lung cancer therapies. - Highlights: • Depletion of SRC-3 enhanced sensitivity of breast and lung cancer cells to HDAC inhibitors. • Overexpression of SRC-3 enhanced cancer cell resistance to HDAC inhibitors. • SRC-3 inhibitor bufalin increased cancer cell apoptosis induced by HDAC inhibitors. • Bufalin synergized with HDAC inhibitor attenuated AKT activation and reduced Bcl-2 levels in human cancer cell.

  8. Cytogenetic study of Ascaris trypsin inhibitor in cultured human ...

    Indian Academy of Sciences (India)

    2009-04-01

    Apr 1, 2009 ... The trypsin inhibitor (ATI) isolated from gastrointestinal nematode Ascaris suum was tested in vitro for induction of chro- mosome aberrations and ... and 72 h test of SCE) with exogenous metabolic activation. ATI was tested in dose ... atogenically on white leghorn chick embryo (Blaszkowska. 1998a, 2001).

  9. Determination of activities of human carbonic anhydrase II inhibitors ...

    African Journals Online (AJOL)

    Purpose: To evaluate the activities of new curcumin analogs as carbonic anhydrase II (CA-II) inhibitor. Methods: Carbonic anhydrase II (CA-II) inhibition was determined by each ligand capability to inhibit the esterase activity of CA-II using 4-NPA as a substrate in 96-well plates. Dimethyl sulfoxide was used to dissolve each ...

  10. 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....

  11. Structure-activity relationships of substituted oxyoxalamides as inhibitors of the human soluble epoxide hydrolase.

    Science.gov (United States)

    Kim, In-Hae; Lee, In-Hee; Nishiwaki, Hisashi; Hammock, Bruce D; Nishi, Kosuke

    2014-02-01

    We explored both structure-activity relationships among substituted oxyoxalamides used as the primary pharmacophore of inhibitors of the human sEH and as a secondary pharmacophore to improve water solubility of inhibitors. When the oxyoxalamide function was modified with a variety of alkyls or substituted alkyls, compound 6 with a 2-adamantyl group and a benzyl group was found to be a potent sEH inhibitor, suggesting that the substituted oxyoxalamide function is a promising primary pharmacophore for the human sEH, and compound 6 can be a novel lead structure for the development of further improved oxyoxalamide or other related derivatives. In addition, introduction of substituted oxyoxalamide to inhibitors with an amide or urea primary pharmacophore produced significant improvements in inhibition potency and water solubility. In particular, the N,N,O-trimethyloxyoxalamide group in amide or urea inhibitors (26 and 31) was most effective among those tested for both inhibition and solubility. The results indicate that substituted oxyoxalamide function incorporated into amide or urea inhibitors is a useful secondary pharmacophore, and the resulting structures will be an important basis for the development of bioavailable sEH inhibitors. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Identification of a specific inhibitor for DNA ligase I in human cells.

    OpenAIRE

    Yang, S W; Becker, F F; Chan, J Y

    1992-01-01

    A protein inhibitor for human DNA ligase I has recently been identified. It was copurified with a fraction of the enzymes from HeLa cells through several steps of chromatography. The inhibitor was first identified by the absence of ligation activity of the associated enzyme, while it retained the ability to form the ligase-[32P]AMP adducts. The inhibitor was eluted as a single peak at approximately 0.25-0.30 M NaCl from a Mono S column. It inhibited the ligation of both double-stranded and si...

  13. Co-expression of BMPs and BMP-inhibitors in human fractures and non-unions.

    Science.gov (United States)

    Kloen, Peter; Lauzier, Dominique; Hamdy, Reggie C

    2012-07-01

    Bone morphogenetic proteins (BMPs) are increasingly being used clinically to enhance fracture repair and healing of non-unions. However, the potential efficacy of supraphysiological dosing for clinical results warrants further clarification of the BMP signaling pathway in human fracture healing. As BMP signaling can be fine-tuned at numerous levels, the role of BMP-inhibitors has become a major focus. The aim of the present study was to document co-expression of BMPs, pSmad 1/5/8, and BMP-inhibitors in human fracture callus and human non-unions. Using human tissue of fracture callus (n=14) and non-unions (n=4) we documented expression of BMPs (BMP2, BMP3 and BMP7), pSmad 1/5/8 and the BMP-inhibitors noggin, gremlin, chordin, Smad-6, Smad-7 and BAMBI. Co-expression of pSmad 1/5/8, BMPs and BMP-inhibitors was noted in the osteoblasts of fracture callus as well as of non-unions. Expression of BMP-inhibitors was generally stronger in non-unions than in fracture callus. The most pertinent differences were noted in the cartilaginous tissue components. Expression of BMP2 in chondrocytes was markedly decreased in non-unions compared to fracture callus and that of BMP7 was almost completely absent. Expression of BMP-inhibitors was almost the same in osteoblasts, chondrocytes and fibroblasts of fracture callus and well as in non-unions. Interestingly, although BMP ligands were present in the chondrocytes and fibroblasts of non-unions, they did not co-express pSmad 1/5/8 suggesting that BMP signaling may have been inhibited at some point before Smad 1/5/8 phosphorylation. These results suggest co-expression of BMP, pSmad 1/5/8 and BMP-inhibitors occurs in human fracture callus as well as non-unions but the relative expression of BMPs vs. BMP-inhibitors was different between these two tissue types. In contrast to our expectations, the expression of BMP inhibitors was comparable between fracture callus and non-unions, whereas the expression of BMPs was notably lower in the

  14. Analysis of Gene Expression in Human Dermal Fibroblasts Treated with Senescence-Modulating COX Inhibitors

    Directory of Open Access Journals (Sweden)

    Jeong A. Han

    2017-06-01

    Full Text Available We have previously reported that NS-398, a cyclooxygenase-2 (COX-2–selective inhibitor, inhibited replicative cellular senescence in human dermal fibroblasts and skin aging in hairless mice. In contrast, celecoxib, another COX-2–selective inhibitor, and aspirin, a non-selective COX inhibitor, accelerated the senescence and aging. To figure out causal factors for the senescence-modulating effect of the inhibitors, we here performed cDNA microarray experiment and subsequent Gene Set Enrichment Analysis. The data showed that several senescence-related gene sets were regulated by the inhibitor treatment. NS-398 up-regulated gene sets involved in the tumor necrosis factor β receptor pathway and the fructose and mannose metabolism, whereas it down-regulated a gene set involved in protein secretion. Celecoxib up-regulated gene sets involved in G2M checkpoint and E2F targets. Aspirin up-regulated the gene set involved in protein secretion, and down-regulated gene sets involved in RNA transcription. These results suggest that COX inhibitors modulate cellular senescence by different mechanisms and will provide useful information to understand senescence-modulating mechanisms of COX inhibitors.

  15. Trypsin inhibitor complexes with human and bovine serum albumins: TEM and spectroscopic analysis.

    Science.gov (United States)

    Hebia, C; Bekale, L; Chanphai, P; Agbebavi, J; Tajmir-Riahi, H A

    2014-01-05

    We report the binding of trypsin inhibitor (TI) with human serum albumin (HSA) and bovine serum albumin (BSA) at physiological conditions, using FTIR, CD, UV-Visible spectroscopic methods and transmission electron microscopy (TEM). Structural analysis showed that trypsin inhibitor binds HSA and BSA via hydrophilic and hydrophobic contacts with overall binding constants of KTI-HSA=1.4 (±0.5)×10(4)M(-1) and KTI-BSA=1.1 (±0.4)×10(6)M(-1). Trypsin inhibitor complexation induces minor reduction of the protein α-helix and a major increase in β-sheet structure. TEM images show that trypsin inhibitor complex formation leads to the protein aggregation and fibrillation. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. 2-Aminoimidazole Amino Acids as Inhibitors of the Binuclear Manganese Metalloenzyme Human Arginase I

    Energy Technology Data Exchange (ETDEWEB)

    Ilies, M.; Di Costanzo, L; North, M; Scott, J; Christianson, D

    2010-01-01

    Arginase, a key metalloenzyme of the urea cycle that converts L-arginine into L-ornithine and urea, is presently considered a pharmaceutical target for the management of diseases associated with aberrant L-arginine homeostasis, such as asthma, cardiovascular diseases, and erectile dysfunction. We now report the design, synthesis, and evaluation of a series of 2-aminoimidazole amino acid inhibitors in which the 2-aminoimidazole moiety serves as a guanidine mimetic. These compounds represent a new class of arginase inhibitors. The most potent inhibitor identified in this study, 2-(S)-amino-5-(2-aminoimidazol-1-yl)pentanoic acid (A1P, 10), binds to human arginase I with K{sub d} = 2 {micro}M and significantly attenuates airways hyperresponsiveness in a murine model of allergic airways inflammation. These findings suggest that 2-aminoimidazole amino acids represent new leads for the development of arginase inhibitors with promising pharmacological profiles.

  17. Mapping of inhibitors and activity data to the human kinome and exploring promiscuity from a ligand and target perspective.

    Science.gov (United States)

    Hu, Ye; Kunimoto, Ryo; Bajorath, Jürgen

    2017-06-01

    An up-to-date collection of publicly available kinase inhibitors and activity data was mapped to the human kinome to comprehensively analyze current small molecule-kinase interactions. Compound distributions across the kinome were explored, structural relationships between inhibitors determined, and the tendency to form activity cliffs assessed. Furthermore, promiscuity was analyzed at the level of inhibitors and kinases, and a number of kinase targets with distinct preferences for single- or multitarget inhibitors were identified. Taken together, the results of current analysis provide a detailed view of kinase-inhibitor interaction characteristics across the human kinome. © 2016 John Wiley & Sons A/S.

  18. Synergistic interactions between HDAC and sirtuin inhibitors in human leukemia cells.

    Science.gov (United States)

    Cea, Michele; Soncini, Debora; Fruscione, Floriana; Raffaghello, Lizzia; Garuti, Anna; Emionite, Laura; Moran, Eva; Magnone, Mirko; Zoppoli, Gabriele; Reverberi, Daniele; Caffa, Irene; Salis, Annalisa; Cagnetta, Antonia; Bergamaschi, Micaela; Casciaro, Salvatore; Pierri, Ivana; Damonte, Gianluca; Ansaldi, Filippo; Gobbi, Marco; Pistoia, Vito; Ballestrero, Alberto; Patrone, Franco; Bruzzone, Santina; Nencioni, Alessio

    2011-01-01

    Aberrant histone deacetylase (HDAC) activity is frequent in human leukemias. However, while classical, NAD(+)-independent HDACs are an established therapeutic target, the relevance of NAD(+)-dependent HDACs (sirtuins) in leukemia treatment remains unclear. Here, we assessed the antileukemic activity of sirtuin inhibitors and of the NAD(+)-lowering drug FK866, alone and in combination with traditional HDAC inhibitors. Primary leukemia cells, leukemia cell lines, healthy leukocytes and hematopoietic progenitors were treated with sirtuin inhibitors (sirtinol, cambinol, EX527) and with FK866, with or without addition of the HDAC inhibitors valproic acid, sodium butyrate, and vorinostat. Cell death was quantified by propidium iodide cell staining and subsequent flow-cytometry. Apoptosis induction was monitored by cell staining with FITC-Annexin-V/propidium iodide or with TMRE followed by flow-cytometric analysis, and by measuring caspase3/7 activity. Intracellular Bax was detected by flow-cytometry and western blotting. Cellular NAD(+) levels were measured by enzymatic cycling assays. Bax was overexpressed by retroviral transduction. Bax and SIRT1 were silenced by RNA-interference. Sirtuin inhibitors and FK866 synergistically enhanced HDAC inhibitor activity in leukemia cells, but not in healthy leukocytes and hematopoietic progenitors. In leukemia cells, HDAC inhibitors were found to induce upregulation of Bax, a pro-apoptotic Bcl2 family-member whose translocation to mitochondria is normally prevented by SIRT1. As a result, leukemia cells become sensitized to sirtuin inhibitor-induced apoptosis. In conclusion, NAD(+)-independent HDACs and sirtuins cooperate in leukemia cells to avoid apoptosis. Combining sirtuin with HDAC inhibitors results in synergistic antileukemic activity that could be therapeutically exploited.

  19. Synergistic interactions between HDAC and sirtuin inhibitors in human leukemia cells.

    Directory of Open Access Journals (Sweden)

    Michele Cea

    Full Text Available Aberrant histone deacetylase (HDAC activity is frequent in human leukemias. However, while classical, NAD(+-independent HDACs are an established therapeutic target, the relevance of NAD(+-dependent HDACs (sirtuins in leukemia treatment remains unclear. Here, we assessed the antileukemic activity of sirtuin inhibitors and of the NAD(+-lowering drug FK866, alone and in combination with traditional HDAC inhibitors. Primary leukemia cells, leukemia cell lines, healthy leukocytes and hematopoietic progenitors were treated with sirtuin inhibitors (sirtinol, cambinol, EX527 and with FK866, with or without addition of the HDAC inhibitors valproic acid, sodium butyrate, and vorinostat. Cell death was quantified by propidium iodide cell staining and subsequent flow-cytometry. Apoptosis induction was monitored by cell staining with FITC-Annexin-V/propidium iodide or with TMRE followed by flow-cytometric analysis, and by measuring caspase3/7 activity. Intracellular Bax was detected by flow-cytometry and western blotting. Cellular NAD(+ levels were measured by enzymatic cycling assays. Bax was overexpressed by retroviral transduction. Bax and SIRT1 were silenced by RNA-interference. Sirtuin inhibitors and FK866 synergistically enhanced HDAC inhibitor activity in leukemia cells, but not in healthy leukocytes and hematopoietic progenitors. In leukemia cells, HDAC inhibitors were found to induce upregulation of Bax, a pro-apoptotic Bcl2 family-member whose translocation to mitochondria is normally prevented by SIRT1. As a result, leukemia cells become sensitized to sirtuin inhibitor-induced apoptosis. In conclusion, NAD(+-independent HDACs and sirtuins cooperate in leukemia cells to avoid apoptosis. Combining sirtuin with HDAC inhibitors results in synergistic antileukemic activity that could be therapeutically exploited.

  20. ROCK inhibitor enhances adhesion and wound healing of human corneal endothelial cells.

    Directory of Open Access Journals (Sweden)

    Aurélien Pipparelli

    Full Text Available Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and "pump" functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34, the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.

  1. Recent progress in the discovery of natural inhibitors against human carboxylesterases.

    Science.gov (United States)

    Wang, Dan-Dan; Zou, Li-Wei; Jin, Qiang; Hou, Jie; Ge, Guang-Bo; Yang, Ling

    2017-03-01

    Mammalian carboxylesterases (CEs) are important serine hydrolases catalyzing the hydrolysis of ester- or amide-containing compounds into the corresponding alcohols and carboxylic acids. In human, two primary carboxylesterases including hCE1 and hCE2 have been identified and extensively studied in the past decade. hCE1 is known to play crucial roles in the metabolism of a wide variety of endogenous esters, clinical drugs and insecticides, while hCE2 plays a key role in the metabolic activation of anticancer agents including irinotecan and capecitabine. The key roles of hCEs in both human health and xenobiotic metabolism arouse great interest in the discovery of potent and selective hCEs inhibitors to modulate endobiotic metabolism or to improve the outcomes of patients administrated with ester drugs. This review covers the significance and recent progress in the discovery of natural inhibitors against hCEs. The tools for screening and characterization of inhibitors against human CEs, including traditional LC-based approaches and the newly developed optical substrate-based assays, are summarized and discussed for the first time. Furthermore, the structural information and inhibitory capacities of all reported hCEs inhibitors including fatty acids, flavonoids, tanshinones and triterpenoids have been systematically summarized. All information and knowledge presented in this review will be very helpful for medicinal chemists to develop more potent and highly selective inhibitors against hCEs for potential biomedical applications. Copyright © 2017. Published by Elsevier B.V.

  2. Cytogenetic study of Ascaris trypsin inhibitor in cultured human ...

    Indian Academy of Sciences (India)

    2009-04-01

    Apr 1, 2009 ... Lee D. E. and Xie C. Y. 1995 IgE regulation by nematodes: the body fluid of Ascaris contains a B-cell mitogen. J. Allergy Clin. Immunol. 96, 1246–1254. Lipski J. J., Berninger R. W., Hyman L. R. and Talama R. C. 1979. Presence of alpha-1-antitrypsin on mitogen-stimulated human lymphocytes. J. Immunol.

  3. Effect of the human follicle-stimulating hormone-binding inhibitor ...

    Indian Academy of Sciences (India)

    The follicle-stimulating hormone (FSH)-binding inhibitor (FSHBI), purified by our laboratory from human ovarian follicular fluid, has been shown to suppress ovulation and induce follicular atresia/apoptosis in mice as well as impair fertility in marmosets, the new world monkeys. The octapeptide, a peptide corresponding to ...

  4. Synthesis and biological evaluation of NAD analogs as human pyridine nucleotide adenylyltransferase inhibitors.

    Science.gov (United States)

    Franchetti, Palmarisa; Petrelli, R; Cappellacci, L; Pasqualini, M; Vita, P; Sorci, L; Mazzola, F; Raffaelli, N; Magni, Giulio

    2005-01-01

    NAD analogs modified at the ribose adenylyl moiety, named N-2'-MeAD and Na-2'-MeAD, were synthesized as ligands of pyridine nucleotide (NMN/NaMN) adenylyltransferase (NMNAT). Both dinucleotides resulted selective inhibitors against human NMNAT-3 isoenzyme.

  5. Cellular inhibitor of apoptosis protein 2 controls human colonic epithelial restitution, migration, and Rac1 activation

    DEFF Research Database (Denmark)

    Seidelin, Jakob Benedict; Larsen, Sylvester; Linnemann, Dorte

    2015-01-01

    Identification of pathways involved in wound healing is important for understanding the pathogenesis of various intestinal diseases. Cellular inhibitor of apoptosis protein 2 (cIAP2) regulates proliferation and migration in nonepithelial cells and is expressed in human colonocytes. The aim...

  6. A patent review on the development of human cytochrome P450 inhibitors.

    Science.gov (United States)

    Francis, Sheena; Delgoda, Rupika

    2014-06-01

    CYP, a ubiquitous superfamily of enzymes expressed in major organs in humans, plays a key role in biosynthesis of steroids and metabolism of xenobiotics. Inhibitors of these vital enzymes provide, as tools, the opportunity to gain an insight to their role in a myriad of bioactivity and to intervene as therapeutics in disease. This article reviews granted patents for human CYP inhibitors from the US and European territories within the past decade. Granted patents, albeit mostly embodying evidence from in vitro and limited preclinical trials, demonstrate good potential for use in industry and the clinic following future human trials. Indeed, only a handful is on the market or under clinical evaluation. Diagnostic monoclonal antibodies (mAbs) show high specificity for CYP families 1, 2, and 3, while potent inhibitors of CYPs 17, 19, 24, 26, 3A4 activities, in use with or without other drugs, display potential in treating prostate and breast cancers, dermatology, and improved retroviral therapy, although some may have challenges in delivery to target tissues. The involvement of this superfamily of enzymes in cellular functions, a multitude of disease states, and pharmacogenetics make them ideal candidates to better understand contemporary human health issues and identification of targeted, specific, and potent inhibitors is a useful strategy to employ, toward achieving that wider goal.

  7. Molecular modeling study for inhibition mechanism of human chymase and its application in inhibitor design.

    Directory of Open Access Journals (Sweden)

    Mahreen Arooj

    Full Text Available Human chymase catalyzes the hydrolysis of peptide bonds. Three chymase inhibitors with very similar chemical structures but highly different inhibitory profiles towards the hydrolase function of chymase were selected with the aim of elucidating the origin of disparities in their biological activities. As a substrate (angiotensin-I bound crystal structure is not available, molecular docking was performed to dock the substrate into the active site. Molecular dynamics simulations of chymase complexes with inhibitors and substrate were performed to calculate the binding orientation of inhibitors and substrate as well as to characterize conformational changes in the active site. The results elucidate details of the 3D chymase structure as well as the importance of K40 in hydrolase function. Binding mode analysis showed that substitution of a heavier Cl atom at the phenyl ring of most active inhibitor produced a great deal of variation in its orientation causing the phosphinate group to interact strongly with residue K40. Dynamics simulations revealed the conformational variation in region of V36-F41 upon substrate and inhibitor binding induced a shift in the location of K40 thus changing its interactions with them. Chymase complexes with the most active compound and substrate were used for development of a hybrid pharmacophore model which was applied in databases screening. Finally, hits which bound well at the active site, exhibited key interactions and favorable electronic properties were identified as possible inhibitors for chymase. This study not only elucidates inhibitory mechanism of chymase inhibitors but also provides key structural insights which will aid in the rational design of novel potent inhibitors of the enzyme. In general, the strategy applied in the current study could be a promising computational approach and may be generally applicable to drug design for other enzymes.

  8. Regulation of human skin mast cell histamine release by PDE inhibitors.

    Science.gov (United States)

    Eskandari, N; Bastan, R; Peachell, P T

    2015-01-01

    Mast cell and basophiles are thought to be central to inflammation that has an allergic basis as allergens activate these cells in an IgE-dependent manner to generate mediators such as histamine, eicosanoids and cytokines. Phosphodiesterase (PDE) is known to exist as multiple molecular forms of enzyme that metabolise the second messengers. Studies of our own have shown that, of a variety of isoform-selective drugs, the PDE4-selective inhibitors, such as rolipram, attenuate the IgE-mediated release of histamine from human basophiles but not from human lung mast cells (HLMC). The main aim of the present study was to characterise the type and role of PDEs regulating human skin mast cells by using selective and non-selective PDE inhibitors. Cells were pre-treated for 15 min with these agents and then challenged with an optimal releasing concentration of anti IgE (1:300) for a further 25 min for the release of histamine. The data show that all the selective PDE-inhibitor compounds (10(-5)M) were ineffective whereas the non-selective PDE inhibitor, theophylline (10(-3)M), inhibited histamine release from HSMC (74 ± 4% inhibition; peffect on histamine release from HLMC whereas, in basophiles, compounds with activity at PDE 4 (rolipram, denbufylline, Ro-2017, Org 30029) were effective inhibitors of histamine release. The data suggest that unlike most inflammatory cells, PDE-selective inhibitors are ineffective stabilisers of HSMC activity which is similar to HLMC. Copyright © 2013 SEICAP. Published by Elsevier Espana. All rights reserved.

  9. Identification of the first highly selective inhibitor of human GABA transporter GAT3

    DEFF Research Database (Denmark)

    Damgaard, Maria; Al-Khawaja, Anas; Vogensen, Stine B.

    2015-01-01

    Screening a library of small-molecule compounds using a cell line expressing human GABA transporter 3 (hGAT3) in a [(3)H]GABA uptake assay identified isatin derivatives as a new class of hGAT3 inhibitors. A subsequent structure-activity relationship (SAR) study led to the identification of hGAT3-...... site that matched the observed selectivity, inhibition kinetics, and SAR of the compound series. These compounds are the most potent GAT3 inhibitors reported to date that provide selectivity for GAT3 over other GABA transporter subtypes....

  10. Crystal Structure of Inhibitor-Bound Human 5-lipoxygenase-activating Protein

    Energy Technology Data Exchange (ETDEWEB)

    Ferguson,A.; McKeever, B.; Xu, S.; Wisniewski, D.; Miller, D.; Yamin, T.; Spencer, R.; Chu, L.; Ujjainwalla, F.; et al.

    2007-01-01

    Leukotrienes are proinflammatory products of arachidonic acid oxidation by 5-lipoxygenase that have been shown to be involved in respiratory and cardiovascular diseases. The integral membrane protein FLAP is essential for leukotriene biosynthesis. We describe the x-ray crystal structures of human FLAP in complex with two leukotriene biosynthesis inhibitors at 4.0 and 4.2 angstrom resolution, respectively. The structures show that inhibitors bind in membrane-embedded pockets of FLAP, which suggests how these inhibitors prevent arachidonic acid from binding to FLAP and subsequently being transferred to 5-lipoxygenase, thereby preventing leukotriene biosynthesis. This structural information provides a platform for the development of therapeutics for respiratory and cardiovascular diseases.

  11. A novel histone deacetylase inhibitor Chidamide induces apoptosis of human colon cancer cells

    International Nuclear Information System (INIS)

    Liu, Lin; Chen, Baoan; Qin, Shukui; Li, Suyi; He, Xiangming; Qiu, Shaomin; Zhao, Wei; Zhao, Hong

    2010-01-01

    Many studies have demonstrated that histone deacetylase (HDAC) inhibitors induce various tumor cells to undergo apoptosis, and such inhibitors have been used in different clinical trials against different human cancers. In this study, we designed and synthesized a novel HDAC inhibitor, Chidamide. We showed that Chidamide was able to increase the acetylation levels of histone H3 and to inhibit the PI3K/Akt and MAPK/Ras signaling pathways, which resulted in arresting colon cancer cells at the G1 phase of the cell cycle and promoting apoptosis. As a result, the proliferation of colon cancer cells was suppressed in vitro. Our data support the potential application of Chidamide as an anticancer agent in treating colon cancer. Future studies are needed to demonstrate its in vivo efficacy.

  12. Computational drug design strategies applied to the modelling of human immunodeficiency virus-1 reverse transcriptase inhibitors

    Directory of Open Access Journals (Sweden)

    Lucianna Helene Santos

    2015-11-01

    Full Text Available Reverse transcriptase (RT is a multifunctional enzyme in the human immunodeficiency virus (HIV-1 life cycle and represents a primary target for drug discovery efforts against HIV-1 infection. Two classes of RT inhibitors, the nucleoside RT inhibitors (NRTIs and the nonnucleoside transcriptase inhibitors are prominently used in the highly active antiretroviral therapy in combination with other anti-HIV drugs. However, the rapid emergence of drug-resistant viral strains has limited the successful rate of the anti-HIV agents. Computational methods are a significant part of the drug design process and indispensable to study drug resistance. In this review, recent advances in computer-aided drug design for the rational design of new compounds against HIV-1 RT using methods such as molecular docking, molecular dynamics, free energy calculations, quantitative structure-activity relationships, pharmacophore modelling and absorption, distribution, metabolism, excretion and toxicity prediction are discussed. Successful applications of these methodologies are also highlighted.

  13. Azobenzene-based inhibitors of human carbonic anhydrase II

    Directory of Open Access Journals (Sweden)

    Leander Simon Runtsch

    2015-07-01

    Full Text Available Aryl sulfonamides are a widely used drug class for the inhibition of carbonic anhydrases. In the context of our program of photochromic pharmacophores we were interested in the exploration of azobenzene-containing sulfonamides to block the catalytic activity of human carbonic anhydrase II (hCAII. Herein, we report the synthesis and in vitro evaluation of a small library of nine photochromic sulfonamides towards hCAII. All molecules are azobenzene-4-sulfonamides, which are substituted by different functional groups in the 4´-position and were characterized by X-ray crystallography. We aimed to investigate the influence of electron-donating or electron-withdrawing substituents on the inhibitory constant Ki. With the aid of an hCAII crystal structure bound to one of the synthesized azobenzenes, we found that the electronic structure does not strongly affect inhibition. Taken together, all compounds are strong blockers of hCAII with Ki = 25–65 nM that are potentially photochromic and thus combine studies from chemical synthesis, crystallography and enzyme kinetics.

  14. Crystal structure of two new bifunctional nonsubstrate type thrombin inhibitors complexed with human alpha-thrombin.

    Science.gov (United States)

    Féthière, J; Tsuda, Y; Coulombe, R; Konishi, Y; Cygler, M

    1996-06-01

    The crystal structures of two new thrombin inhibitors, P498 and P500, complexed with human alpha-thrombin have been determined at 2.0 A resolution and refined to crystallographic R-factors of 0.170 and 0.169, respectively. These compounds, with picomolar binding constants, belong to a family of potent bifunctional inhibitors that bind thrombin at two remote sites: the active site and the fibrinogen recognition exosite (FRE). The inhibitors incorporate a nonsubstrate type active site binding fragment: Dansyl-Arg-(D)Pipecolic acid (Dns-Arg-(D)Pip), reminiscent of the active-site directed inhibitors MD-805 and MQPA, rendering them resistant to thrombin-induced hydrolysis. The FRE binding fragment of these inhibitors corresponds to the hirudin55-65 sequence. They differ in the chemical nature of the nonpeptidyl linker bridging these two functional activities. In both cases, the active site binding fragment is well defined in the electron density. The DnsH1, ArgH2, and (D)PipH3 groups occupy the S3, S1, and S2 subsites of thrombin, respectively, in a way similar to that observed in the thrombin-MQPA complexes. Binding in the active site of thrombin is characterized by numerous van der Waals contacts and ring-ring system interactions. Unlike in the substrate-like inhibitors, ArgH2 enters the S1 specificity pocket from the P2 position and adopts a bent conformation to make an hydrogen bond to the carboxylate of Asp189. In this noncanonical position, its carbonyl points away from the oxyanion hole, which is now occupied by well-ordered solvent molecules. The linkers fit in the groove extending from the active site to the FRE. The C-terminal fragments of both inhibitors bind in the same way as analogous FRE binding elements in previously described complexes.

  15. Bioassays and in silico methods in the identification of human DNA topoisomerase IIa inhibitors.

    Science.gov (United States)

    Bergant, Kaja; Janezic, Matej; Perdih, Andrej

    2018-03-06

    The family of DNA topoisomerases comprises a group of enzymes that catalyse the induction of topological changes to DNA. These enzymes play a role in the cell replication machinery and are, therefore, important targets for anticancer drugs - with human DNA topoisomerase IIα being one of the most prominent. Active compounds targeting this enzyme are classified into two groups with diverse mechanisms of action: DNA poisons act by stabilizing a covalent cleavage complex between DNA and the topoisomerase enzyme, transforming it into a cellular toxin, while the second diverse group of catalytic inhibitors, provides novel inhibition avenues for tackling this enzyme due to frequent occurrence of side effects observed during the DNA poison therapy. Based on a comprehensive literature search we present an overview of available bioassays and in silico methods in the identification of human DNA topoisomerase IIα inhibitors. A comprehensive outline of the available methods and approaches that explore in detail the in vitro mechanistic and functional aspects of the topoisomerase IIα inhibition of both topo IIα inhibitor groups is presented. The utilized in vitro cell-based assays and in vivo studies to further explore the validated topo IIα inhibitors in subsequent preclinical stages of the drug discovery are discussed. The potential of in silico methods in topoisomerase IIα inhibitor discovery is outlined. A list of practical guidelines was compiled to aid new as well experienced researchers in how to optimally approach the design of targeted inhibitors and validation in the preclinical drug development stages. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. HOMOLOGY MODELING AND DOCKING STUDIES OF HUMAN CHITOTRIOSIDASE WITH ITS NATURAL INHIBITORS

    Directory of Open Access Journals (Sweden)

    Deepsikha Roy

    2015-06-01

    Full Text Available Chitinase inhibitors have been found to have anti-inflammatory potential against asthma, allergic diseases and various other disorders. In this study various naturally occurring chitinase inhibitors against human chitinase (chitotriosidases, CHIT1 were studied with the help of protein-ligand docking. The structure of CHIT1 was modeled by homology modeling tool and validated with the help of various computational tools. Following validation, secondary structure, function and solvent accessibility of the protein was analyzed. A molecular dynamics (MD simulation study was conducted by GROMACS simulation package to study the stability of the structure. This was further followed by docking studies with natural inhibitors like allosamidin, argifin and argadin against CHIT1 by GLIDE docking software. Argadin was observed to have the highest affinity (G-score =-10.955 towards CHIT1 and allosamidin scored the lowest GLIDE score (G-score =-7.741. The structural behavior of the best inhibitor protein complex (CHIT1- argadin was validated through molecular dynamics simulation studies. A structure based virtual screening on the basis of the binding modes of these inhibitor was performed and best scoring hits were identified. The sequence analysis can be further used for the designing of potent drugs against diseases caused by CHIT1, thereby aiding knowledge in the field of research

  17. Glucocorticoids and Polyamine Inhibitors Synergize to Kill Human Leukemic CEM Cells

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    Aaron L. Miller

    2002-01-01

    Full Text Available Glucocorticoids are well-known apoptotic agents in certain classes of lymphoid cell malignancies. Reduction of intracellular polyamine levels by use of inhibitors that block polyamine synthesis slows or inhibits growth of many cells in vitro. Several such inhibitors have shown efficacy in clinical trials, though the toxicity of some compounds has limited their usefulness. We have tested the effects of combinations of the glucocorticoid dexamethasone. (20Dex and two polyamine inhibitors, difluoromethylornithine. (20DFMO and methyl glyoxal bis guanylhydrazone. (20MGBG, on the clonal line of human acute lymphoblastic leukemia cells, CEM-C7-14. Dex alone kills these cells, though only after a delay of at least 24 hours. We also evaluated a partially glucocorticoid-resistant c-Myc-expressing CEM-C7-14 clone. We show that Dex downregulates ornithine decarboxylase. (20ODC, the rate-limiting enzyme in polyamine synthesis. Pretreatment with the ODC inhibitor DFMO, followed by addition of Dex, enhances steroid-evoked kill slightly. The combination of pretreatment with sublethal concentrations of both DFMO and the inhibitor of S-adenosylmethionine decarboxylase, MGBG, followed by addition of Dex, results in strong synergistic cell kill. Both the rapidity and extent of cell kill are enhanced compared to the effects of Dex alone. These results suggest that use of such combinations in vivo may result in apoptosis of malignant cells with lower overall toxicity.

  18. Glucocorticoids and Polyamine Inhibitors Synergize to Kill Human Leukemic CEM Cells1

    Science.gov (United States)

    Miller, Aaron L; Johnson, Betty H; Medh, Rheem D; Townsend, Courtney M; Thompson, E Brad

    2002-01-01

    Abstract Glucocorticoids are well-known apoptotic agents in certain classes of lymphoid cell malignancies. Reduction of intracellular polyamine levels by use of inhibitors that block polyamine synthesis slows or inhibits growth of many cells in vitro. Several such inhibitors have shown efficacy in clinical trials, though the toxicity of some compounds has limited their usefulness. We have tested the effects of combinations of the glucocorticoid dexamethasone (Dex) and two polyamine inhibitors, difluoromethylornithine (DFMO) and methyl glyoxal bis guanylhydrazone (MGBG), on the clonal line of human acute lymphoblastic leukemia cells, CEM-C7-14. Dex alone kills these cells, though only after a delay of at least 24 hours. We also evaluated a partially glucocorticoid-resistant c-Myc-expressing CEM-C7-14 clone. We show that Dex downregulates ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis. Pretreatment with the ODC inhibitor DFMO, followed by addition of Dex, enhances steroid-evoked kill slightly. The combination of pretreatment with sublethal concentrations of both DFMO and the inhibitor of S-adenosylmethionine decarboxylase, MGBG, followed by addition of Dex, results in strong synergistic cell kill. Both the rapidity and extent of cell kill are enhanced compared to the effects of Dex alone. These results suggest that use of such combinations in vivo may result in apoptosis of malignant cells with lower overall toxicity. PMID:11922393

  19. Zoniporide: a potent and highly selective inhibitor of human Na(+)/H(+) exchanger-1.

    Science.gov (United States)

    Marala, Ravi B; Brown, Janice A; Kong, Jimmy X; Tracey, W Ross; Knight, Delvin R; Wester, Ronald T; Sun, Dexue; Kennedy, Scott P; Hamanaka, Ernest S; Ruggeri, Roger B; Hill, Roger J

    2002-09-06

    We evaluated the in vitro pharmacological profile of a novel, potent and highly selective Na(+)/H(+) exchanger-1 (NHE-1) inhibitor, [1-(Quinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine hydrochloride monohydrate (zoniporide or CP-597,396). The potency and selectivity of zoniporide were determined via inhibition of 22Na(+) uptake by PS-120 fibroblast cell lines overexpressing human NHE-1, -2 or rat NHE-3. Additionally, potency for endogenous NHE-1 was confirmed via ex vivo human platelet swelling assay (PSA), in which platelet swelling was induced by exposure to sodium propionate. The pharmacological profile of zoniporide was compared with that of eniporide and cariporide. Zoniporide inhibited 22Na(+) uptake in fibroblasts expressing human NHE-1 in a concentration-dependent manner (IC(50) = 14 nM) and was highly selective (157-fold and 15,700-fold vs. human NHE-2 and rat NHE-3, respectively). Zoniporide was 1.64- to 2.6-fold more potent at human NHE-1 than either eniporide or cariporide (IC(50) = 23 and 36 nM, respectively). Zoniporide was also more selective at inhibiting human NHE-1 vs. human NHE-2 than either eniporide or cariporide (157-fold selective compared with 27- and 49-fold, respectively). All three compounds inhibited human platelet swelling with IC(50) values in low nanomolar range. From these results, we conclude that zoniporide represents a novel, potent and highly selective NHE-1 inhibitor. Copyright 2002 Elsevier Science B.V.

  20. Recombinant Human Plasminogen Activator Inhibitor-1 Accelerates Odontoblastic Differentiation of Human Stem Cells from Apical Papilla.

    Science.gov (United States)

    Jin, Bin; Choung, Pill-Hoon

    2016-05-01

    Dental caries, the most prevalent oral disease in dental patients, involves the phases of demineralization and destruction of tooth hard tissues like enamel, dentin, and cementum. Dentin is a major component of the root and is also the innermost layer that protects the tooth nerve, exposure of which results in pain. In this study, we used human stem cells from apical papilla (hSCAP), which are early progenitor cells, to examine the effects of recombinant human plasminogen activator inhibitor-1 (rhPAI-1) on odontogenic differentiation in vitro and in vivo. We demonstrated that rhPAI-1 promoted the proliferation and odontogenic differentiation of hSCAP and increased the expression levels of odontoblast-associated markers. We also observed that rhPAI-1 upregulated the expression of Smad4, nuclear factor I-C (NFI-C), Runx2, and osterix (OSX) during odontogenic differentiation. Notably, transplantation of rhPAI-1-treated hSCAP effectively induced odontoblastic differentiation and dentinal formation. And the differentiated odontoblast-like cells showed numerous odontoblast processes inserted in dentin tubules and arranged collagen fibers. Furthermore, odontoblast-associated markers were more highly expressed in the rhPAI-1-induced differentiated odontoblast-like cells compared with the control group. These markers were also more highly expressed in the newly formed dentin-like tissue of the rhPAI-1-treated group compared with the control group. Consistent with our in vitro results, the expression levels of Smad4, NFI-C, and OSX were also increased in the rhPAI-1-treated group compared with the control group. Taken together, these results suggest that rhPAI-1 promotes odontoblast differentiation and dentin formation of hSCAP, and Smad4/NFI-C/OSX may play critical roles in the rhPAI-1-induced odontogenic differentiation. Thus, dental stem cells from apical papilla combined with rhPAI-1 could lead to dentin regeneration in clinical implications.

  1. Purification and biochemical characterisation of human and murine stem cell inhibitors (SCI).

    Science.gov (United States)

    Graham, G J; Freshney, M G; Donaldson, D; Pragnell, I B

    1992-01-01

    We have recently characterised an inhibitor of haemopoietic stem cell proliferation (SCI/MIP-1 alpha) and report here on its purification and initial biological and biochemical characterisation. The activity can be detected by direct addition to the CFU-A stem cell assay and this simple test for inhibitory activity has greatly facilitated the purification of the molecule. The purification involves a combination of Mono Q ion exchange chromatography, heparin-sepharose affinity chromatography and Blue Sepharose affinity chromatography. The purified stem cell inhibitor is an 8 kD peptide which is identical to the previously described peptide macrophage inflammatory protein 1 alpha. The peptide has a natural tendency to form large self-aggregates and appears, in physiological buffers, to have a native molecular weight of around 90 kD. SCI is a heat stable, protease sensitive protein which is half maximally active at between 10 and 25 pM in the CFU-A assay. The self-aggregates can be disrupted by dilute solutions of acetic acid and it appears that disruption increases the specific activity of SCI preparations. We also report the characterisation of the human homologue of the stem cell inhibitor (human SCI/MIP-1 alpha) which is 74% identical to murine MIP-1 alpha and which shares all the above features of the murine inhibitor.

  2. Identification of lympho-epithelial Kazal-type inhibitor 2 in human skin as a kallikrein-related peptidase 5-specific protease inhibitor.

    Directory of Open Access Journals (Sweden)

    Ulf Meyer-Hoffert

    Full Text Available Kallikreins-related peptidases (KLKs are serine proteases and have been implicated in the desquamation process of the skin. Their activity is tightly controlled by epidermal protease inhibitors like the lympho-epithelial Kazal-type inhibitor (LEKTI. Defects of the LEKTI-encoding gene serine protease inhibitor Kazal type (Spink5 lead to the absence of LEKTI and result in the genodermatose Netherton syndrome, which mimics the common skin disease atopic dermatitis. Since many KLKs are expressed in human skin with KLK5 being considered as one of the most important KLKs in skin desquamation, we proposed that more inhibitors are present in human skin. Herein, we purified from human stratum corneum by HPLC techniques a new KLK5-inhibiting peptide encoded by a member of the Spink family, designated as Spink9 located on chromosome 5p33.1. This peptide is highly homologous to LEKTI and was termed LEKTI-2. Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin. Spink9 mRNA expression was detected in human skin samples and in cultured keratinocytes. LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5. At sites of plantar hyperkeratosis, LEKTI-2 expression was increased. We suggest that LEKTI-2 contributes to the regulation of the desquamation process in human skin by specifically inhibiting KLK5.

  3. Apoptosis inhibitor 5 (API-5; AAC-11; FIF) is upregulated in human carcinomas in vivo

    Czech Academy of Sciences Publication Activity Database

    Kočí, Lenka; Chlebová, K.; Hýžďalová, Martina; Hofmanová, Jiřina; Jíra, M.; Kysela, P.; Kozubík, Alois; Kala, Z.; Krejčí, Pavel

    2012-01-01

    Roč. 3, č. 4 (2012), s. 913-916 ISSN 1792-1074 R&D Projects: GA ČR(CZ) GA305/09/1526; GA ČR(CZ) GD303/09/H048; GA ČR(CZ) GAP301/11/1730 Institutional research plan: CEZ:AV0Z50040702 Keywords : apoptosis inhibitor 5 * apoptosis * human carcinoma Subject RIV: BO - Biophysics Impact factor: 0.237, year: 2012

  4. Recombinant human C1-inhibitor prevents acute antibody-mediated rejection in alloimmunized baboons.

    Science.gov (United States)

    Tillou, Xavier; Poirier, Nicolas; Le Bas-Bernardet, Stéphanie; Hervouet, Jeremy; Minault, David; Renaudin, Karine; Vistoli, Fabio; Karam, Georges; Daha, Mohamed; Soulillou, Jean Paul; Blancho, Gilles

    2010-07-01

    Acute antibody-mediated rejection is an unsolved issue in transplantation, especially in the context of pretransplant immunization. The deleterious effect of preformed cytotoxic anti-HLA antibodies through complement activation is well proven, but very little is known concerning complement blockade to prevent/cure this rejection. Here, we used a baboon model of preimmunization to explore the prevention of acute antibody-mediated rejection by an early inhibition of the classical complement pathway using human recombinant C1-inhibitor. Baboons were immunized against peripheral blood mononuclear cells from allogeneic donors and, once a specific and stable immunization had been established, they received a kidney from the same donor. Rejection occurred at day 2 posttransplant in untreated presensitized recipients, with characteristic histological lesions and complement deposition. As recombinant human C1-inhibitor blocks in vitro cytotoxicity induced by donor-specific antibodies, other alloimmunized baboons received the drug thrice daily intravenously during the first 5 days after transplant. Rejection was prevented during this treatment but occurred after discontinuation of treatment. We show here that early blockade of complement activation by recombinant human C1-inhibitor can prevent acute antibody-mediated rejection in presensitized recipients. This treatment could also be useful in other forms of acute antibody-mediated rejection caused by induced antibodies.

  5. Mechanism-based Inhibitors of the Human Sirtuin 5 Deacylase: Structure-Activity Relationship, Biostructural, and Kinetic Insight

    DEFF Research Database (Denmark)

    Rajabi, Nima; Auth, Marina; Troelsen, Kathrin Rentzius

    2017-01-01

    to date. We provide rationalization of the mode of binding by solving co-crystal structures of selected inhibitors in complex with both human and zebrafish SIRT5, which provide insight for future optimization of inhibitors with more "drug-like" properties. Importantly, enzyme kinetic evaluation revealed...

  6. Discovery of Potent and Selective Inhibitors of Human Platelet type 12-Lipoxygenase

    Science.gov (United States)

    Kenyon, Victor; Rai, Ganesha; Jadhav, Ajit; Schultz, Lena; Armstrong, Michelle; Jameson, J. Brian; Perry, Steven; Joshi, Netra; Bougie, James M.; Leister, William; Taylor-Fishwick, David A.; Nadler, Jerry L.; Holinstat, Michael; Simeonov, Anton; Maloney, David J.; Holman, Theodore R.

    2011-01-01

    We report the discovery of novel small molecule inhibitors of platelet type 12-human lipoxygenase, which display nanomolar activity against the purified enzyme, using a quantitative high throughput screen (qHTS) on a library of 153,607 compounds. These compounds also exhibit excellent specificity, >50-fold selectivity vs. the paralogs, 5-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity vs. ovine cyclooxygenase-1 and human cyclooxygenase-2. Kinetic experiments indicate this chemotype is a non-competitive inhibitor that does not reduce the active site iron. Moreover, chiral HPLC separation of two of the racemic lead molecules revealed a strong preference for the (–)-enantiomers (IC50 of 0.43 +/- 0.04 and 0.38 +/- 0.05 μM) compared to the (+)-enantiomers (IC50 of >25 μM for both), indicating a fine degree of selectivity in the active site due to chiral geometry. In addition, these compounds demonstrate efficacy in cellular models, which underscores their relevance to disease modification. PMID:21739938

  7. FDA-approved drugs and other compounds tested as inhibitors of human glutathione transferase P1-1.

    Science.gov (United States)

    Musdal, Yaman; Hegazy, Usama M; Aksoy, Yasemin; Mannervik, Bengt

    2013-09-05

    Glutathione transferase P1-1 (GST P1-1) is often overexpressed in tumor cells and is regarded as a contributor to their drug resistance. Inhibitors of GST P1-1 are expected to counteract drug resistance and may therefore serve as adjuvants in the chemotherapy of cancer by increasing the efficacy of cytostatic drugs. Finding useful inhibitors among compounds used for other indications would be a shortcut to clinical applications and a search for GST P1-1 inhibitors among approved drugs and other compounds was therefore conducted. We tested 1040 FDA-approved compounds as inhibitors of the catalytic activity of purified human GST P1-1 in vitro. We identified chlorophyllide, merbromine, hexachlorophene, and ethacrynic acid as the most effective GST P1-1 inhibitors with IC50 values in the low micromolar range. For comparison, these compounds were even more potent in the inhibition of human GST A3-3, an enzyme implicated in steroid hormone biosynthesis. In distinction from the other inhibitors, which showed conventional inhibition patterns, the competitive inhibitor ethacrynic acid elicited strong kinetic cooperativity in the glutathione saturation of GST P1-1. Apparently, ethacrynic acid serves as an allosteric inhibitor of the enzyme. In their own right, the compounds investigated are less potent than desired for adjuvants in cancer chemotherapy, but the structures of the most potent inhibitors could serve as leads for the synthesis of more efficient adjuvants. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  8. Dysregulation of protease and protease inhibitors in a mouse model of human pelvic organ prolapse.

    Directory of Open Access Journals (Sweden)

    Madhusudhan Budatha

    Full Text Available Mice deficient for the fibulin-5 gene (Fbln5(-/- develop pelvic organ prolapse (POP due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5(-/- mice, herein named V1 (25 kDa. V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5(-/- mice. PRSS3 was (a localized in epithelial secretions, (b detected in media of vaginal organ culture from both Fbln5(-/- and wild type mice, and (c cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin and Elafin] was dysregulated in Fbln5(-/- epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice.

  9. DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity

    International Nuclear Information System (INIS)

    Kim, Hak Jae; Kim, Jin Ho; Chie, Eui Kyu; Da Young, Park; Kim, In Ah; Kim, Il Han

    2012-01-01

    Histone modifications and DNA methylation are two major factors in epigenetic phenomenon. Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies thus far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers. The principal objective of this study was to evaluate the effects of DNMT inhibitors on the radiosensitivity of human cancer cell lines, and to elucidate the mechanisms relevant to that process. A549 (lung cancer) and U373MG (glioblastoma) cells were exposed to radiation with or without six DNMT inhibitors (5-azacytidine, 5-aza-2'-deoxycytidine, zebularine, hydralazine, epigallocatechin gallate, and psammaplin A) for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays. Cell cycle and apoptosis were analyzed via flow cytometry. Expressions of DNMT1, 3A/3B, and cleaved caspase-3 were detected via Western blotting. Expression of γH2AX, a marker of radiation-induced DNA double-strand break, was examined by immunocytochemistry. Pretreatment with psammaplin A, 5-aza-2'-deoxycytidine, and zebularine radiosensitized both A549 and U373MG cells. Pretreatment with psammaplin A increased the sub-G1 fraction of A549 cells, as compared to cells exposed to radiation alone. Prolongation of γH2AX expression was observed in the cells treated with DNMT inhibitors prior to radiation as compared with those treated by radiation alone. Psammaplin A, 5-aza-2'-deoxycytidine, and zebularine induce radiosensitivity in both A549 and U373MG cell lines, and suggest that this effect might be associated with the inhibition of DNA repair

  10. Sensitization of multidrug-resistant human cancer cells to Hsp90 inhibitors by down-regulation of SIRT1

    Science.gov (United States)

    Kim, Hak-Bong; Lee, Su-Hoon; Um, Jee-Hyun; Oh, Won Keun; Kim, Dong-Wan; Kang, Chi-Dug; Kim, Sun-Hee

    2015-01-01

    The effectiveness of Hsp90 inhibitors as anticancer agents was limited in multidrug-resistant (MDR) human cancer cells due to induction of heat shock proteins (Hsps) such as Hsp70/Hsp27 and P-glycoprotein (P-gp)-mediated efflux. In the present study, we showed that resistance to Hsp90 inhibitors of MDR human cancer cells could be overcome with SIRT1 inhibition. SIRT1 knock-down or SIRT1 inhibitors (amurensin G and EX527) effectively suppressed the resistance to Hsp90 inhibitors (17-AAG and AUY922) in several MDR variants of human lymphoblastic leukemia and human breast cancer cell lines. SIRT1 inhibition down-regulated the expression of heat shock factor 1 (HSF1) and subsequently Hsps and facilitated Hsp90 multichaperone complex disruption via hyperacetylation of Hsp90/Hsp70. These findings were followed by acceleration of ubiquitin ligase CHIP-mediated mutant p53 (mut p53) degradation and subsequent down-regulation of P-gp in 17-AAG-treated MDR cancer cells expressing P-gp and mut p53 after inhibition of SIRT1. Therefore, combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be a more effective therapeutic approach for Hsp90 inhibitor-resistant MDR cells via down-regulation of HSF1/Hsps, mut p53 and P-gp. PMID:26416354

  11. Expression and alternative splicing of the cyclin-dependent kinase inhibitor-3 gene in human cancer.

    Science.gov (United States)

    Cress, W Douglas; Yu, Peng; Wu, Jie

    2017-10-01

    The cyclin-dependent kinase inhibitor-3 (CDKN3) gene encodes a dual-specificity protein tyrosine phosphatase that dephosphorylates CDK1/CDK2 and other proteins. CDKN3 is often overexpressed in human cancer, and this overexpression correlates with reduced survival in several types of cancer. CDKN3 transcript variants and mutations have also been reported. The mechanism of CDKN3 overexpression and the role of CDKN3 transcript variants in human cancer are not entirely clear. Here, we review the literature and provide additional data to assess the correlation of CDKN3 expression with patient survival. Besides the full-length CDKN3 encoding transcript and a major transcript that skips exon 2 express in normal and cancer cells, minor aberrant transcript variants have been reported. Aberrant CDKN3 transcripts were postulated to encode dominant-negative inhibitors of CDKN3 as an explanation for overexpression of the perceived tumor suppressor gene in human cancer. However, while CDKN3 is often overexpressed in human cancer, aberrant CDKN3 transcripts occur infrequently and at lower levels. CDKN3 mutations and copy number alternation are rare in human cancer, implying that neither loss of CDKN3 activity nor constitutive gain of CDKN3 expression offer an advantage to tumorigenesis. Recently, it was found that CDKN3 transcript and protein levels fluctuate during the cell cycle, peaking in mitosis. Given that rapidly growing tumors have more mitotic cells, the high level of mitotic CDKN3 expression is the most plausible mechanism of frequent CDKN3 overexpression in human cancer. This finding clarifies the mechanism of CDKN3 overexpression in human cancer and questions the view of CDKN3 as a tumor suppressor. Copyright © 2017 Elsevier Ltd. All rights reserved.

  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. Further insight into the roles of the glycans attached to human blood protein C inhibitor

    DEFF Research Database (Denmark)

    Sun, Wei; Parry, Simon; Ubhayasekera, Wimal

    2010-01-01

    Protein C inhibitor (PCI) is a 57-kDa glycoprotein that exists in many tissues and secretions in human. As a member of the serpin superfamily of proteins it displays unusually broad protease specificity. PCI is implicated in the regulation of a wide range of processes, including blood coagulation......, fertilization, prevention of tumors and pathogen defence. It has been reported that PCI isolated from human blood plasma is highly heterogeneous, and that this heterogeneity is caused by differences in N-glycan structures, N-glycosylation occupancy, and the presence of two forms that differ by the presence...... or absence of 6 amino acids at the amino-terminus. In this study we have verified that such heterogeneity exists in PCI purified from single individuals, and that individuals of two different ethnicities possess a similar PCI pattern, verifying that the micro-heterogeneity is conserved among humans...

  14. Evaluation of histone deacetylase inhibitors (HDACi) as therapeutic leads for human African trypanosomiasis (HAT).

    Science.gov (United States)

    Carrillo, Angela K; Guiguemde, W Armand; Guy, R Kiplin

    2015-08-15

    Two of the histone deacetylases, TbDAC1 and TbDAC3, have been reported to be essential genes in trypanosomes. Therefore, we tested the activity of a panel of human histone deacetylase inhibitors (HDACi) for their ability to block proliferation of Trypanosoma brucei brucei. Among the HDACi's, the hydroxamic acid derivatives panobinostat and belinostat exhibited potency that appeared to make them viable candidates for development due to their reported pharmacokinetic characteristics. However, cellular pharmacodynamic analysis demonstrated that these drugs were unable to kill cultured parasites at exposures seen in patients at their tolerated doses and additionally failed to show any synergistic effects in combination with pentamidine, suramin, melarsoprol, or nifurtimox. Analysis of the potency of the entire HDACi panel revealed no correlations between potency against any human HDAC isoform and inhibition of T. brucei proliferation, suggesting that the trypanosome histone deacetylases possess a unique specificity. These studies confirmed that HDAC inhibitors have potential as leads against human African trypanosomiasis but that none of the current clinical candidates can be directly repurposed. Therefore, development of HDACi's with appropriate specificity and potency may be a viable route to a new class of anti-trypanosomal drugs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Potent and selective inhibitors of human immunodeficiency virus protease structurally related to L-694,746.

    Science.gov (United States)

    Franchetti, P; Perlini, P; Abu Sheikha, G; Cappellacci, L; Grifantini, M; Loi, A G; De Montis, A; Pani, A; Marongiu, M E; La Colla, P

    1998-07-01

    A series of human immunodeficiency virus (HIV) protease inhibitors, which are analogues of N-[2(R)-hydroxy-1(S)- indanyl]-5(S)-[(tert-butyloxycarbonyl)amino]-4(S)-hydroxy-6-phenyl-2-(R) - [[4-(carboxymethoxy)phenyl]methyl]hexanamide (L-694,746), a metabolite of the anti-HIV agent L-689,502, were synthesized. In these compounds, the acetic group linked to the para position of the P1' phenyl in the reference inhibitor was replaced either by the bioisosteric phosphonomethoxy group and its diisopropyl/dibenzyl derivatives, or the 1H-tetrazol-5-yl-methoxy group and its 1-benzyl derivative. In enzyme assays, phosphonomethoxy and tetrazolmethoxy analogues proved to be potent inhibitors of the HIV-1 protease, with IC50 values as low as 0.04 nM. When tested for anti-HIV-1 activity in cell-based assays, most of the new derivatives proved active, with benzyl derivatives being more active than their highly polar, unsubstituted counterparts. The dibenzylphosphonomethoxy analogue was the most active compound, with an EC50 value of 10 nM and a selectivity index of 20,000. When compounds were examined for their capability to reduce p24 levels in both acutely and chronically infected MT-4 and H9/IIIB cells, all of them were found to be active at concentrations close to those capable of preventing HIV-1-induced cytopathic effect.

  16. Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1

    Energy Technology Data Exchange (ETDEWEB)

    Lewis-Ballester, Ariel; Pham, Khoa N.; Batabyal, Dipanwita; Karkashon, Shay; Bonanno, Jeffrey B.; Poulos, Thomas L.; Yeh, Syun-Ru (Einstein); (UCI)

    2017-11-22

    Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination of an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.

  17. Crystal structure correlations with the intrinsic thermodynamics of human carbonic anhydrase inhibitor binding

    Science.gov (United States)

    Smirnov, Alexey; Zubrienė, Asta; Manakova, Elena; Gražulis, Saulius

    2018-01-01

    The structure-thermodynamics correlation analysis was performed for a series of fluorine- and chlorine-substituted benzenesulfonamide inhibitors binding to several human carbonic anhydrase (CA) isoforms. The total of 24 crystal structures of 16 inhibitors bound to isoforms CA I, CA II, CA XII, and CA XIII provided the structural information of selective recognition between a compound and CA isoform. The binding thermodynamics of all structures was determined by the analysis of binding-linked protonation events, yielding the intrinsic parameters, i.e., the enthalpy, entropy, and Gibbs energy of binding. Inhibitor binding was compared within structurally similar pairs that differ by para- or meta-substituents enabling to obtain the contributing energies of ligand fragments. The pairs were divided into two groups. First, similar binders—the pairs that keep the same orientation of the benzene ring exhibited classical hydrophobic effect, a less exothermic enthalpy and a more favorable entropy upon addition of the hydrophobic fragments. Second, dissimilar binders—the pairs of binders that demonstrated altered positions of the benzene rings exhibited the non-classical hydrophobic effect, a more favorable enthalpy and variable entropy contribution. A deeper understanding of the energies contributing to the protein-ligand recognition should lead toward the eventual goal of rational drug design where chemical structures of ligands could be designed based on the target protein structure. PMID:29503769

  18. Beyond topoisomerase inhibition: antitumor 1,4-naphthoquinones as potential inhibitors of human monoamine oxidase.

    Science.gov (United States)

    Coelho-Cerqueira, Eduardo; Netz, Paulo A; do Canto, Vanessa P; Pinto, Angelo C; Follmer, Cristian

    2014-04-01

    Monoamine oxidase (MAO) action has been involved in the regulation of neurotransmitters levels, cell signaling, cellular growth, and differentiation as well as in the balance of the intracellular polyamine levels. Although so far obscure, MAO inhibitors are believed to have some effect on tumors progression. 1,4-naphthoquinone (1,4-NQ) has been pointed out as a potential pharmacophore for inhibition of both MAO and DNA topoisomerase activities, this latter associated with antitumor activity. Herein, we demonstrated that certain antitumor 1,4-NQs, including spermidine-1,4-NQ, lapachol, and nor-lapachol display inhibitory activity on human MAO-A and MAO-B. Kinetic studies indicated that these compounds are reversible and competitive MAO inhibitors, being the enzyme selectivity greatly affected by substitutions on 1,4-NQ ring. Molecular docking studies suggested that the most potent MAO inhibitors are capable to bind to the MAO active site in close proximity of flavin moiety. Furthermore, ability to inhibit both MAO-A and MAO-B can be potentialized by the formation of hydrogen bonds between these compounds and FAD and/or the residues in the active site. Although spermidine-1,4-NQs exhibit antitumor action primarily by inhibiting topoisomerase via DNA intercalation, our findings suggest that their effect on MAO activity should be taken into account when their application in cancer therapy is considered. © 2013 John Wiley & Sons A/S.

  19. Selective Sparing of Human Tregs by Pharmacologic Inhibitors of the Phosphatidylinositol 3-Kinase and MEK Pathways

    Science.gov (United States)

    Zwang, N. A.; Zhang, R.; Germana, S.; Fan, M. Y.; Hastings, W. D.; Cao, A.; Turka, L. A.

    2016-01-01

    Phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase/extracellular signal-regulated (MEK) signaling are central to the survival and proliferation of many cell types. Multiple lines of investigation in murine models have shown that control of the PI3K pathway is particularly important for regulatory T cell (Treg) stability and function. PI3K and MEK inhibitors are being introduced into the clinic, and we hypothesized that pharmacologic inhibition of PI3K, and possibly MEK, in mixed cultures of human mononuclear cells would preferentially affect CD4+ and CD8+ lymphocytes compared with Tregs. We tested this hypothesis using four readouts: proliferation, activation, functional suppression, and signaling. Results showed that Tregs were less susceptible to inhibition by both δ and α isoform–specific PI3K inhibitors and by an MEK inhibitor compared with their conventional CD4+ and CD8+ counterparts. These studies suggest less functional reliance on PI3K and MEK signaling in Tregs compared with conventional CD4+ and CD8+ lymphocytes. Therefore, the PI3K and MEK pathways are attractive pharmacologic targets for transplantation and treatment of autoimmunity. PMID:27017850

  20. Targeting Cyclin-Dependent Kinases in Human Cancers: From Small Molecules to Peptide Inhibitors

    Directory of Open Access Journals (Sweden)

    Marion Peyressatre

    2015-01-01

    Full Text Available Cyclin-dependent kinases (CDK/Cyclins form a family of heterodimeric kinases that play central roles in regulation of cell cycle progression, transcription and other major biological processes including neuronal differentiation and metabolism. Constitutive or deregulated hyperactivity of these kinases due to amplification, overexpression or mutation of cyclins or CDK, contributes to proliferation of cancer cells, and aberrant activity of these kinases has been reported in a wide variety of human cancers. These kinases therefore constitute biomarkers of proliferation and attractive pharmacological targets for development of anticancer therapeutics. The structural features of several of these kinases have been elucidated and their molecular mechanisms of regulation characterized in depth, providing clues for development of drugs and inhibitors to disrupt their function. However, like most other kinases, they constitute a challenging class of therapeutic targets due to their highly conserved structural features and ATP-binding pocket. Notwithstanding, several classes of inhibitors have been discovered from natural sources, and small molecule derivatives have been synthesized through rational, structure-guided approaches or identified in high throughput screens. The larger part of these inhibitors target ATP pockets, but a growing number of peptides targeting protein/protein interfaces are being proposed, and a small number of compounds targeting allosteric sites have been reported.

  1. Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors

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    Gozzo A.J.

    2003-01-01

    Full Text Available Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 µg/ml on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates reduced (1.2 to 3.0 times the catalytic efficiency of kallikrein (in a nanomolar range on the hydrolysis of plasminogen (0.3 to 1.8 µM and increased (1.9 to 7.7 times the enzyme efficiency in factor XII (0.1 to 10 µM activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times kallikrein inhibition by antithrombin (1.4 µM, while chondroitin 4- and 6-sulfates reduced it (1.3 times. Heparin and heparan sulfate increased (1.4 times the enzyme inhibition by the C1-inhibitor (150 nM.

  2. Susceptibility of Human Endogenous Retrovirus Type K to Reverse Transcriptase Inhibitors.

    Science.gov (United States)

    Contreras-Galindo, Rafael; Dube, Derek; Fujinaga, Koh; Kaplan, Mark H; Markovitz, David M

    2017-12-01

    Human endogenous retroviruses (HERVs) make up 8% of the human genome. The HERV type K (HERV-K) HML-2 (HK2) family contains proviruses that are the most recent entrants into the human germ line and are transcriptionally active. In HIV-1 infection and cancer, HK2 genes produce retroviral particles that appear to be infectious, yet the replication capacity of these viruses and potential pathogenicity has been difficult to ascertain. In this report, we screened the efficacy of commercially available reverse transcriptase inhibitors (RTIs) at inhibiting the enzymatic activity of HK2 RT and HK2 genomic replication. Interestingly, only one provirus, K103, was found to encode a functional RT among those examined. Several nucleoside analogue RTIs (NRTIs) blocked K103 RT activity and consistently inhibited the replication of HK2 genomes. The NRTIs zidovudine (AZT), stavudine (d4T), didanosine (ddI), and lamivudine (3TC), and the nucleotide RTI inhibitor tenofovir (TDF), show efficacy in blocking K103 RT. HIV-1-specific nonnucleoside RTIs (NNRTIs), protease inhibitors (PIs), and integrase inhibitors (IIs) did not affect HK2, except for the NNRTI etravirine (ETV). The inhibition of HK2 infectivity by NRTIs appears to take place at either the reverse transcription step of the viral genome prior to HK2 viral particle formation and/or in the infected cells. Inhibition of HK2 by these drugs will be useful in suppressing HK2 infectivity if these viruses prove to be pathogenic in cancer, neurological disorders, or other diseases associated with HK2. The present studies also elucidate a key aspect of the life cycle of HK2, specifically addressing how they do, and/or did, replicate. IMPORTANCE Endogenous retroviruses are relics of ancestral virus infections in the human genome. The most recent of these infections was caused by HK2. While HK2 often remains silent in the genome, this group of viruses is activated in HIV-1-infected and cancer cells. Recent evidence suggests that these

  3. New Small-Molecule Inhibitor Class Targeting Human Immunodeficiency Virus Type 1 Virion Maturation▿

    Science.gov (United States)

    Blair, Wade S.; Cao, Joan; Fok-Seang, Juin; Griffin, Paul; Isaacson, Jason; Jackson, R. Lynn; Murray, Edward; Patick, Amy K.; Peng, Qinghai; Perros, Manos; Pickford, Chris; Wu, Hua; Butler, Scott L.

    2009-01-01

    A new small-molecule inhibitor class that targets virion maturation was identified from a human immunodeficiency virus type 1 (HIV-1) antiviral screen. PF-46396, a representative molecule, exhibits antiviral activity against HIV-1 laboratory strains and clinical isolates in T-cell lines and peripheral blood mononuclear cells (PBMCs). PF-46396 specifically inhibits the processing of capsid (CA)/spacer peptide 1 (SP1) (p25), resulting in the accumulation of CA/SP1 (p25) precursor proteins and blocked maturation of the viral core particle. Viral variants resistant to PF-46396 contain a single amino acid substitution in HIV-1 CA sequences (CAI201V), distal to the CA/SP1 cleavage site in the primary structure, which we demonstrate is sufficient to confer significant resistance to PF-46396 and 3-O-(3′,3′-dimethylsuccinyl) betulinic acid (DSB), a previously described maturation inhibitor. Conversely, a single amino substitution in SP1 (SP1A1V), which was previously associated with DSB in vitro resistance, was sufficient to confer resistance to DSB and PF-46396. Further, the CAI201V substitution restored CA/SP1 processing in HIV-1-infected cells treated with PF-46396 or DSB. Our results demonstrate that PF-46396 acts through a mechanism that is similar to DSB to inhibit the maturation of HIV-1 virions. To our knowledge, PF-46396 represents the first small-molecule HIV-1 maturation inhibitor that is distinct in chemical class from betulinic acid-derived maturation inhibitors (e.g., DSB), demonstrating that molecules of diverse chemical classes can inhibit this mechanism. PMID:19805571

  4. Human Kunitz-type protease inhibitor engineered for enhanced matrix retention extends longevity of fibrin biomaterials.

    Science.gov (United States)

    Briquez, Priscilla S; Lorentz, Kristen M; Larsson, Hans M; Frey, Peter; Hubbell, Jeffrey A

    2017-08-01

    Aprotinin is a broad-spectrum serine protease inhibitor used in the clinic as an anti-fibrinolytic agent in fibrin-based tissue sealants. However, upon re-exposure, some patients suffer from hypersensitivity immune reactions likely related to the bovine origin of aprotinin. Here, we aimed to develop a human-derived substitute to aprotinin. Based on sequence homology analyses, we identified the Kunitz-type protease inhibitor (KPI) domain of human amyloid-β A4 precursor protein as being a potential candidate. While KPI has a lower intrinsic anti-fibrinolytic activity than aprotinin, we reasoned that its efficacy is additionally limited by its fast release from fibrin material, just as aprotinin's is. Thus, we engineered KPI variants for controlled retention in fibrin biomaterials, using either covalent binding through incorporation of a substrate for the coagulation transglutaminase Factor XIIIa or through engineering of extracellular matrix protein super-affinity domains for sequestration into fibrin. We showed that both engineered KPI variants significantly slowed plasmin-mediated fibrinolysis in vitro, outperforming aprotinin. In vivo, our best engineered KPI variant (incorporating the transglutaminase substrate) extended fibrin matrix longevity by 50%, at a dose at which aprotinin did not show efficacy, thus qualifying it as a competitive substitute of aprotinin in fibrin sealants. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  5. The non-coding RNA TERRA is a natural ligand and direct inhibitor of human telomerase.

    Science.gov (United States)

    Redon, Sophie; Reichenbach, Patrick; Lingner, Joachim

    2010-09-01

    Telomeres, the physical ends of eukaryotes chromosomes are transcribed into telomeric repeat containing RNA (TERRA), a large non-coding RNA of unknown function, which forms an integral part of telomeric heterochromatin. TERRA molecules resemble in sequence the telomeric DNA substrate as they contain 5'-UUAGGG-3' repeats near their 3'-end which are complementary to the template sequence of telomerase RNA. Here we demonstrate that endogenous TERRA is bound to human telomerase in cell extracts. Using in vitro reconstituted telomerase and synthetic TERRA molecules we demonstrate that the 5'-UUAGGG-3' repeats of TERRA base pair with the RNA template of the telomerase RNA moiety (TR). In addition TERRA contacts the telomerase reverse transcriptase (TERT) protein subunit independently of hTR. In vitro studies further demonstrate that TERRA is not used as a telomerase substrate. Instead, TERRA acts as a potent competitive inhibitor for telomeric DNA in addition to exerting an uncompetitive mode of inhibition. Our data identify TERRA as a telomerase ligand and natural direct inhibitor of human telomerase. Telomerase regulation by the telomere substrate may be mediated via its transcription.

  6. Fumarate analogs act as allosteric inhibitors of the human mitochondrial NAD(P)+-dependent malic enzyme.

    Science.gov (United States)

    Hsieh, Ju-Yi; Liu, Jyung-Hurng; Yang, Pai-Chun; Lin, Chi-Li; Liu, Guang-Yaw; Hung, Hui-Chih

    2014-01-01

    Human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME) is allosterically activated by the four-carbon trans dicarboxylic acid, fumarate. Previous studies have suggested that the dicarboxylic acid in a trans conformation around the carbon-carbon double bond is required for the allosteric activation of the enzyme. In this paper, the allosteric effects of fumarate analogs on m-NAD(P)-ME are investigated. Two fumarate-insensitive mutants, m-NAD(P)-ME_R67A/R91A and m-NAD(P)-ME_K57S/E59N/K73E/D102S, as well as c-NADP-ME, were used as the negative controls. Among these analogs, mesaconate, trans-aconitate, monomethyl fumarate and monoethyl fumarate were allosteric activators of the enzyme, while oxaloacetate, diethyl oxalacetate, and dimethyl fumarate were found to be allosteric inhibitors of human m-NAD(P)-ME. The IC50 value for diethyl oxalacetate was approximately 2.5 mM. This paper suggests that the allosteric inhibitors may impede the conformational change from open form to closed form and therefore inhibit m-NAD(P)-ME enzyme activity.

  7. Fumarate analogs act as allosteric inhibitors of the human mitochondrial NAD(P+-dependent malic enzyme.

    Directory of Open Access Journals (Sweden)

    Ju-Yi Hsieh

    Full Text Available Human mitochondrial NAD(P+-dependent malic enzyme (m-NAD(P-ME is allosterically activated by the four-carbon trans dicarboxylic acid, fumarate. Previous studies have suggested that the dicarboxylic acid in a trans conformation around the carbon-carbon double bond is required for the allosteric activation of the enzyme. In this paper, the allosteric effects of fumarate analogs on m-NAD(P-ME are investigated. Two fumarate-insensitive mutants, m-NAD(P-ME_R67A/R91A and m-NAD(P-ME_K57S/E59N/K73E/D102S, as well as c-NADP-ME, were used as the negative controls. Among these analogs, mesaconate, trans-aconitate, monomethyl fumarate and monoethyl fumarate were allosteric activators of the enzyme, while oxaloacetate, diethyl oxalacetate, and dimethyl fumarate were found to be allosteric inhibitors of human m-NAD(P-ME. The IC50 value for diethyl oxalacetate was approximately 2.5 mM. This paper suggests that the allosteric inhibitors may impede the conformational change from open form to closed form and therefore inhibit m-NAD(P-ME enzyme activity.

  8. High throughput Screening to Identify Natural Human Monoamine Oxidase B Inhibitors

    Science.gov (United States)

    Mazzio, E; Deiab, S; Park, K; Soliman, KFA

    2012-01-01

    Age-related increase in monoamine oxidase B (MAO-B) may contribute to CNS neurodegenerative diseases. Moreover, MAO-B inhibitors are used in the treatment of idiopathic Parkinson disease as preliminary monotherapy or adjunct therapy with L-dopa. To date, meager natural sources of MAO-B inhibitors have been identified, and the relative strength, potency and rank of many plants relative to standard drugs such as Selegiline (L-deprenyl, Eldepryl) are not known. In this work, we developed and utilized a high throughput enzyme microarray format to screen and evaluate 905 natural product extracts (0.025–.7 mg/ml) to inhibit human MAO-B derived from BTI-TN-5B1-4 cells infected with recombinant baculovirus. The protein sequence of purified enzyme was confirmed using 1D gel electrophoresis-matrix assisted laser desorption ionization-time-of-flight-tandem mass spectroscopy, and enzyme activity was confirmed by [1] substrate conversion (3-mM benzylamine) to H202 and [2] benzaldehyde. Of the 905 natural extracts tested, the lowest IC50s [Comfrey, Bringraj, Skullcap, Kava-kava, Wild Indigo, Gentian and Green Tea. In conclusion, the data reflect relative potency information by rank of commonly used herbs and plants that contain human MAO-B inhibitory properties in their natural form. PMID:22887993

  9. Mechanisms of molecular recognition: crystal structure analysis of human and rat transthyretin inhibitor complexes.

    Science.gov (United States)

    Cody, Vivian

    2002-12-01

    Structure-activity data show that many pharmacological agents are strong competitive inhibitors for thyroxine (T4) binding to transthyretin (TTR) and that this competition can interfere with their normal pharmacological actions. TTR is a tetrameric serum protein responsible for the transport of 20% of the circulating T4 in man, while in lower vertebrates such as rats it is the only carrier. The sequence of rat TTR is 85% homologous to the human protein. Crystallographic analyses of ligand co-crystal complexes of human and rat TTR have been studied to understand the molecular basis for binding selectivity of competitor binding to TTR. Analysis of TTR crystal complexes with several classes of competitors (hormone metabolites, flavonoids, fluorescent probes, analgesics and cardiac agents) revealed multiple modes of binding with both forward and reverse ligand binding orientations. These ligands also have different binding positions along the length of the channel with the smallest ligands located deeper within the hormone domain. Data for the human TTR complex with the bromoflavone EMD21388 incubated at different times revealed variable binding positions and occupancies dependent upon incubation time. Comparison of the structures of T4 thyroacetic acid in complex with both human and rat TTR revealed forward and reverse binding, but also showed different modes of binding in the rat compared to the human complex. These data highlight the importance of hydrogen bonding with Lys-15 and Ser-117 and provide insight into ligand binding affinity and negative cooperativity.

  10. Potent human uric acid transporter 1 inhibitors: in vitro and in vivo metabolism and pharmacokinetic studies

    Directory of Open Access Journals (Sweden)

    Wempe MF

    2012-11-01

    Full Text Available Michael F Wempe,1 Janet W Lightner,2 Bettina Miller,1 Timothy J Iwen,1 Peter J Rice,1 Shin Wakui,3 Naohiko Anzai,4 Promsuk Jutabha,4 Hitoshi Endou51Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA; 2Department of Pharmacology, East Tennessee State University, Johnson City, TN, USA; 3Department of Toxicology, Azabu University School of Veterinary Medicine, Chuo Sagamihara, Kanagawa, Japan; 4Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, Mibu, Shimotsuga, Tochigi, Japan; 5Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Mitaka, Tokyo, JapanAbstract: Human uric acid transporter 1 (hURAT1; SLC22A12 is a very important urate anion exchanger. Elevated urate levels are known to play a pivotal role in cardiovascular diseases, chronic renal disease, diabetes, and hypertension. Therefore, the development of potent uric acid transport inhibitors may lead to novel therapeutic agents to combat these human diseases. The current study investigates small molecular weight compounds and their ability to inhibit 14C-urate uptake in oocytes expressing hURAT1. Using the most promising drug candidates generated from our structure–activity relationship findings, we subsequently conducted in vitro hepatic metabolism and pharmacokinetic (PK studies in male Sprague-Dawley rats. Compounds were incubated with rat liver microsomes containing cofactors nicotinamide adenine dinucleotide phosphate and uridine 5'-diphosphoglucuronic acid. In vitro metabolism and PK samples were analyzed using liquid chromatography/mass spectrometry-mass spectrometry methods. Independently, six different inhibitors were orally (capsule dosing or intravenously (orbital sinus administered to fasting male Sprague-Dawley rats. Blood samples were collected and analyzed; these data were used to compare in vitro and in vivo metabolism and to

  11. Evaluation of six proton pump inhibitors as inhibitors of various human cytochromes P450: focus on cytochrome P450 2C19.

    Science.gov (United States)

    Zvyaga, Tatyana; Chang, Shu-Ying; Chen, Cliff; Yang, Zheng; Vuppugalla, Ragini; Hurley, Jeremy; Thorndike, Denise; Wagner, Andrew; Chimalakonda, Anjaneya; Rodrigues, A David

    2012-09-01

    Six proton pump inhibitors (PPIs), omeprazole, lansoprazole, esomeprazole, dexlansoprazole, pantoprazole, and rabeprazole, were shown to be weak inhibitors of cytochromes P450 (CYP3A4, -2B6, -2D6, -2C9, -2C8, and -1A2) in human liver microsomes. In most cases, IC₅₀ values were greater than 40 μM, except for dexlansoprazole and lansoprazole with CYP1A2 (IC₅₀ = ∼8 μM) and esomeprazole with CYP2C8 (IC₅₀ = 31 μM). With the exception of CYP2C19 inhibition by omeprazole and esomeprazole (IC₅₀ ratio, 2.5 to 5.9), there was no evidence for a marked time-dependent shift in IC₅₀ (IC₅₀ ratio, ≤ 2) after a 30-min preincubation with NADPH. In the absence of preincubation, lansoprazole (IC₅₀ = 0.73 μM) and esomeprazole (IC₅₀ = 3.7 μM) were the most potent CYP2C19 inhibitors, followed by dexlansoprazole and omeprazole (IC₅₀ = ∼7.0 μM). Rabeprazole and pantoprazole (IC₅₀ = ≥ 25 μM) were the weakest. A similar ranking was obtained with recombinant CYP2C19. Despite the IC₅₀ ranking, after consideration of plasma levels (static and dynamic), protein binding, and metabolism-dependent inhibition, it is concluded that omeprazole and esomeprazole are the most potent CYP2C19 inhibitors. This was confirmed after the incubation of the individual PPIs with human primary hepatocytes (in the presence of human serum) and by monitoring their impact on diazepam N-demethylase activity at a low concentration of diazepam (2 μM). Data described herein are consistent with reports that PPIs are mostly weak inhibitors of cytochromes P450 in vivo. However, two members of the PPI class (esomeprazole and omeprazole) are more likely to serve as clinically relevant inhibitors of CYP2C19.

  12. Discovery of a novel dual fungal CYP51/human 5-lipoxygenase inhibitor: implications for anti-fungal therapy.

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    Eric K Hoobler

    Full Text Available We report the discovery of a novel dual inhibitor targeting fungal sterol 14α-demethylase (CYP51 or Erg11 and human 5-lipoxygenase (5-LOX with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. A series of potent 5-LOX inhibitors containing a phenylenediamine core, were synthesized that exhibit nanomolar potency and >30-fold selectivity against the LOX paralogs, platelet-type 12-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity against ovine cyclooxygenase-1 and human cyclooxygnease-2. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual inhibitor against human 5-LOX (IC50 = 700 nM and CYP51 (IC50 = 43 nM in vitro. It was tested in whole blood and found to down-regulate LTB4 synthesis, displaying 45% inhibition at 10 µM. In addition, ketaminazole selectively inhibited yeast CYP51 relative to human CYP51 by 17-fold, which is greater selectivity than that of ketoconazole and could confer a therapeutic advantage. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have therapeutic uses against fungal infections that have an anti-inflammatory component.

  13. Cholesterol synthesis inhibitor RO 48-8071 suppresses transcriptional activity of human estrogen and androgen receptor.

    Science.gov (United States)

    Mafuvadze, Benford; Liang, Yayun; Hyder, Salman M

    2014-10-01

    Breast cancer cells express enzymes that convert cholesterol, the synthetic precursor of steroid hormones, into estrogens and androgens, which then drive breast cancer cell proliferation. In the present study, we sought to determine whether oxidosqualene cyclase (OSC), an enzyme in the cholesterol biosynthetic pathway, may be targeted to suppress progression of breast cancer cells. In previous studies, we showed that the OSC inhibitor RO 48-8071 (RO) may be a ligand which could potentially be used to control the progression of estrogen receptor-α (ERα)-positive breast cancer cells. Herein, we showed, by real-time PCR analysis of mRNA from human breast cancer biopsies, no significant differences in OSC expression at various stages of disease, or between tumor and normal mammary cells. Since the growth of hormone-responsive tumors is ERα-dependent, we conducted experiments to determine whether RO affects ERα. Using mammalian cells engineered to express human ERα or ERβ protein, together with an ER-responsive luciferase promoter, we found that RO dose-dependently inhibited 17β-estradiol (E2)-induced ERα responsive luciferase activity (IC50 value, ~10 µM), under conditions that were non-toxic to the cells. RO was less effective against ERβ-induced luciferase activity. Androgen receptor (AR) mediated transcriptional activity was also reduced by RO. Notably, while ERα activity was reduced by atorvastatin, the HMG-CoA reductase inhibitor did not influence AR activity, showing that RO possesses broader antitumor properties. Treatment of human BT-474 breast cancer cells with RO reduced levels of estrogen-induced PR protein, confirming that RO blocks ERα activity in tumor cells. Our findings demonstrate that an important means by which RO suppresses hormone-dependent growth of breast cancer cells is through its ability to arrest the biological activity of ERα. This warrants further investigation of RO as a potential therapeutic agent for use against hormone

  14. PARP Inhibitors in Clinical Use Induce Genomic Instability in Normal Human Cells.

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    Shuhei Ito

    Full Text Available Poly(ADP-ribose polymerases (PARPs are the first proteins involved in cellular DNA repair pathways to be targeted by specific inhibitors for clinical benefit. Tumors harboring genetic defects in homologous recombination (HR, a DNA double-strand break (DSB repair pathway, are hypersensitive to PARP inhibitors (PARPi. Early phase clinical trials with PARPi have been promising in patients with advanced BRCA1 or BRCA2-associated breast, ovary and prostate cancer and have led to limited approval for treatment of BRCA-deficient ovary cancer. Unlike HR-defective cells, HR-proficient cells manifest very low cytotoxicity when exposed to PARPi, although they mount a DNA damage response. However, the genotoxic effects on normal human cells when agents including PARPi disturb proficient cellular repair processes have not been substantially investigated. We quantified cytogenetic alterations of human cells, including primary lymphoid cells and non-tumorigenic and tumorigenic epithelial cell lines, exposed to PARPi at clinically relevant doses by both sister chromatid exchange (SCE assays and chromosome spreading. As expected, both olaparib and veliparib effectively inhibited poly-ADP-ribosylation (PAR, and caused marked hypersensitivity in HR-deficient cells. Significant dose-dependent increases in SCEs were observed in normal and non-tumorigenic cells with minimal residual PAR activity. Clinically relevant doses of the FDA-approved olaparib led to a marked increase of SCEs (5-10-fold and chromatid aberrations (2-6-fold. Furthermore, olaparib potentiated SCE induction by cisplatin in normal human cells. Our data have important implications for therapies with regard to sustained genotoxicity to normal cells. Genomic instability arising from PARPi warrants consideration, especially if these agents will be used in people with early stage cancers, in prevention strategies or for non-oncologic indications.

  15. Selective and irreversible inhibitors of aphid acetylcholinesterases: steps toward human-safe insecticides.

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    Yuan-Ping Pang

    Full Text Available Aphids, among the most destructive insects to world agriculture, are mainly controlled by organophosphate insecticides that disable the catalytic serine residue of acetylcholinesterase (AChE. Because these agents also affect vertebrate AChEs, they are toxic to non-target species including humans and birds. We previously reported that a cysteine residue (Cys, found at the AChE active site in aphids and other insects but not mammals, might serve as a target for insect-selective pesticides. However, aphids have two different AChEs (termed AP and AO, and only AP-AChE carries the unique Cys. The absence of the active-site Cys in AO-AChE might raise concerns about the utility of targeting that residue. Herein we report the development of a methanethiosulfonate-containing small molecule that, at 6.0 microM, irreversibly inhibits 99% of all AChE activity extracted from the greenbug aphid (Schizaphis graminum without any measurable inhibition of the human AChE. Reactivation studies using beta-mercaptoethanol confirm that the irreversible inhibition resulted from the conjugation of the inhibitor to the unique Cys. These results suggest that AO-AChE does not contribute significantly to the overall AChE activity in aphids, thus offering new insight into the relative functional importance of the two insect AChEs. More importantly, by demonstrating that the Cys-targeting inhibitor can abolish AChE activity in aphids, we can conclude that the unique Cys may be a viable target for species-selective agents to control aphids without causing human toxicity and resistance problems.

  16. Human pregnane X receptor is activated by dibenzazepine carbamate-based inhibitors of constitutive androstane receptor.

    Science.gov (United States)

    Jeske, Judith; Windshügel, Björn; Thasler, Wolfgang E; Schwab, Matthias; Burk, Oliver

    2017-06-01

    Unintentional activation of xenosensing nuclear receptors pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR) by clinical drug use is known to produce severe side effects in patients, which may be overcome by co-administering antagonists. However, especially antagonizing CAR is hampered by the lack of specific inhibitors, which do not activate PXR. Recently, compounds based on a dibenzazepine carbamate scaffold were identified as potent CAR inhibitors. However, their potential to activate PXR was not thoroughly investigated, even if the lead compound was named "CAR inhibitor not PXR activator 1" (CINPA1). Thus, we performed a comprehensive analysis of the interaction of CINPA1 and four analogs with PXR. Cellular assays were used to investigate intra- and intermolecular interactions and transactivation activity of PXR as a function of the compounds. Modulation of PXR target gene expression was analyzed in primary human hepatocytes. Ligand binding to PXR was investigated by molecular docking and limited proteolytic digestion. We show here that CINPA1 induced the assembly of the PXR ligand-binding domain, released co-repressors from and recruited co-activators to the receptor. CINPA1 and its analogs induced the PXR-dependent activation of a CYP3A4 reporter gene and CINPA1 induced the expression of endogenous cytochrome P450 genes in primary hepatocytes, while not consistently inhibiting CAR-mediated induction. Molecular docking revealed favorable binding of CINPA1 and analogs to the PXR ligand-binding pocket, which was confirmed in vitro. Altogether, our data provide consistent evidence that compounds with a dibenzazepine carbamate scaffold, such as CINPA1 and its four analogs, bind to and activate PXR.

  17. Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells

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    Minyong Kang

    2017-02-01

    Full Text Available Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1, chloroquine (CQ and 3-methyladenine (3-MA were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8 assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating

  18. The histone deacetylase inhibitor suberoylanilide hydroxamic acid attenuates human astrocyte neurotoxicity induced by interferon-γ

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    Hashioka Sadayuki

    2012-05-01

    Full Text Available Abstract Backgrounds Increasing evidence shows that the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA possesses potent anti-inflammatory and immunomodulatory properties. It is tempting to evaluate the potential of SAHA as a therapeutic agent in various neuroinflammatory and neurodegenerative disorders. Methods We examined the effects of SAHA on interferon (IFN-γ-induced neurotoxicity of human astrocytes and on IFN-γ-induced phosphorylation of signal transducer and activator of transcription (STAT 3 in human astrocytes. We also studied the effects of SAHA on the astrocytic production of two representative IFN-γ-inducible inflammatory molecules, namely IFN-γ-inducible T cell α chemoattractant (I-TAC and intercellular adhesion molecule-1 (ICAM-1. Results SAHA significantly attenuated the toxicity of astrocytes activated by IFN-γ towards SH-SY5Y human neuronal cells. In the IFN-γ-activated astrocytes, SAHA reduced the STAT3 phosphorylation. SAHA also inhibited the IFN-γ-induced astrocytic production of I-TAC, but not ICAM-1. These results indicate that SAHA suppresses IFN-γ-induced neurotoxicity of human astrocytes through inhibition of the STAT3 signaling pathway. Conclusion Due to its anti-neurotoxic and anti-inflammatory properties, SAHA appears to have the therapeutic or preventive potential for a wide range of neuroinflammatory disorders associated with activated astrocytes.

  19. Discovery and optimization of piperazine-1-thiourea-based human phosphoglycerate dehydrogenase inhibitors.

    Science.gov (United States)

    Rohde, Jason M; Brimacombe, Kyle R; Liu, Li; Pacold, Michael E; Yasgar, Adam; Cheff, Dorian M; Lee, Tobie D; Rai, Ganesha; Baljinnyam, Bolormaa; Li, Zhuyin; Simeonov, Anton; Hall, Matthew D; Shen, Min; Sabatini, David M; Boxer, Matthew B

    2018-05-01

    Proliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate dehydrogenase (PHGDH) controls flux through the biosynthetic pathway for this important amino acid and represents a putative target in oncology. To discover inhibitors of PHGDH, a coupled biochemical assay was developed and optimized to enable high-throughput screening for inhibitors of human PHGDH. Feedback inhibition was minimized by coupling PHGDH activity to two downstream enzymes (PSAT1 and PSPH), providing a marked improvement in enzymatic turnover. Further coupling of NADH to a diaphorase/resazurin system enabled a red-shifted detection readout, minimizing interference due to compound autofluorescence. With this protocol, over 400,000 small molecules were screened for PHGDH inhibition, and following hit validation and triage work, a piperazine-1-thiourea was identified. Following rounds of medicinal chemistry and SAR exploration, two probes (NCT-502 and NCT-503) were identified. These molecules demonstrated improved target activity and encouraging ADME properties, enabling in vitro assessment of the biological importance of PHGDH, and its role in the fate of serine in PHGDH-dependent cancer cells. This manuscript reports the assay development and medicinal chemistry leading to the development of NCT-502 and -503 reported in Pacold et al. (2016). Copyright © 2018 Elsevier Ltd. All rights reserved.

  20. The tyrosine kinase inhibitor dasatinib induces a marked adipogenic differentiation of human multipotent mesenchymal stromal cells.

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    Adriana Borriello

    Full Text Available BACKGROUND: The introduction of specific BCR-ABL inhibitors in chronic myelogenous leukemia therapy has entirely mutated the prognosis of this hematologic cancer from being a fatal disorder to becoming a chronic disease. Due to the probable long lasting treatment with tyrosine-kinase inhibitors (TKIs, the knowledge of their effects on normal cells is of pivotal importance. DESIGN AND METHODS: We investigated the effects of dasatinib treatment on human bone marrow-derived mesenchymal stromal cells (MSCs. RESULTS: Our findings demonstrate, for the first time, that dasatinib induces MSCs adipocytic differentiation. Particularly, when the TKI is added to the medium inducing osteogenic differentiation, a high MSCs percentage acquires adipocytic morphology and overexpresses adipocytic specific genes, including PPARγ, CEBPα, LPL and SREBP1c. Dasatinib also inhibits the activity of alkaline phosphatase, an osteogenic marker, and remarkably reduces matrix mineralization. The increase of PPARγ is also confirmed at protein level. The component of osteogenic medium required for dasatinib-induced adipogenesis is dexamethasone. Intriguingly, the increase of adipocytic markers is also observed in MSCs treated with dasatinib alone. The TKI effect is phenotype-specific, since fibroblasts do not undergo adipocytic differentiation or PPARγ increase. CONCLUSIONS: Our data demonstrate that dasatinib treatment affects bone marrow MSCs commitment and suggest that TKIs therapy might modify normal phenotypes with potential significant negative consequences.

  1. Use of human Dihydroorotate Dehydrogenase (hDHODH) Inhibitors in Autoimmune Diseases and New Perspectives in Cancer Therapy.

    Science.gov (United States)

    Lolli, Marco L; Sainas, Stefano; Pippione, Agnese C; Giorgis, Marta; Boschi, Donatella; Dosio, Franco

    2018-01-01

    Human dihydroorotate dehydrogenase (hDHODH, EC 1.3.5.2), a flavindependent mitochondrial enzyme involved in de novo pyrimidine biosynthesis, is a validated therapeutic target for the treatment of autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis. However, human DHODH inhibitors have also been investigated as treatment for cancer, parasite infections (i.e. malaria) and viruses as well as in the agrochemicals industry. An overview of current knowledge of hDHODH inhibitors and their potential uses in diseases where hDHODH is involved. This review focuses on recent advances in the development and application of hDHODH inhibitors, specifically covering the patent field, starting from a brief description of enzyme topography and of the strategies usually followed in designing its selective inhibitors. The most important and well-described novelty is the fact that the discovery, in the autumn of 2016, that hDHODH inhibitors are able to induce in vivo myeloid differentiation has led to the possibility of developing novel hDHODH based treatments for Acute Myelogenous Leukemia (AML). The review will describe a variety of specific inhibitor classes and conclude on recent and future therapeutic perspectives for this target. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  2. Synthetic Routes to N-9 Alkylated 8-Oxoguanines; Weak Inhibitors of the Human DNA Glycosylase OGG1

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    Tushar R. Mahajan

    2015-09-01

    Full Text Available The human 8-oxoguanine DNA glycosylase OGG1 is involved in base excision repair (BER, one of several DNA repair mechanisms that may counteract the effects of chemo- and radiation therapy for the treatment of cancer. We envisage that potent inhibitors of OGG1 may be found among the 9-alkyl-8-oxoguanines. Thus we explored synthetic routes to 8-oxoguanines and examined these as OGG1 inhibitors. The best reaction sequence started from 6-chloroguanine and involved N-9 alkylation, C-8 bromination, and finally simultaneous hydrolysis of both halides. Bromination before N-alkylation should only be considered when the N-substituent is not compatible with bromination conditions. The 8-oxoguanines were found to be weak inhibitors of OGG1. 6-Chloro-8-oxopurines, byproducts in the hydrolysis of 2,6-halopurines, turned out to be slightly better inhibitors than the corresponding 8-oxoguanines.

  3. Nitrobenzylthioinosine (NBT), a nucleoside transport inhibitor, protects against Shiga toxin cytotoxicity in human microvascular endothelial cells.

    Science.gov (United States)

    Ohmi, K; Kiyokawa, N; Sekino, T; Suzuki, T; Mimori, K; Taguchi, T; Nakajima, H; Katagiri, Y U; Fujimoto, J; Nakao, H; Takeda, T

    2001-01-01

    Infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) cause microvascular endothelial cell damage, resulting in hemorrhagic colitis and hemolytic uremic syndrome. The prevention of endothelial cell damage is therefore a crucial step in overcoming this disorder. Here, we report that nitrobenzylthioinosine (NBT), a nucleoside transport inhibitor, has a protective effect against the cytotoxicity of Stxs in human microvascular endothelial cells (HMVECs). The relative viability of cells treated with 1.5-15 pM of Stx1 was reduced to 10-20% of that without Stx1. However, the viability of cells treated with NBT (10-100 microM) remained higher than 80%, even in the presence of Stx1. NBT also protected against Stx1 cytotoxicity in sodium butyrate-treated hypersensitive HMVECs. The protective effect of NBT against Stx cytotoxicity may be due to the depletion of ATP in the cells, thereby inhibiting the entry of Stx1.

  4. The M358R variant of α{sub 1}-proteinase inhibitor inhibits coagulation factor VIIa

    Energy Technology Data Exchange (ETDEWEB)

    Sheffield, William P., E-mail: sheffiel@mcmaster.ca [Canadian Blood Services, Centre for Innovation, Hamilton, Ontario (Canada); Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario (Canada); Bhakta, Varsha [Canadian Blood Services, Centre for Innovation, Hamilton, Ontario (Canada)

    2016-02-12

    The naturally occurring M358R mutation of the plasma serpin α{sub 1}-proteinase inhibitor (API) changes both its cleavable reactive centre bond to Arg–Ser and the efficacy with which it inhibits different proteases, reducing the rate of inhibition of neutrophil elastase, and enhancing that of thrombin, factor XIa, and kallikrein, by several orders of magnitude. Although another plasma serpin with an Arg–Ser reactive centre, antithrombin (AT), has been shown to inhibit factor VIIa (FVIIa), no published data are available with respect to FVIIa inhibition by API M358R. Recombinant bacterially-expressed API M358R and plasma-derived AT were therefore compared using gel-based and kinetic assays of FVIIa integrity and activity. Under pseudo-first order conditions of excess serpin over protease, both AT and API M358R formed denaturation-resistant inhibitory complexes with FVIIa in reactions accelerated by TF; AT, but not API M358R, also required heparin for maximal activity. The second order rate constant for heparin-independent API M358R-mediated FVIIa inhibition was determined to be 7.8 ± 0.8 × 10{sup 2} M{sup −1}sec{sup −1}. We conclude that API M358R inhibits FVIIa by forming inhibitory complexes of the serpin type more rapidly than AT in the absence of heparin. The likely 20-fold excess of API M358R over AT in patient plasma during inflammation raises the possibility that it could contribute to the hemorrhagic tendencies manifested by rare individuals expressing this mutant serpin. - Highlights: • The inhibitory specificity of the serpin alpha-1-proteinase inhibitor (API) is sharply altered in the M358R variant. • API M358R forms denaturation-resistant complexes with coagulation factor VIIa at a rate accelerated by tissue factor but unaffected by heparin. • Complex formation was shown by gel-based assays and quantified kinetically by inhibition of FVIIa-dependent amidolysis.

  5. Comparative studies on the human serum albumin binding of the clinically approved EGFR inhibitors gefitinib, erlotinib, afatinib, osimertinib and the investigational inhibitor KP2187.

    Science.gov (United States)

    Dömötör, Orsolya; Pelivan, Karla; Borics, Attila; Keppler, Bernhard K; Kowol, Christian R; Enyedy, Éva A

    2018-03-11

    Binding interactions between human serum albumin (HSA) and four approved epidermal growth factor receptor (EGFR) inhibitors gefitinib (GEF), erlotinib (ERL), afatinib (AFA), osimertinib (OSI), as well as the experimental drug KP2187, were investigated by means of spectrofluorometric and molecular modelling methods. Steady-state and time resolved spectrofluorometric techniques were carried out, including direct quenching of protein fluorescence and site marker displacement measurements. Proton dissociation processes and solvent dependent fluorescence properties were investigated as well. The EGFR inhibitors were predominantly presented in their single protonated form (HL + ) at physiological pH except ERL, which is charge-neutral. Significant solvent dependent fluorescence properties were found for GEF, ERL and KP2187, namely their emission spectra show strong dependence on the polarity and the hydrogen bonding ability of the solvents. The inhibitors proved to be bound at site I of HSA (in subdomain IIA) in a weak-to-moderate fashion (logK' 3.9-4.9) using spectrofluorometry. OSI (logK' 4.3) and KP2187 can additionally bind in site II (in subdomain IIIA), while GEF, ERL and AFA clearly show no interaction here. Docking methods qualitatively confirmed binding site preferences of compounds GEF and KP2187, and indicated that they probably bind to HSA in their neutral forms. Binding constants calculated on the basis of the various experimental data indicate a weak-to-moderate binding on HSA, only OSI exhibits somewhat higher affinity towards this protein. However, model calculations performed at physiological blood concentrations of HSA resulted in high (ca. 90%) bound fractions for the inhibitors, highlighting the importance of plasma protein binding. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Recombinant human tissue factor pathway inhibitor exerts anticoagulant, anti-inflammatory and antimicrobial effects in murine pneumococcal pneumonia

    NARCIS (Netherlands)

    van den Boogaard, F. E.; Brands, X.; Schultz, M. J.; Levi, M. [=Marcel M.; Roelofs, J. J. T. H.; van 't Veer, C.; van der Poll, T.

    2011-01-01

    Background: Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia and a major cause of sepsis. Recombinant human tissue factor pathway inhibitor (rh-TFPI) attenuates sepsis-induced coagulation and has been evaluated in clinical trials involving patients

  7. A phase I/IIa study with succinylated human serum albumin (Suc-HSA), a candidate HIV-1 fusion inhibitor

    NARCIS (Netherlands)

    Vermeulen, Joost N.; Meijer, Dirk K. F.; Over, Jan; Lange, Joep M. A.; Proost, Johannes H.; Bakker, Hester I.; Beljaars, Leonie; Wit, Ferdinand W. N. M.; Prins, Jan M.

    2007-01-01

    BACKGROUND: Succinylated human serum albumin (Suc-HAS) is a negatively charged neo-glycoprotein that binds to the positively charged V3-loop of HIV-1 gp120, acting as HIV-1-fusion inhibitor in vitro (IC50: 0.5-5.0 microg/ml). Suc-HSA was safe in rats and monkeys, and showed antiretroviral effect in

  8. A Phase I/IIa study with succinylated human serum albumin (Suc-HSA), a candidate HIV-1 fusion inhibitor

    NARCIS (Netherlands)

    Vermeulen, Joost N.; Meijer, Dirk K. F.; Over, Jan; Lange, Joep M. A.; Proost, Johannes H.; Bakker, Hester I.; Beljaars, Leonie; Wit, Ferdinand W. N. M.; Prins, Jan M.

    2007-01-01

    Background: Succinylated human serum albumin (Sue-HAS) is a negatively charged neo-glycoprotein that binds to the positively charged V3-loop of HIV-1 gp120, acting as HIV-1-fusion inhibitor in vitro (IC50: 0.5-5.0 mu g/ml). Sue-HSA was safe in rats and monkeys, and showed antiretroviral effect in a

  9. Effects of histone deacetylase inhibitors on regenerative cell responses in human dental pulp cells.

    Science.gov (United States)

    Luo, Z; Wang, Z; He, X; Liu, N; Liu, B; Sun, L; Wang, J; Ma, F; Duncan, H; He, W; Cooper, P

    2017-04-04

    To investigate the growth, migratory and adhesive effects of trichostatin A (TSA) and valproic acid (VPA), two histone deacetylase inhibitors (HDACis), on human dental pulp stem cells (hDPSCs). To verify that TSA or VPA functions as an HDAC inhibitor, the expressions of histones H3 and H4 were examined using Western blotting analysis. hDPSC growth and metabolic activity was evaluated by MTT viability analysis at different time-points and by cell count experiments. The expression of cell cycle regulatory proteins and apoptosis-associated proteins was examined by Western blot analysis. Migration effects were investigated using wound healing and transwell migration assays. An adhesion assay was also performed in the presence and absence of HDACis. The levels of chemokines and adhesion molecules relevant to repair in hDPSCs were also assessed by qRT-PCR and Western blot analysis. The data were analysed, where appropriate, using Student's t-test or one-way anova followed by the Student-Newman-Keuls test using SPSS software. Trichostatin A and VPA enhanced acetylation of histones H3 and H4 (P  0.05). At the same time, the expression of Cdx2 and cyclin A was upregulated by 2 nmol L -1 TSA and 1 mmol L -1 VPA (P < 0.05). Higher TSA or VPA concentrations induced apoptosis in hDPSCs in the cell count and apoptosis experiments (P < 0.05). Moreover, TSA and VPA significantly depressed the expression of Cdx2 and cyclin A (P < 0.05), whilst it significantly improved the level of p21 (P < 0.05). TSA and VPA promoted migration and adhesion of hDPSCs (P < 0.05). The levels of chemokines and adhesion molecules were significantly upregulated after exposure of hDPSCs to 20 nmol L -1 TSA or 1 mmol L -1 VPA (P < 0.05). Histone deacetylase inhibitors at specific concentrations promoted proliferation, migration and adhesion of hDPSCs, which may contribute to novel regenerative therapies for pulpal disease treatment. © 2017 International Endodontic Journal. Published

  10. Long-term culture of human odontoma-derived cells with a Rho kinase inhibitor.

    Science.gov (United States)

    Uzawa, Katsuhiro; Kasamatsu, Atsushi; Saito, Tomoaki; Takahara, Toshikazu; Minakawa, Yasuyuki; Koike, Kazuyuki; Yamatoji, Masanobu; Nakashima, Dai; Higo, Morihiro; Sakamoto, Yosuke; Shiiba, Masashi; Tanzawa, Hideki

    2016-09-10

    Because of cellular senescence/apoptosis, no effective culture systems are available to maintain replication of cells from odontogenic tumors especially for odontoma, and, thus, the ability to isolate human odontoma-derived cells (hODCs) for functional studies is needed. The current study was undertaken to develop an approach to isolate hODCs and fully characterize the cells in vitro. The hODCs were cultured successfully with a Rho-associated protein kinase inhibitor (Y-27632) for an extended period with stabilized lengths of the telomeres to sustain a similar phenotype/property as the primary tumoral cells. While the hODCs showed stable long-term expansion with expression of major dental epithelial markers including dentin sialophosphoprotein (DSPP) even in the three-dimensional microenvironment, they lack the specific markers for the characteristics of stem cells. Moreover, cells from dental pulp showed significant up-regulation of DSPP when co-cultured with the hODCs, while control fibroblasts with the hODCs did not. Taken together, we propose that the hODCs can be isolated and expanded over the long term with Y-27632 to investigate not only the development of the hODCs but also other types of benign human tumors. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. A comprehensive review of the pharmacodynamics of the SGLT2 inhibitor empagliflozin in animals and humans.

    Science.gov (United States)

    Michel, Martin C; Mayoux, Eric; Vallon, Volker

    2015-08-01

    Empagliflozin (formerly known as BI 10773) is a potent, competitive, and selective inhibitor of the sodium glucose transporter SGLT2, which mediates glucose reabsorption in the early proximal tubule and most of the glucose reabsorption by the kidney, overall. Accordingly, empagliflozin treatment increased urinary glucose excretion. This has been observed across multiple species including humans and was reported under euglycemic conditions, in obesity and, most importantly, in type 2 diabetic patients and multiple animal models of type 2 diabetes and of type 1 diabetes. This led to a reduction in blood glucose, smaller blood glucose excursions during oral glucose tolerance tests, and, upon chronic treatment, a reduction in HbA1c in animal models and patients. In rodents, such effects were observed in early and late phases of experimental diabetes and were associated with preservation of pancreatic β-cell function. Combination studies in animals demonstrated that beneficial metabolic effects of empagliflozin may also manifest when added to other types of anti-hyperglycemic treatments including linagliptin and pioglitazone. While some anti-hyperglycemic drugs lead to weight gain, empagliflozin treatment was associated with reduced body weight in normoglycemic obese and non-obese animals despite an increased food intake, largely due to a loss of adipose tissue; on the other hand, empagliflozin preserved body weight in models of type 1 diabetes. Empagliflozin improved endothelial dysfunction in diabetic rats and arterial stiffness, reduced blood pressure in diabetic patients, and attenuated early signs of nephropathy in diabetic animal models. Taken together, the SGLT2 inhibitor empagliflozin improves glucose metabolism by enhancing urinary glucose excretion; upon chronic administration, at least in animal models, the reductions in blood glucose levels are associated with beneficial effects on cardiovascular and renal complications of diabetes.

  12. Immunosafety of recombinant human C1-inhibitor in hereditary angioedema: evaluation of ige antibodies.

    Science.gov (United States)

    Hack, C Erik; Relan, Anurag; Baboeram, Aartie; Oortwijn, Beatrijs; Versteeg, Serge; van Ree, Ronald; Pijpstra, Rienk

    2013-04-01

    Recombinant human C1-inhibitor (rhC1INH) purified from milk of transgenic rabbits is used for the treatment of acute attacks in patients with hereditary angioedema (HAE) due to C1-inhibitor (C1INH) deficiency. The objective was to investigate the risk of rhC1INH inducing IgE antibodies or eliciting anaphylactic reactions. In subjects treated with rhC1INH, we retrospectively analysed the frequency and clinical relevance of pre-exposure and potentially newly induced IgE antibodies against rabbit and other animal allergens including cow's milk by the ImmunoCAP(®) Specific IgE blood test system. 130 HAE patients and 14 healthy subjects received 300 administrations of rhC1INH, 65 subjects (47.4 %) on one occasion; 72 (52.6 %) on at least two occasions (range 2-12; median 2). Five subjects had pre-existing anti-rabbit epithelium IgE; the subject with the highest levels and a previously undisclosed rabbit allergy developed an anaphylactic reaction upon first exposure to rhC1INH, whereas the other four subjects with lower pre-existing IgE levels (Class 1-3), did not. No other anaphylactic reactions were identified in any of the subjects exposed to rhC1INH. Analysis of post-exposure samples revealed that the risk of inducing new or boosting existing IgE responses to rabbit or cow's milk allergens was negligible. The propensity of rhC1INH to induce IgE antibodies following repeated administration of rhC1INH is low. Subjects with substantially elevated anti-rabbit epithelium IgE antibodies and/or clinical allergy to rabbits may have an increased risk for an allergic reaction. No other risk factors for allergic reactions to rhC1INH have been identified.

  13. A Novel Ras Inhibitor (MDC-1016 Reduces Human Pancreatic Tumor Growth in Mice

    Directory of Open Access Journals (Sweden)

    Gerardo G Mackenzie

    2013-10-01

    Full Text Available Pancreatic cancer has one of the poorest prognoses among all cancers partly because of its persistent resistance to chemotherapy. The currently limited treatment options for pancreatic cancer underscore the need for more efficient agents. Because activating Kras mutations initiate and maintain pancreatic cancer, inhibition of this pathway should have a major therapeutic impact. We synthesized phospho-farnesylthiosalicylic acid (PFTS; MDC-1016 and evaluated its efficacy, safety, and metabolism in preclinical models of pancreatic cancer. PFTS inhibited the growth of human pancreatic cancer cells in culture in a concentration- and time-dependent manner. In an MIA PaCa-2 xenograft mouse model, PFTS at a dose of 50 and 100 mg/kg significantly reduced tumor growth by 62% and 65% (P < .05 vs vehicle control. Furthermore, PFTS prevented pancreatitis-accelerated acinar-to-ductal metaplasia in mice with activated Kras. PFTS appeared to be safe, with the animals showing no signs of toxicity during treatment. Following oral administration, PFTS was rapidly absorbed, metabolized to FTS and FTS glucuronide, and distributed through the blood to body organs. Mechanistically, PFTS inhibited Ras-GTP, the active form of Ras, both in vitro and in vivo, leading to the inhibition of downstream effector pathways c-RAF/mitogen-activated protein-extracellular signal-regulated kinase (ERK kinase (MEK/ERK1/2 kinase and phosphatidylinositol 3-kinase/AKT. In addition, PFTS proved to be a strong combination partner with phospho-valproic acid, a novel signal transducer and activator of transcription 3 (STAT3 inhibitor, displaying synergy in the inhibition of pancreatic cancer growth. In conclusion, PFTS, a direct Ras inhibitor, is an efficacious agent for the treatment of pancreatic cancer in preclinical models, deserving further evaluation.

  14. HDAC inhibitor trichostatin A promotes proliferation and odontoblast differentiation of human dental pulp stem cells.

    Science.gov (United States)

    Jin, Hexiu; Park, Joo-Young; Choi, Hwajung; Choung, Pill-Hoon

    2013-03-01

    Trichostatin A (TSA) is a potent histone deacetylase (HDAC) inhibitor with a broad spectrum of epigenetic activities known to regulate diverse cellular mechanisms, including differentiation of mesenchymal stem cells. In this study, we demonstrate that TSA promotes proliferation and odontoblast differentiation of human dental pulp stem cells (hDPSCs) in vitro and has the ability to enhance dentin formation and odontoblast differentiation in vivo during tooth development. We observed that TSA increased the expression of proliferating cell nuclear antigen and cyclin D1 in hDPSCs at a certain concentration and the activation of JNK/c-Jun pathway was essential for TSA-dependent hDPSC proliferation. Further, TSA accelerated mineral nodule formation in vitro and increased gene expression of dentin sialophosphoprotein, dentin matrix protein 1, bone sialoprotein, and osteocalcin. In addition, TSA significantly upregulated the levels of phospho-Smad2/3, Smad4, and nuclear factor I-C, while the specific inhibitor of Smad3 inhibits TSA enhancing mineralization differentiation of hDPSCs. HDAC3 is downregulated by TSA treatment, suggesting a possible mediator of TSA-dependent pathways among the members of HDAC family. Moreover, TSA-injected embryos exhibited increased dentin thickness, larger dentin areas, and higher odontoblast numbers in their postnatal molars with stronger dentin sialoprotein expression in immunohistochemical staining. These findings indicate that TSA may serve a key role in proliferation and odontoblast differentiation of hDPSCs in dental developmental stages and can be used as an accelerator in dental hard tissue engineering.

  15. Thermodynamics parameters for binding of halogenated benzotriazole inhibitors of human protein kinase CK2α.

    Science.gov (United States)

    Winiewska, Maria; Kucińska, Katarzyna; Makowska, Małgorzata; Poznański, Jarosław; Shugar, David

    2015-10-01

    The interaction of human CK2α (hCK2α) with nine halogenated benzotriazoles, TBBt and its analogues representing all possible patterns of halogenation on the benzene ring of benzotriazole, was studied by biophysical methods. Thermal stability of protein-ligand complexes, monitored by calorimetric (DSC) and optical (DSF) methods, showed that the increase in the mid-point temperature for unfolding of protein-ligand complexes (i.e. potency of ligand binding to hCK2α) follow the inhibitory activities determined by biochemical assays. The dissociation constant for the ATP-hCK2α complex was estimated with the aid of microscale thermophoresis (MST) as 4.3±1.8 μM, and MST-derived dissociation constants determined for halogenated benzotriazoles, when converted according to known ATP concentrations, perfectly reconstruct IC50 values determined by the biochemical assays. Ligand-dependent quenching of tyrosine fluorescence, together with molecular modeling and DSC-derived heats of unfolding, support the hypothesis that halogenated benzotriazoles bind in at least two alternative orientations, and those that are efficient hCK2α inhibitors bind in the orientation which TBBt adopts in its complex with maize CK2α. DSC-derived apparent heat for ligand binding (ΔΔHbind) is driven by intermolecular electrostatic interactions between Lys68 and the triazole ring of the ligand, as indicated by a good correlation between ΔΔHbind and ligand pKa. Overall results, additionally supported by molecular modeling, confirm that a balance of hydrophobic and electrostatic interactions contribute predominantly (~40 kJ/mol), relative to possible intermolecular halogen/hydrogen bonding (less than 10 kJ/mol), in binding of halogenated benzotriazoles to the ATP-binding site of hCK2α. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Structure of a Myeloid cell leukemia-1 (Mcl-1) inhibitor bound to drug site 3 of Human Serum Albumin.

    Science.gov (United States)

    Zhao, Bin; Sensintaffar, John; Bian, Zhiguo; Belmar, Johannes; Lee, Taekyu; Olejniczak, Edward T; Fesik, Stephen W

    2017-06-15

    Amplification of the gene encoding Myeloid cell leukemia-1 (Mcl-1) is one of the most common genetic aberrations in human cancer and is associated with high tumor grade and poor survival. Recently, we reported on the discovery of high affinity Mcl-1 inhibitors that elicit mechanism-based cell activity. These inhibitors are lipophilic and contain an acidic functionality which is a common chemical profile for compounds that bind to albumin in plasma. Indeed, these Mcl-1 inhibitors exhibited reduced in vitro cell activity in the presence of serum. Here we describe the structure of a lead Mcl-1 inhibitor when bound to Human Serum Albumin (HSA). Unlike many acidic lipophilic compounds that bind to drug site 1 or 2, we found that this Mcl-1 inhibitor binds predominantly to drug site 3. Site 3 of HSA may be able to accommodate larger, more rigid compounds that do not fit into the smaller drug site 1 or 2. Structural studies of molecules that bind to this third site may provide insight into how some higher molecular weight compounds bind to albumin and could be used to aid in the design of compounds with reduced albumin binding. Copyright © 2017. Published by Elsevier Ltd.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-07-19

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

  18. Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation

    DEFF Research Database (Denmark)

    Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina

    2015-01-01

    ) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced...... has an important role in controlling the conformational equilibrium of human SERT....

  19. Identification of a hexapeptide inhibitor of the human immunodeficiency virus integrase protein by using a combinatorial chemical library.

    OpenAIRE

    Puras Lutzke, R A; Eppens, N A; Weber, P A; Houghten, R A; Plasterk, R H

    1995-01-01

    Integration of human immunodeficiency virus (HIV) DNA into the human genome requires the virus-encoded integrase (IN) protein, and therefore the IN protein is a suitable target for antiviral strategies. To find a potent HIV IN inhibitor, we screened a "synthetic peptide combinatorial library." We identified a hexapeptide with the sequence HCKFWW that inhibits IN-mediated 3'-processing and integration with an IC50 of 2 microM. The peptide is active on IN proteins from other retroviruses such a...

  20. Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase.

    Science.gov (United States)

    Waldman, Barbara Criscuolo; Wang, Yibin; Kilaru, Kasturi; Yang, Zhengguan; Bhasin, Alaukik; Wyatt, Michael D; Waldman, Alan S

    2008-10-01

    Thymidylate deprivation brings about "thymineless death" in prokaryotes and eukaryotes. Although the precise mechanism for thymineless death has remained elusive, inhibition of the enzyme thymidylate synthase (TS), which catalyzes the de novo synthesis of TMP, has served for many years as a basis for chemotherapeutic strategies. Numerous studies have identified a variety of cellular responses to thymidylate deprivation, including disruption of DNA replication and induction of DNA breaks. Since stalled or collapsed replication forks and strand breaks are generally viewed as being recombinogenic, it is not surprising that a link has been demonstrated between recombination induction and thymidylate deprivation in bacteria and lower eukaryotes. A similar connection between recombination and TS inhibition has been suggested by studies done in mammalian cells, but the relationship between recombination and TS inhibition in mammalian cells had not been demonstrated rigorously. To gain insight into the mechanism of thymineless death in mammalian cells, in this work we undertook a direct investigation of recombination in human cells treated with raltitrexed (RTX), a folate analog that is a specific inhibitor of TS. Using a model system to study intrachromosomal homologous recombination in cultured fibroblasts, we provide definitive evidence that treatment with RTX can stimulate accurate recombination events in human cells. Gene conversions not associated with crossovers were specifically enhanced several-fold by RTX. Additional experiments demonstrated that recombination events provoked by a double-strand break (DSB) were not impacted by treatment with RTX, nor was error-prone DSB repair via nonhomologous end-joining. Our work provides evidence that thymineless death in human cells is not mediated by corruption of DSB repair processes and suggests that an increase in chromosomal recombination may be an important element of cellular responses leading to thymineless death.

  1. Scabies mite inactive serine proteases are potent inhibitors of the human complement lectin pathway.

    Directory of Open Access Journals (Sweden)

    Simone L Reynolds

    2014-05-01

    Full Text Available Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classified as one of the six most prevalent epidermal parasitic skin diseases infecting populations living in poverty by the World Health Organisation. The role of the complement system, a pivotal component of human innate immunity, as an important defence against invading pathogens has been well documented and many parasites have an arsenal of anti-complement defences. We previously reported on a family of scabies mite proteolytically inactive serine protease paralogues (SMIPP-Ss thought to be implicated in host defence evasion. We have since shown that two family members, SMIPP-S D1 and I1 have the ability to bind the human complement components C1q, mannose binding lectin (MBL and properdin and are capable of inhibiting all three human complement pathways. This investigation focused on inhibition of the lectin pathway of complement activation as it is likely to be the primary pathway affecting scabies mites. Activation of the lectin pathway relies on the activation of MBL, and as SMIPP-S D1 and I1 have previously been shown to bind MBL, the nature of this interaction was examined using binding and mutagenesis studies. SMIPP-S D1 bound MBL in complex with MBL-associated serine proteases (MASPs and released the MASP-2 enzyme from the complex. SMIPP-S I1 was also able to bind MBL in complex with MASPs, but MASP-1 and MASP-2 remained in the complex. Despite these differences in mechanism, both molecules inhibited activation of complement components downstream of MBL. Mutagenesis studies revealed that both SMIPP-Ss used an alternative site of the molecule from the residual active site region to inhibit the lectin pathway. We propose that SMIPP-Ss are potent lectin pathway inhibitors and that this mechanism represents an important tool in the immune evasion repertoire of the parasitic mite and a potential target for therapeutics.

  2. Human inter-α-inhibitor is a substrate for factor XIIIa and tissue transglutaminase

    DEFF Research Database (Denmark)

    Sonne-Schmidt, Carsten Scavenius; Sanggaard, Kristian Wejse; Nikolajsen, Camilla Lund

    2011-01-01

    In this study, we show that inter-α-inhibitor is a substrate for both factor XIIIa and tissue transglutaminase. These enzymes catalyze the incorporation of dansylcadaverine and biotin-pentylamine, revealing that inter-α-inhibitor contains reactive Gln residues within all three subunits. These fin......In this study, we show that inter-α-inhibitor is a substrate for both factor XIIIa and tissue transglutaminase. These enzymes catalyze the incorporation of dansylcadaverine and biotin-pentylamine, revealing that inter-α-inhibitor contains reactive Gln residues within all three subunits...

  3. Apoptosis inhibitor 5 (API-5; AAC-11; FIF) is upregulated in human carcinomas in vivo.

    Science.gov (United States)

    Koci, Lenka; Chlebova, Katarina; Hyzdalova, Martina; Hofmanova, Jirina; Jira, Miroslav; Kysela, Petr; Kozubik, Alois; Kala, Zdenek; Krejci, Pavel

    2012-04-01

    Apoptosis inhibitor 5 (API-5) is a 55 kDa nuclear protein with potent anti-apoptotic signaling in tumor cells in vitro. In this study, we analyzed the expression of the API-5 protein in vivo in a broad spectrum of human carcinomas, including those of the colon, lung, liver, kidney, pancreas, stomach and esophagus using tumor tissues obtained during tumor resection. The results showed significant upregulation of API-5 expression in biopsies of lung (23%, n=13) and colorectal tumors (33%, n=27) in comparison with biopsies from the adjacent normal tissue. Colon cancer biopsies were used to study the cell populations with an upregulated level of expression of API-5 more closely. Using a magnetic bead-based selection for the epithelial cell marker EpCAM, we purified epithelial cells from the tumor and control tissues and analyzed these cells for API-5 expression by western immunoblotting. We observed that EpCAM-positive tumor cells expressed API-5 in all three colorectal cancer cases tested, in contrast to the control EpCAM-positive and EpCAM-negative cells isolated from the control or tumor tissues. These data suggest that the expression of the API-5 protein is upregulated in tumor epithelial cells and may serve as a prognostic marker in colorectal cancer.

  4. Expression of ODC Antizyme Inhibitor 2 (AZIN2 in Human Secretory Cells and Tissues.

    Directory of Open Access Journals (Sweden)

    Tiina Rasila

    Full Text Available Ornithine decarboxylase (ODC antizyme inhibitor 2 (AZIN2, originally called ODCp, is a regulator of polyamine synthesis that we originally identified and cloned. High expression of ODCp mRNA was found in brain and testis. We reported that AZIN2 is involved in regulation of cellular vesicle transport and / or secretion, but the ultimate physiological role(s of AZIN2 is still poorly understood. In this study we used a peptide antibody (K3 to human AZIN2 and by immunohistochemistry mapped its expression in various normal tissues. We found high expression in the nervous system, in type 2 pneumocytes in the lung, in megakaryocytes, in gastric parietal cells co-localized with H,K-ATPase beta subunit, in selected enteroendocrine cells, in acinar cells of sweat glands, in podocytes, in macula densa cells and epithelium of collecting ducts in the kidney. The high expression of AZIN2 in various cells with secretory or vesicle transport activity indicates that the polyamine metabolism regulated by AZIN2 is more significantly involved in these events than previously appreciated.

  5. Biochemical characterization and structure determination of a potent, selective antibody inhibitor of human MMP9.

    Science.gov (United States)

    Appleby, Todd C; Greenstein, Andrew E; Hung, Magdeleine; Liclican, Albert; Velasquez, Maile; Villaseñor, Armando G; Wang, Ruth; Wong, Melanie H; Liu, Xiaohong; Papalia, Giuseppe A; Schultz, Brian E; Sakowicz, Roman; Smith, Victoria; Kwon, Hyock Joo

    2017-04-21

    Matrix metalloproteinase 9 (MMP9) is a member of a large family of proteases that are secreted as inactive zymogens. It is a key regulator of the extracellular matrix, involved in the degradation of various extracellular matrix proteins. MMP9 plays a pathological role in a variety of inflammatory and oncology disorders and has long been considered an attractive therapeutic target. GS-5745, a potent, highly selective humanized monoclonal antibody inhibitor of MMP9, has shown promise in treating ulcerative colitis and gastric cancer. Here we describe the crystal structure of GS-5745·MMP9 complex and biochemical studies to elucidate the mechanism of inhibition of MMP9 by GS-5745. GS-5745 binds MMP9 distal to the active site, near the junction between the prodomain and catalytic domain, and inhibits MMP9 by two mechanisms. Binding to pro-MMP9 prevents MMP9 activation, whereas binding to active MMP9 allosterically inhibits activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. EGFR inhibitor C225 increases the radiosensitivity of human lung squamous cancer cells

    Directory of Open Access Journals (Sweden)

    Yang Ruijie

    2010-10-01

    Full Text Available Abstract Background The purpose of the present study is to investigate the direct biological effects of the epidermal growth factor receptor (EGFR inhibitor C225 on the radiosensitivity of human lung squamous cancer cell-H520. H520 cells were treated with different dosage of 60Co γ ray irradiation (1.953 Gy/min in the presence or absence of C225. The cellular proliferation, colony forming capacity, apoptosis, the cell cycle distribution as well as caspase-3 were analyzed in vitro. Results We found that C225 treatment significantly increased radiosensitivity of H-520 cells to irradiation, and led to cell cycle arrest in G1 phase, whereas 60Co γ ray irradiation mainly caused G2 phase arrest. H-520 cells thus displayed both the G1 and G2 phase arrest upon treatment with C225 in combination with 60Co γ ray irradiation. Moreover, C225 treatment significantly increased the apoptosis percentage of H-520 cells (13.91% ± 1.88% compared with the control group (5.75% ± 0.64%, P Conclusion In this regard, C225 treatment may make H-520 cells more sensitive to irradiation through the enhancement of caspase-3 mediated tumor cell apoptosis and cell cycle arrest.

  7. Resorcinol-, catechol- and saligenin-based bronchodilating β2-agonists as inhibitors of human cholinesterase activity.

    Science.gov (United States)

    Bosak, Anita; Knežević, Anamarija; Gazić Smilović, Ivana; Šinko, Goran; Kovarik, Zrinka

    2017-12-01

    We investigated the influence of bronchodilating β2-agonists on the activity of human acetylcholinesterase (AChE) and usual, atypical and fluoride-resistant butyrylcholinesterase (BChE). We determined the inhibition potency of racemate and enantiomers of fenoterol as a resorcinol derivative, isoetharine and epinephrine as catechol derivatives and salbutamol and salmeterol as saligenin derivatives. All of the tested compounds reversibly inhibited cholinesterases with K i constants ranging from 9.4 μM to 6.4 mM and had the highest inhibition potency towards usual BChE, but generally none of the cholinesterases displayed any stereoselectivity. Kinetic and docking results revealed that the inhibition potency of the studied compounds could be related to the size of the hydroxyaminoethyl chain on the benzene ring. The additional π-π interaction of salmeterol's benzene ring and Trp286 and hydrogen bond with His447 probably enhanced inhibition by salmeterol which was singled out as the most potent inhibitor of all the cholinesterases.

  8. Antipsychotic agents screened as human carbonic anhydrase I and II inhibitors.

    Science.gov (United States)

    Erzengin, Mahmut; Bilen, Cigdem; Ergun, Adem; Gencer, Nahit

    2014-02-01

    The antipsychotic drugs currently used to treat schizophrenia can be divided into two distinct classes, typical and atypical antipsychotics. Many drug molecules are enzyme inhibitors that bind reversibly or irreversibly to their target through intermolecular interactions. That's why enzyme inhibition studies are an important issue for drug design and biochemical applications. In this study, in vitro inhibition effect of some antipsychotic drugs on the purified carbonic anhydrase (CA) I and II isoenzymes were investigated by using CO2 as a substrate. CA I and II were purified from human erythrocytes by a simple one step procedure using Sepharose 4B-L-tyrosine-sulfonamide affinity column. The results showed that all the drugs inhibited the cytosolic carbonic anhydrases enzyme activity in a concentration-dependent fashion. Among the studied drugs, aripiprazole and pramipexole were found to be the most active one for hCA I (IC50: 3.64 and 5.37 μM) and hCA II (IC50: 4.16 and 4.81 μM) activity, respectively.

  9. Plant-derived protease inhibitors LC-pi (Lavatera cashmeriana) inhibit human lung cancer cell proliferation in vitro.

    Science.gov (United States)

    Rakashanda, Syed; Qazi, Asif Khurshid; Majeed, Rabiya; Andrabi, Syed Mubashir; Hamid, Abid; Sharma, P R; Amin, Shajrul

    2015-01-01

    The objective of this study was to check the anticancer activity of purified protease inhibitors of Lavatera cashmeriana viz LC-pi I, II, III, and IV (Lavatera cashmeriana protease inhibitors) on A549 (lung) cell. It was found that LC-pi I and II significantly inhibited the proliferation of A549 cells with IC₅₀ value of 54 μg/ml and 38 μg/ml, respectively, whereas inhibition by LC-pi III and IV was negligible. LC-pi I and II were further found to inhibit formation of colonies in a dose-dependent manner. Also, both inhibitors were found to induce apoptosis causing chromatin condensation and DNA fragmentation, without loss of mitochondrial membrane potential. Cell cycle revealed a significant increase of subG₀/G₁ phase cells that are apoptotic cells. We also demonstrated a dose-dependent decrease in migration of A549 cells on cell migration assay by both inhibitors. Taken together, we demonstrate that LC-pi I and II inhibited proliferation through arresting cells before apoptosis, inducing apoptosis and inhibiting cell migration in human lung cancer cells, but the study warrants further investigation. Our results support the notion that plant protease inhibitors may have the potential to advance as chemopreventive agents.

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

    Science.gov (United States)

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

    2009-01-01

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

  11. Thermodynamic parameters for binding of some halogenated inhibitors of human protein kinase CK2

    Energy Technology Data Exchange (ETDEWEB)

    Winiewska, Maria; Makowska, Małgorzata [Institute of Biochemistry and Biophysics PAS, Warszawa (Poland); Maj, Piotr [Institute of Biochemistry and Biophysics PAS, Warszawa (Poland); Nencki Institute of Experimental Biology PAS, Warszawa (Poland); Wielechowska, Monika; Bretner, Maria [Warsaw University of Technology, Faculty of Chemistry, Warszawa (Poland); Poznański, Jarosław, E-mail: jarek@ibb.waw.pl [Institute of Biochemistry and Biophysics PAS, Warszawa (Poland); Shugar, David [Institute of Biochemistry and Biophysics PAS, Warszawa (Poland)

    2015-01-02

    Highlights: • Two new compounds being potential human CK2a inhibitors are studied. • Their IC50 values were determined in vitro. • The heats of binding and kbind were estimated using DSC. • The increased stability of protein–ligand complexes was followed by fluorescence. • Methylated TBBt derivative (MeBr3Br) is almost as active as TBBt. - Abstract: The interaction of human CK2α with a series of tetrabromobenzotriazole (TBBt) and tetrabromobenzimidazole (TBBz) analogs, in which one of the bromine atoms proximal to the triazole/imidazole ring is replaced by a methyl group, was studied by biochemical (IC{sub 50}) and biophysical methods (thermal stability of protein–ligand complex monitored by DSC and fluorescence). Two newly synthesized tri-bromo derivatives display inhibitory activity comparable to that of the reference compounds, TBBt and TBBz, respectively. DSC analysis of the stability of protein–ligand complexes shows that the heat of ligand binding (H{sub bind}) is driven by intermolecular electrostatic interactions involving the triazole/imidazole ring, as indicated by a strong correlation between H{sub bind} and ligand pK{sub a}. Screening, based on fluorescence-monitored thermal unfolding of protein–ligand complexes, gave comparable results, clearly identifying ligands that most strongly bind to the protein. Overall results, additionally supported by molecular modeling, confirm that a balance of hydrophobic and electrostatic interactions contribute predominantly, relative to possible intermolecular halogen bonding, in binding of the ligands to the CK2α ATP-binding site.

  12. 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.

  13. Carbohydrate as covalent crosslink in human inter-alpha-trypsin inhibitor

    DEFF Research Database (Denmark)

    Jessen, T E; Faarvang, K L; Ploug, M

    1988-01-01

    The primary structure of inter-alpha-trypsin inhibitor is partially elucidated, but controversy about the construction of the polypeptide backbone still exists. We present evidence suggesting that inter-alpha-trypsin inhibitor represents a novel plasma protein structure with two separate polypept...... polypeptide chains covalently crosslinked only by carbohydrate (chondroitin sulphate)....

  14. Characterization of transgenic mice that secrete functional human protein C inhibitor into the circulation

    NARCIS (Netherlands)

    Wagenaar, G. T.; van Vuuren, A. J.; Girma, M.; Tiekstra, M. J.; Kwast, L.; Koster, J. G.; Rijneveld, A. W.; Elisen, M. G.; van der Poll, T.; Meijers, J. C.

    2000-01-01

    Protein C inhibitor (PCI) is a heparin binding serine protease inhibitor in plasma, which exerts procoagulant activity by inhibiting thrombomodulin-bound thrombin or activated protein C (APC). Since the role of PCI in vivo is largely unknown we generated genetically modified mice with expression of

  15. Crystal Structure of Novel Metallocarboxypeptidase Inhibitor from Marine Mollusk Nerita versicolor in Complex with Human Carboxypeptidase A4*

    Science.gov (United States)

    Covaleda, Giovanni; Alonso del Rivero, Maday; Chávez, María A.; Avilés, Francesc X.; Reverter, David

    2012-01-01

    NvCI is a novel exogenous proteinaceous inhibitor of metallocarboxypeptidases from the marine snail Nerita versicolor. The complex between human carboxypeptidase A4 and NvCI has been crystallized and determined at 1.7 Å resolution. The NvCI structure defines a distinctive protein fold basically composed of a two-stranded antiparallel β-sheet connected by three loops and the inhibitory C-terminal tail and stabilized by three disulfide bridges. NvCI is a tight-binding inhibitor that interacts with the active site of the enzyme in a substrate-like manner. NvCI displays an extended and novel interface with human carboxypeptidase A4, responsible for inhibitory constants in the picomolar range for some members of the M14A subfamily of carboxypeptidases. This makes NvCI the strongest inhibitor reported so far for this family. The structural homology displayed by the C-terminal tails of different carboxypeptidase inhibitors represents a relevant example of convergent evolution. PMID:22294694

  16. Crystal structure of novel metallocarboxypeptidase inhibitor from marine mollusk Nerita versicolor in complex with human carboxypeptidase A4.

    Science.gov (United States)

    Covaleda, Giovanni; del Rivero, Maday Alonso; Chávez, María A; Avilés, Francesc X; Reverter, David

    2012-03-16

    NvCI is a novel exogenous proteinaceous inhibitor of metallocarboxypeptidases from the marine snail Nerita versicolor. The complex between human carboxypeptidase A4 and NvCI has been crystallized and determined at 1.7 Å resolution. The NvCI structure defines a distinctive protein fold basically composed of a two-stranded antiparallel β-sheet connected by three loops and the inhibitory C-terminal tail and stabilized by three disulfide bridges. NvCI is a tight-binding inhibitor that interacts with the active site of the enzyme in a substrate-like manner. NvCI displays an extended and novel interface with human carboxypeptidase A4, responsible for inhibitory constants in the picomolar range for some members of the M14A subfamily of carboxypeptidases. This makes NvCI the strongest inhibitor reported so far for this family. The structural homology displayed by the C-terminal tails of different carboxypeptidase inhibitors represents a relevant example of convergent evolution.

  17. Characterization of recombinant human C1 inhibitor secreted in milk of transgenic rabbits.

    Science.gov (United States)

    van Veen, Harrie A; Koiter, Jaco; Vogelezang, Carla J M; van Wessel, Noucha; van Dam, Tijtje; Velterop, Ingeborg; van Houdt, Kristina; Kupers, Luc; Horbach, Danielle; Salaheddine, Mourad; Nuijens, Jan H; Mannesse, Maurice L M

    2012-12-31

    C1 inhibitor (C1INH) is a single-chain glycoprotein that inhibits activation of the contact system of coagulation and the complement system. C1INH isolated from human blood plasma (pd-hC1INH) is used for the management of hereditary angioedema (HAE), a disease caused by heterozygous deficiency of C1INH, and is a promise for treatment of ischemia-reperfusion injuries like acute myocardial or cerebral infarction. To obtain large quantities of C1INH, recombinant human C1INH (rhC1INH) was expressed in the milk of transgenic rabbits (12 g/l) harboring genomic human C1INH sequences fused to 5' bovine αS(1) casein promoter sequences. Recombinant hC1INH was isolated from milk to a specific activity of 6.1 U/mg and a purity of 99%; by size-exclusion chromatography the 1% impurities consisted of multimers and N-terminal cleaved C1INH species. Mass spectrometric analysis of purified rhC1INH revealed a relative molecular mass (M(r)) of 67,200. Differences in M(r) on SDS PAGE and mass spectrometric analysis between rhC1INH and pd-hC1INH are explained by differential glycosylation (calculated carbohydrate contents of 21% and 28%, respectively), since protein sequencing analysis of rhC1INH revealed intact N- and C-termini. Host-related impurity analysis by ELISA revealed trace amounts of rabbit protein (approximately 10 ppm) in purified batches, but not endogenous rabbit C1INH. The kinetics of inhibition of the target proteases C1s, Factor XIIa, kallikrein and Factor XIa by rhC1INH and pd-hC1INH, indicated comparable inhibitory potency and specificity. Recently, rhC1INH (Ruconest(®)) has been approved by the European Medicines Agency for the treatment of acute attacks of HAE. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. An organometallic inhibitor for the human repair enzyme 7,8-dihydro-8-oxoguanosine triphosphatase.

    Science.gov (United States)

    Streib, Manuel; Kräling, Katja; Richter, Kristin; Xie, Xiulan; Steuber, Holger; Meggers, Eric

    2014-01-03

    The probe-based discovery of the first small-molecule inhibitor of the repair enzyme 8-oxo-dGTPase (MTH1) is presented, which is an unconventional cyclometalated ruthenium half-sandwich complex. The organometallic inhibitor with low-nanomolar activity displays astonishing specificity, as verified in tests with an extended panel of protein kinases and other ATP binding proteins. The binding of the organometallic inhibitor to MTH1 is investigated by protein crystallography. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Plasma and cerebrospinal fluid pharmacokinetics of the histone deacetylase inhibitor, belinostat (PXD101), in non-human primates

    DEFF Research Database (Denmark)

    Warren, K.E.; McCully, C.; Dvinge, H.

    2008-01-01

    is a novel, potent, pan-HDAC inhibitor with antiproliferative activity on a wide variety of tumor cell lines. We studied the cerebrospinal fluid (CSF) penetration of intravenous (IV) belinostat in a non-human primate model as a surrogate for blood:brain barrier penetration. DESIGN: Five adult rhesus monkeys....... CSF drug exposure was drug exposure and drug exposure. CONCLUSION: IV belinostat is rapidly cleared from plasma and has limited penetration into the CSF Udgivelsesdato: 2008/8...

  20. Cystatins - Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Wallin, Hanna; Bjarnadottir, Maria; Vogel, Lotte

    2010-01-01

    signal peptides) for cellular export following translation. Results indicating existence of systems for significant internalisation of type 2 cystatins from the extracellular to intracellular compartments are reviewed. Data showing that human neuroblastoma cell lines generally secrete high levels...... staining was observed. The simplistic denotation of the type 2 cystatins as extracellular inhibitors is thus challenged, and possible biological functions of the internalised cystatins are discussed. To illustrate the special case of high cellular cystatin content seen in cells of patients with hereditary...

  1. Population pharmacokinetics of recombinant human C1 inhibitor in patients with hereditary angioedema.

    Science.gov (United States)

    Farrell, Colm; Hayes, Siobhan; Relan, Anurag; van Amersfoort, Edwin S; Pijpstra, Rienk; Hack, C Erik

    2013-12-01

    To characterize the pharmacokinetics (PK) of recombinant human C1 inhibitor (rhC1INH) in healthy volunteers and hereditary angioedema (HAE) patients. Plasma levels of C1INH following 294 administrations of rhC1INH in 133 subjects were fitted using nonlinear mixed-effects modelling. The model was used to simulate maximal C1INH levels for the proposed dosing scheme. A one-compartment model with Michaelis-Menten elimination kinetics described the data. Baseline C1INH levels were 0.901 [95% confidence interval (CI): 0.839-0.968] and 0.176 U ml(-1) (95% CI: 0.154-0.200) in healthy volunteers and HAE patients, respectively. The volume of distribution of rhC1INH was 2.86 l (95% CI: 2.68-3.03). The maximal rate of elimination and the concentration corresponding to half this maximal rate were 1.63 U ml(-1) h(-1) (95% CI: 1.41-1.88) and 1.60 U ml(-1) (95% CI: 1.14-2.24), respectively, for healthy volunteers and symptomatic HAE patients. The maximal elimination rate was 36% lower in asymptomatic HAE patients. Peak C1INH levels did not change upon repeated administration of rhC1INH. Bodyweight was found to be an important predictor of the volume of distribution. Simulations of the proposed dosing scheme predicted peak C1INH concentrations above the lower level of the normal range (0.7 U ml(-1)) for at least 94% of all patients. The population PK model for C1INH supports a dosing scheme on a 50 U kg(-1) basis up to 84 kg, with a fixed dose of 4200 U above 84 kg. The PK of rhC1INH following repeat administration are consistent with the PK following the first administration. © 2013 The British Pharmacological Society.

  2. Thrombin-activatable fibrinolysis inhibitor influences disease severity in humans and mice with pneumococcal meningitis.

    Science.gov (United States)

    Mook-Kanamori, B B; Valls Serón, M; Geldhoff, M; Havik, S R; van der Ende, A; Baas, F; van der Poll, T; Meijers, J C M; P Morgan, B; Brouwer, M C; van de Beek, D

    2015-11-01

    Mortality and morbidity in patients with bacterial meningitis result from the proinflammatory response and dysregulation of coagulation and fibrinolysis. Thrombin-activatable fibrinolysis inhibitor (TAFI) is activated by free thrombin or thrombin in complex with thrombomodulin, and plays an antifibrinolytic role during fibrin clot degradation, but also has an anti-inflammatory role by inactivating proinflammatory mediators, such as complement activation products. To assess the role of TAFI in pneumococcal meningitis. We performed a prospective nationwide genetic association study in patients with bacterial meningitis, determined TAFI and complement levels in cerebrospinal fluid (CSF), and assessed the function of TAFI in a pneumococcal meningitis mouse model by using Cpb2 (TAFI) knockout mice. Polymorphisms (reference sequences: rs1926447 and rs3742264) in the CPB2 gene, coding for TAFI, were related to the development of systemic complications in patients with pneumococcal meningitis. Higher protein levels of TAFI in CSF were significantly associated with CSF complement levels (C3a, iC3b, and C5b-9) and with more systemic complications in patients with bacterial meningitis. The risk allele of rs1926447 (TT) was associated with higher levels of TAFI in CSF. In the murine model, consistent with the human data, Cpb2-deficient mice had decreased disease severity, as reflected by lower mortality, and attenuated cytokine levels and bacterial outgrowth in the systemic compartment during disease, without differences in the brain compartment, as compared with wild-type mice. These findings suggest that TAFI plays an important role during pneumococcal meningitis, which is likely to be mediated through inhibition of the complement system, and influences the occurrence of systemic complications and inflammation. © 2015 International Society on Thrombosis and Haemostasis.

  3. Synthetic peptides mimicking the binding site of human acetylcholinesterase for its inhibitor fasciculin 2.

    Science.gov (United States)

    Kafurke, Uwe; Erijman, Ariel; Aizner, Yonatan; Shifman, Julia M; Eichler, Jutta

    2015-09-01

    Molecules capable of mimicking protein binding and/or functional sites present useful tools for a range of biomedical applications, including the inhibition of protein-ligand interactions. Such mimics of protein binding sites can currently be generated through structure-based design and chemical synthesis. Computational protein design could be further used to optimize protein binding site mimetics through rationally designed mutations that improve intermolecular interactions or peptide stability. Here, as a model for the study, we chose an interaction between human acetylcholinesterase (hAChE) and its inhibitor fasciculin-2 (Fas) because the structure and function of this complex is well understood. Structure-based design of mimics of the hAChE binding site for Fas yielded a peptide that binds to Fas at micromolar concentrations. Replacement of hAChE residues known to be essential for its interaction with Fas with alanine, in this peptide, resulted in almost complete loss of binding to Fas. Computational optimization of the hAChE mimetic peptide yielded a variant with slightly improved affinity to Fas, indicating that more rounds of computational optimization will be required to obtain peptide variants with greatly improved affinity for Fas. CD spectra in the absence and presence of Fas point to conformational changes in the peptide upon binding to Fas. Furthermore, binding of the optimized hAChE mimetic peptide to Fas could be inhibited by hAChE, providing evidence for a hAChE-specific peptide-Fas interaction. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

  4. Human monoamine oxidase is inhibited by tobacco smoke: β-carboline alkaloids act as potent and reversible inhibitors

    International Nuclear Information System (INIS)

    Herraiz, Tomas; Chaparro, Carolina

    2005-01-01

    Monoamine oxidase (MAO) is a mitochondrial outer-membrane flavoenzyme involved in brain and peripheral oxidative catabolism of neurotransmitters and xenobiotic amines, including neurotoxic amines, and a well-known target for antidepressant and neuroprotective drugs. Recently, positron emission tomography imaging has shown that smokers have a much lower activity of peripheral and brain MAO-A (30%) and -B (40%) isozymes compared to non-smokers. This MAO inhibition results from a pharmacological effect of smoke, but little is known about its mechanism. Working with mainstream smoke collected from commercial cigarettes we confirmed that cigarette smoke is a potent inhibitor of human MAO-A and -B isozymes. MAO inhibition was partly reversible, competitive for MAO-A, and a mixed-type inhibition for MAO-B. Two β-carboline alkaloids, norharman (β-carboline) and harman (1-methyl-β-carboline), were identified by GC-MS, quantified, and isolated from the mainstream smoke by solid phase extraction and HPLC. Kinetics analysis revealed that β-carbolines from cigarette smoke were competitive, reversible, and potent inhibitors of MAO enzymes. Norharman was an inhibitor of MAO-A (K i = 1.2 ± 0.18 μM) and MAO-B (K i = 1.12 ± 0.19 μM), and harman of MAO-A (K i = 55.54 ± 5.3 nM). β-Carboline alkaloids are psychopharmacologically active compounds that may occur endogenously in human tissues, including the brain. These results suggest that β-carboline alkaloids from cigarette smoke acting as potent reversible inhibitors of MAO enzymes may contribute to the MAO-reduced activity produced by tobacco smoke in smokers. The presence of MAO inhibitors in smoke like β-carbolines and others may help us to understand some of the purported neuropharmacological effects associated with smoking

  5. Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation.

    Science.gov (United States)

    Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch; Sinning, Steffen; Kristensen, Anders Skov; Strømgaard, Kristian; Andersen, Jacob

    2015-06-05

    The serotonin transporter (SERT) terminates serotonergic neurotransmission by performing reuptake of released serotonin, and SERT is the primary target for antidepressants. SERT mediates the reuptake of serotonin through an alternating access mechanism, implying that a central substrate site is connected to both sides of the membrane by permeation pathways, of which only one is accessible at a time. The coordinated conformational changes in SERT associated with substrate translocation are not fully understood. Here, we have identified a Leu to Glu mutation at position 406 (L406E) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced by L406E are not simply charge-related effects. Leu(406) is located >10 Å from the central inhibitor binding site indicating that the mutation affects inhibitor binding in an indirect manner. We found that L406E decreased accessibility to a residue in the cytoplasmic pathway. The shift in equilibrium to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4 has an important role in controlling the conformational equilibrium of human SERT. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Effect of synthetic matrix-metalloproteinase inhibitors on invasive capacity and proliferation of human malignant gliomas in vitro.

    Science.gov (United States)

    Tonn, J C; Kerkau, S; Hanke, A; Bouterfa, H; Mueller, J G; Wagner, S; Vince, G H; Roosen, K

    1999-03-01

    Glioma invasion into the surrounding brain tissue is still a major obstacle for any therapeutical approach. As in other solid tumors, matrix-metalloproteases (MMPs) have been suggested as being involved. The aim of this study was to evaluate whether the use of MMP inhibitors to target the protease-mediated invasion process could be a feasible approach. Two human cell lines (U251 and GaMG) and surgical specimens of 6 patients with malignant gliomas were grown as monolayers and spheroid cultures respectively. MMP- and u-PA-mRNA expression was investigated by semi-quantitative RT-PCR. Invasion was studied in Matrigel-coated Boyden chamber transwell assays for monolayers and in confrontation cultures of tumor spheroids with fetal rat brain aggregates in the presence of the synthetic MMP inhibitors batimastat (BB-94) and marimastat (BB-2516). Cytotoxicity/cytostatic effects of high concentrations of both compounds were assessed by growth curves, MTT assays and flow cytometry in human glioma cell lines. Batimastat and marimastat revealed a cytostatic effect at high concentrations (above 1 microM) without cytotoxicity. Both MMP inhibitors effectively reduced glioma invasion in Boyden-chamber assays at low concentrations of 0.3 microM. In confrontation cultures, concentrations of 10 microM and above were necessary to reduce invasion. This effect was observable with inter-individual heterogeneity in the patient's tumor material. MMP inhibitors effectively reduce glioma invasion, although high concentrations were required in 3-dimensional culture systems. At these concentrations, both compounds revealed a cytostatic, but no cytotoxic effect. Thus, high local concentrations of MMP inhibitors could offer a new therapeutic strategy for the treatment of gliomas.

  7. Restoration of Transforming Growth Factor Beta Signaling by Histone Deacetylase Inhibitors in Human Prostate Carcinoma

    National Research Council Canada - National Science Library

    Qian, Zheng D

    2006-01-01

    The goal of the current grant is to investigate the potential antitumor activity of histone deacetylase inhibitor MS-275 along with the activation of TGFb signaling pathway with the restoration of TGFb receptor II...

  8. Restoration of Transforming Growth Factor Beta Signaling by Histone Deacetylase Inhibitors in Human Prostate Carcinoma

    National Research Council Canada - National Science Library

    Qian, Zheng D

    2005-01-01

    The goal of the current grant is to investigate the potential antitumor activity of histone deacetylase inhibitor MS-275 a with the activation of TGFb signaling pathway with the restoration of TGFbeta receptor II...

  9. Molecular Modulation of Inhibitors of Apoptosis as a Novel Approach for Radiosensitization of Human Prostate Cancer

    National Research Council Canada - National Science Library

    Xu, Liang

    2007-01-01

    .... IAP-inhibitorspotently enhanced radiationinduced tumor growth inhibition. In nude mouse xenograft models, IAP-inhibitors Embelin and SH-130 potently sensitized the DU-145 tumors to X-ray radiation...

  10. Therapeutic relevance of inhibitors of MMPs or of caspases in HD-induced injury in the ex vivo human skin model

    NARCIS (Netherlands)

    Mol, M.A.E.; Berg, R.M. van den; Chau, L.F.

    2004-01-01

    In order to prevent microvesication, direct and indirect inhibitors of matrix metalloproteases (MMPs) can be used to fully prevent HD-induced epidermal-dermal separation in organ-cultured human skin pieces. The MMP inhibitors show no effect on the massive epidermal cell damage caused by HD.

  11. Discovery of potent inhibitors of human β-tryptase from pre-equilibrated dynamic combinatorial libraries.

    Science.gov (United States)

    Jiang, Qian-Qian; Sicking, Wilhelm; Ehlers, Martin; Schmuck, Carsten

    2015-03-01

    Pre-equilibrated dynamic combinatorial libraries based on acyl hydrazone interchange of peptide-derived hydrazides and di- and tri-aldehydes have been used to discover potent inhibitors with nanomolar affinities for β-tryptase. To identify potent inhibitors the activity of the full library containing 95 members was compared with those of sub-libraries in which individual building blocks were missing. The most active library members contain a rigid central aromatic scaffold with three cationic peptide arms. The arms of the best inhibitors also contained a tailor-made GCP oxoanion binding motif attached to a lysine side chain. The most potent tri-armed hydrazones with peptide arms GKWR or GKWK(GCP) were shown to inhibit β-tryptase ( K i ca. 10-20 nM) reversibly, non-competitively and selectively (compared to related serine proteases, e.g. trypsin and chymotrypsin), most likely by binding to the protein surface, also in agreement with molecular modelling calculations. These new inhibitors are one order of magnitude more efficient than related tetravalent inhibitors obtained from previous work on a split-mix-combinatorial library and were identified with significantly less effort, demonstrating the usefulness of this approach for the identification of enzyme inhibitors in general.

  12. Structure-based drug design: the discovery of novel nonpeptide orally active inhibitors of human renin.

    Science.gov (United States)

    Rahuel, J; Rasetti, V; Maibaum, J; Rüeger, H; Göschke, R; Cohen, N C; Stutz, S; Cumin, F; Fuhrer, W; Wood, J M; Grütter, M G

    2000-07-01

    The aspartic proteinase renin plays an important physiological role in the regulation of blood pressure. It catalyses the first step in the conversion of angiotensinogen to the hormone angiotensin II. In the past, potent peptide inhibitors of renin have been developed, but none of these compounds has made it to the end of clinical trials. Our primary aim was to develop novel nonpeptide inhibitors. Based on the available structural information concerning renin-substrate interactions, we synthesized inhibitors in which the peptide portion was replaced by lipophilic moieties that interact with the large hydrophobic S1/S3-binding pocket in renin. Crystal structure analysis of renin-inhibitor complexes combined with computational methods were employed in the medicinal-chemistry optimisation process. Structure analysis revealed that the newly designed inhibitors bind as predicted to the S1/S3 pocket. In addition, however, these compounds interact with a hitherto unrecognised large, distinct, sub-pocket of the enzyme that extends from the S3-binding site towards the hydrophobic core of the enzyme. Binding to this S3(sp) sub-pocket was essential for high binding affinity. This unprecedented binding mode guided the drug-design process in which the mostly hydrophobic interactions within subsite S3(sp) were optimised. Our design approach led to compounds with high in vitro affinity and specificity for renin, favourable bioavailability and excellent oral efficacy in lowering blood pressure in primates. These renin inhibitors are therefore potential therapeutic agents for the treatment of hypertension and related cardiovascular diseases.

  13. Role of protein phosphatases inhibitors on the histamine release and the functional desensitization in human lung mast cells

    Directory of Open Access Journals (Sweden)

    Reza Bastan

    2014-01-01

    Full Text Available Background: The β2-adrenoceptor agonist, isoprenaline, is an effective inhibitor of histamine release from human lung mast cells (HLMC. Since phosphorylations of the β2-adrenoceptors are probably important in inducing desensitization, we sought to investigate the importance of phosphorylation events by targeting protein phosphatases (PPs in mast cells. To this end, the effects of the inhibitor of on the functional desensitization of β-adrenoceptor-mediated responses in mast cells were investigated. Materials and Methods: In this study the effects of PP inhibitors on the inhibition of histamine release from HLMC, on β-agonists in mast cells and on desensitization were investigated. Results: Long-term exposure of mast cells to both isoprenaline and salbutamol substantially reduced the extent to which isoprenaline inhibited histamine release. Pretreatments of up to 24 h with inhibitors alone had no effect on immunoglobulin E-mediated histamine release. Shorter (≤4 h pretreatments had little effect on the activity of isoprenaline and salbutamol to inhibit histamine release from mast cells. Conclusion: Collectively, these data suggest that PP has an important role in regulating mast cell β2-adrenoceptors.

  14. Identification of human flap endonuclease 1 (FEN1) inhibitors using a machine learning based consensus virtual screening.

    Science.gov (United States)

    Deshmukh, Amit Laxmikant; Chandra, Sharat; Singh, Deependra Kumar; Siddiqi, Mohammad Imran; Banerjee, Dibyendu

    2017-07-25

    Human Flap endonuclease1 (FEN1) is an enzyme that is indispensable for DNA replication and repair processes and inhibition of its Flap cleavage activity results in increased cellular sensitivity to DNA damaging agents (cisplatin, temozolomide, MMS, etc.), with the potential to improve cancer prognosis. Reports of the high expression levels of FEN1 in several cancer cells support the idea that FEN1 inhibitors may target cancer cells with minimum side effects to normal cells. In this study, we used large publicly available, high-throughput screening data of small molecule compounds targeted against FEN1. Two machine learning algorithms, Support Vector Machine (SVM) and Random Forest (RF), were utilized to generate four classification models from huge PubChem bioassay data containing probable FEN1 inhibitors and non-inhibitors. We also investigated the influence of randomly selected Zinc-database compounds as negative data on the outcome of classification modelling. The results show that the SVM model with inactive compounds was superior to RF with Matthews's correlation coefficient (MCC) of 0.67 for the test set. A Maybridge database containing approximately 53 000 compounds was screened and top ranking 5 compounds were selected for enzyme and cell-based in vitro screening. The compound JFD00950 was identified as a novel FEN1 inhibitor with in vitro inhibition of flap cleavage activity as well as cytotoxic activity against a colon cancer cell line, DLD-1.

  15. Merkel cell polyomavirus and human papilloma virus in proliferative skin lesions arising in patients treated with BRAF inhibitors.

    Science.gov (United States)

    Falchook, G S; Rady, P; Konopinski, J C; Busaidy, N; Hess, K; Hymes, S; Nguyen, H P; Prieto, V G; Bustinza-Linares, E; Lin, Q; Parkhurst, K L; Hong, D S; Sherman, S; Tyring, S K; Kurzrock, R

    2016-07-01

    The potential role of oncogenic viruses mediating development of proliferative skin lesions in patients treated with RAF inhibitors is poorly understood. The objective of this study was to investigate human papilloma virus (HPV) and Merkel cell polyomavirus (MCPyV) in skin lesions among patients treated with RAF inhibitors with the help of a case series describing prevalence of HPV, MCPyV, and RAS mutations in skin biopsies obtained from patients receiving RAF inhibitors and developing cutaneous lesions. HPV-DNA was amplified by PCR utilizing multiple nested primer systems designed for detection of a broad range of HPV types. MCPyV copy number determination with real time PCR technology was performed by a "Quantification of MCPyV, small t region" kit. Thirty-six patients were tested (squamous cell carcinoma (SCC) = 14; verruca vulgaris = 15; other = 11). Nine of 12 SCCs (75 %) and eight of 13 verruca vulgaris lesions (62 %) tested positive for MCPyV whereas none of the normal skin biopsies obtained from nine of these patients tested positive for MCPyV (p = 0.0007). HPV incidence in cutaneous SCCs was not different compared to normal skin (50 vs. 56 %, p = 0.86). The association between MCPyV and proliferative skin lesions after RAF inhibitor therapy merits further investigation.

  16. The broad-spectrum caspase inhibitor Boc-Asp-CMK induces cell death in human leukaemia cells.

    Science.gov (United States)

    Frydrych, Ivo; Mlejnek, Petr; Dolezel, Petr; Zoumpourlis, Vassilis; Krumpochova, Petra

    2008-08-01

    Synthetic caspase inhibitors and particularly broad-spectrum caspase inhibitors can prevent cells from death or at least slow down cell death process and abrogate some apoptotic hallmarks [Kitanaka, C., Kuchino, Y., 1999. Caspase-independent programmed cell death with necrotic morphology. Cell Death and Differentiation 6, 508-515]. However, not all synthetic caspase inhibitors diminish cell death. We have found that the broad-spectrum caspase inhibitor Boc-Asp-CMK induced cell death at micromolar concentrations in human leukaemia cells. Interestingly, low concentrations of Boc-Asp-CMK induced cell death with apoptotic hallmarks. Increasing concentrations of Boc-Asp-CMK led to necrotic cell death. The switch between apoptosis and necrosis seemed to depend upon the degree of inhibition of executioner caspases, including caspase-3/7 with Boc-Asp-CMK. Interestingly, caspase-3 processing was not inhibited even for the highest concentration of Boc-Asp-CMK used. We assume, that toxic properties of Boc-Asp-CMK can be attributed to the chloromethylketone residuum in its molecule, as its analogue Boc-Asp-FMK with fluoromethylketone residuum was more than 13 times less toxic. Our results further indicated that toxicity of Boc-Asp-CMK might arise from its interference with mitochondrial metabolism.

  17. Small molecule inhibitors uncover synthetic genetic interactions of human flap endonuclease 1 (FEN1 with DNA damage response genes.

    Directory of Open Access Journals (Sweden)

    Thomas A Ward

    Full Text Available Flap endonuclease 1 (FEN1 is a structure selective endonuclease required for proficient DNA replication and the repair of DNA damage. Cellularly active inhibitors of this enzyme have previously been shown to induce a DNA damage response and, ultimately, cell death. High-throughput screens of human cancer cell-lines identify colorectal and gastric cell-lines with microsatellite instability (MSI as enriched for cellular sensitivity to N-hydroxyurea series inhibitors of FEN1, but not the PARP inhibitor olaparib or other inhibitors of the DNA damage response. This sensitivity is due to a synthetic lethal interaction between FEN1 and MRE11A, which is often mutated in MSI cancers through instabilities at a poly(T microsatellite repeat. Disruption of ATM is similarly synthetic lethal with FEN1 inhibition, suggesting that disruption of FEN1 function leads to the accumulation of DNA double-strand breaks. These are likely a result of the accumulation of aberrant replication forks, that accumulate as a consequence of a failure in Okazaki fragment maturation, as inhibition of FEN1 is toxic in cells disrupted for the Fanconi anemia pathway and post-replication repair. Furthermore, RAD51 foci accumulate as a consequence of FEN1 inhibition and the toxicity of FEN1 inhibitors increases in cells disrupted for the homologous recombination pathway, suggesting a role for homologous recombination in the resolution of damage induced by FEN1 inhibition. Finally, FEN1 appears to be required for the repair of damage induced by olaparib and cisplatin within the Fanconi anemia pathway, and may play a role in the repair of damage associated with its own disruption.

  18. Blockade of the ERK pathway enhances the therapeutic efficacy of the histone deacetylase inhibitor MS-275 in human tumor xenograft models

    International Nuclear Information System (INIS)

    Sakamoto, Toshiaki; Ozaki, Kei-ichi; Fujio, Kohsuke; Kajikawa, Shu-hei; Uesato, Shin-ichi; Watanabe, Kazushi; Tanimura, Susumu; Koji, Takehiko; Kohno, Michiaki

    2013-01-01

    Highlights: •Blockade of the ERK pathway enhances the anticancer efficacy of HDAC inhibitors. •MEK inhibitors sensitize human tumor xenografts to HDAC inhibitor cytotoxicity. •Such the enhanced efficacy is achieved by a transient blockade of the ERK pathway. •This drug combination provides a promising therapeutic strategy for cancer patients. -- Abstract: The ERK pathway is up-regulated in various human cancers and represents a prime target for mechanism-based approaches to cancer treatment. Specific blockade of the ERK pathway alone induces mostly cytostatic rather than pro-apoptotic effects, however, resulting in a limited therapeutic efficacy of the ERK kinase (MEK) inhibitors. We previously showed that MEK inhibitors markedly enhance the ability of histone deacetylase (HDAC) inhibitors to induce apoptosis in tumor cells with constitutive ERK pathway activation in vitro. To evaluate the therapeutic efficacy of such drug combinations, we administered the MEK inhibitor PD184352 or AZD6244 together with the HDAC inhibitor MS-275 in nude mice harboring HT-29 or H1650 xenografts. Co-administration of the MEK inhibitor markedly sensitized the human xenografts to MS-275 cytotoxicity. A dose of MS-275 that alone showed only moderate cytotoxicity thus suppressed the growth of tumor xenografts almost completely as well as induced a marked reduction in tumor cellularity when administered with PD184352 or AZD6244. The combination of the two types of inhibitor also induced marked oxidative stress, which appeared to result in DNA damage and massive cell death, specifically in the tumor xenografts. The enhanced therapeutic efficacy of the drug combination was achieved by a relatively transient blockade of the ERK pathway. Administration of both MEK and HDAC inhibitors represents a promising chemotherapeutic strategy with improved safety for cancer patients

  19. The effect of phosphodiesterase-5 inhibitors on cerebral blood flow in humans

    DEFF Research Database (Denmark)

    Pauls, Mathilde Mh; Moynihan, Barry; Barrick, Thomas R

    2018-01-01

    Agents that augment cerebral blood flow (CBF) could be potential treatments for vascular cognitive impairment. Phosphodiesterase-5 inhibitors are vasodilating drugs established in the treatment of erectile dysfunction (ED) and pulmonary hypertension. We reviewed published data on the effects......, ED, type 2 diabetes, stroke, pulmonary hypertension, Becker muscular dystrophy and subarachnoid haemorrhage. Most studies used middle cerebral artery flow velocity to estimate CBF. Few studies employed direct measurements of tissue perfusion. Resting CBF velocity was unaffected by phosphodiesterase-5...... inhibitors, but cerebrovascular regulation was improved in ED, pulmonary hypertension, diabetes, Becker's and a group of healthy volunteers. This evidence suggests that phosphodiesterase-5 inhibitors improve responsiveness of the cerebral vasculature, particularly in disease states associated...

  20. Gibberellin biosynthetic inhibitors make human malaria parasite Plasmodium falciparum cells swell and rupture to death.

    Directory of Open Access Journals (Sweden)

    Tomoko Toyama

    Full Text Available Malaria remains as one of the most devastating infectious disease, and continues to exact an enormous toll in medical cost and days of labor lost especially in the tropics. Effective malaria control and eventual eradication remain a huge challenge, with efficacious antimalarials as important intervention/management tool. Clearly new alternative drugs that are more affordable and with fewer side effects are desirable. After preliminary in vitro assays with plant growth regulators and inhibitors, here, we focus on biosynthetic inhibitors of gibberellin, a plant hormone with many important roles in plant growth, and show their inhibitory effect on the growth of both apicomplexa, Plasmodium falciparum and Toxoplasma gondii. Treatment of P. falciparum cultures with the gibberellin biosynthetic inhibitors resulted in marked morphological changes that can be reversed to a certain degree under hyperosmotic environment. These unique observations suggest that changes in the parasite membrane permeability may explain the pleiotropic effects observed within the intracellular parasites.

  1. Immunogenicity assessment of recombinant human c1-inhibitor: an integrated analysis of clinical studies.

    Science.gov (United States)

    Hack, C Erik; Mannesse, Maurice; Baboeram, Aartie; Oortwijn, Beatrijs; Relan, Anurag

    2012-10-01

    Recombinant human C1-inhibitor (rhC1INH) is used to treat acute angioedema attacks in hereditary angioedema (HAE) due to a genetic C1INH deficiency. Recombinant proteins in general may induce antibody responses and therefore evaluation of such responses in the target population is an essential step in the clinical development program of a recombinant protein. Here we report the assessment of the immunogenicity of rhC1INH in symptomatic HAE patients. Blood samples collected before and after administration of rhC1INH were tested for antibodies against plasma-derived (pd) or rhC1INH, or against host-related impurities (HRI). Above cut-off screening results were confirmed with displacement assays, and also tested for neutralizing anti-C1INH antibodies. Finally, the relation of antibodies to clinical efficacy and safety of rhC1INH was analyzed. Data from 155 HAE patients who received 424 treatments with rhC1INH were analyzed. 1.5% of all pre-exposure tests and 1.3% of all post-exposure tests were above the cut-off level in the screening assay for anti-C1INH antibodies. Six patients (3.9%) had anti-rhC1INH antibodies positive in the confirmatory assay. In two patients, confirmed antibodies were pre-existing with no increase post-exposure; in three patients, the antibodies occurred on a single occasion post-exposure; and in one patient, on subsequent occasions post-exposure. Neutralizing anti-pdC1INH antibodies were not found. Anti-HRI antibodies in the screening assay occurred in <0.7% of the tests before exposure to rhC1INH, in <1.9% after first exposure and in <3.1% after repeat treatment with rhC1INH. Five patients had anti-HRI antibodies positive in the confirmatory assay. In one patient, the antibodies were pre-existing, whereas in three of the 155 rhC1INH-treated patients (1.9%), confirmed anti-HRI antibodies occurred at more time points. Antibody findings were not associated with altered efficacy of rhC1INH or adverse events. These results indicate a reassuring

  2. Simvastatin suppresses dexamethasone-induced secretion of plasminogen activator inhibitor-1 in human bone marrow adipocytes

    Directory of Open Access Journals (Sweden)

    Baba Hideo

    2011-04-01

    Full Text Available Abstract Background Osteonecrosis of the femoral head is a common complication of high-dose glucocorticoid treatment. Intravascular thrombosis is thought to be associated with the ischemic state of the femoral head. Plasminogen activator inhibitor-1 (PAI-1 is an adipokine, which are physiologically active substances secreted from visceral and subcutaneous adipocytes. PAI-1 suppresses fibrinolysis by binding tissue-type plasminogen activator. Several reports have described the relationship between PAI-1 and steroid-induced osteonecrosis of the femoral head, and the preventive effects of lipid-lowering agents (statins against steroid-induced osteonecrosis of the femoral head. We previously reported that adipokines and dexamethasone induced PAI-1 secretion from bone marrow adipocytes. The purpose of the present study is to examine the effects of simvastatin on PAI-1 secretion from human bone marrow adipocytes in vitro. Methods Primary bone marrow adipocytes were extracted from collagenase-treated bone marrow fluid obtained from the femoral necks of 40 patients (6 men, 34 women; age range, 52-81 years undergoing hip joint replacement surgery. After suspended culture with or without dexamethasone or simvastatin, PAI-1 mRNA expression was assessed by real-time RT-PCR. Total PAI-1 protein secretion in culture medium was assessed by enzyme-linked immunosorbent assay. Results PAI-1 mRNA expression was up-regulated by 388% (P = 0.002 with dexamethasone, and down-regulated by 45% (P = 0.002 with simvastatin, as compared to control levels. Dexamethasone increased total PAI-1 secretion by 166% (P = 0.001 and simvastatin decreased total PAI-1 secretion by 64% (P = 0.002. No significant changes were observed in adiponectin mRNA expression and secretion by dexamethasone and simvastatin, while pre-treatment with simvastatin reversed dexamethasone induced PAI-1 secretion by 89%, as compared to control levels. Conclusion The present study confirmed the suppressive

  3. A synthetic cryptochrome inhibitor induces anti-proliferative effects and increases chemosensitivity in human breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Chun, Sung Kook [Department of Brain & Cognitive Sciences, Daegu-Gyeongbuk Institute of Science & Technology, Daegu, 711-873 (Korea, Republic of); Department of Biological Sciences, Seoul National University, Seoul, 151-747 (Korea, Republic of); Department of Brain & Cognitive Sciences, Seoul National University, Seoul, 151-747 (Korea, Republic of); Chung, Sooyoung [Department of Biological Sciences, Seoul National University, Seoul, 151-747 (Korea, Republic of); Department of Biomedical Sciences, College of Medicine, Korea University, Seoul, 136-705 (Korea, Republic of); Kim, Hee-Dae [Department of Biological Sciences, Seoul National University, Seoul, 151-747 (Korea, Republic of); Lee, Ju Hyung [Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749 (Korea, Republic of); Jang, Jaebong [College of Pharmacy, Seoul National University, Seoul, 151-742 (Korea, Republic of); Kim, Jeongah; Kim, Doyeon [Department of Brain & Cognitive Sciences, Daegu-Gyeongbuk Institute of Science & Technology, Daegu, 711-873 (Korea, Republic of); Department of Biological Sciences, Seoul National University, Seoul, 151-747 (Korea, Republic of); Department of Brain & Cognitive Sciences, Seoul National University, Seoul, 151-747 (Korea, Republic of); Son, Gi Hoon [Department of Biomedical Sciences, College of Medicine, Korea University, Seoul, 136-705 (Korea, Republic of); Oh, Young J. [Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749 (Korea, Republic of); Suh, Young-Ger [College of Pharmacy, Seoul National University, Seoul, 151-742 (Korea, Republic of); Lee, Cheol Soon [Gachon Clinical Trials Center, Gachon University, Incheon, 417-842 (Korea, Republic of); and others

    2015-11-13

    Disruption of circadian rhythm is a major cause of breast cancer in humans. Cryptochrome (CRY), a circadian transcription factor, is a risk factor for initiation of breast cancer, and it is differentially expressed between normal and breast cancer tissues. Here, we evaluated the anti-proliferative and pro-apoptotic activity of KS15, a recently discovered small-molecule inhibitor of CRY, in human breast cancer cells. First, we investigated whether KS15 treatment could promote E-box-mediated transcription by inhibiting the activity of CRY in MCF-7 human breast cancer cells. Protein and mRNA levels of regulators of cell cycle and apoptosis, as well as core clock genes, were differentially modulated in response to KS15. Next, we investigated whether KS15 could inhibit proliferation and increase sensitivity to anti-tumor drugs in MCF-7 cells. We found that KS15 decreased the speed of cell growth and increased the chemosensitivity of MCF-7 cells to doxorubicin and tamoxifen, but had no effect on MCF-10A cells. These findings suggested that pharmacological inhibition of CRY by KS15 exerts an anti-proliferative effect and increases sensitivity to anti-tumor drugs in a specific type of breast cancer. - Highlights: • Cryptochrome inhibitor (KS15) has anti-tumor activity to human breast cancer cells. • KS15 induces differential changes in cell cycle regulators and pro-apoptotic genes. • KS15 inhibits MCF-7 cell growth and enhances susceptibility to anti-tumor drugs.

  4. A synthetic cryptochrome inhibitor induces anti-proliferative effects and increases chemosensitivity in human breast cancer cells

    International Nuclear Information System (INIS)

    Chun, Sung Kook; Chung, Sooyoung; Kim, Hee-Dae; Lee, Ju Hyung; Jang, Jaebong; Kim, Jeongah; Kim, Doyeon; Son, Gi Hoon; Oh, Young J.; Suh, Young-Ger; Lee, Cheol Soon

    2015-01-01

    Disruption of circadian rhythm is a major cause of breast cancer in humans. Cryptochrome (CRY), a circadian transcription factor, is a risk factor for initiation of breast cancer, and it is differentially expressed between normal and breast cancer tissues. Here, we evaluated the anti-proliferative and pro-apoptotic activity of KS15, a recently discovered small-molecule inhibitor of CRY, in human breast cancer cells. First, we investigated whether KS15 treatment could promote E-box-mediated transcription by inhibiting the activity of CRY in MCF-7 human breast cancer cells. Protein and mRNA levels of regulators of cell cycle and apoptosis, as well as core clock genes, were differentially modulated in response to KS15. Next, we investigated whether KS15 could inhibit proliferation and increase sensitivity to anti-tumor drugs in MCF-7 cells. We found that KS15 decreased the speed of cell growth and increased the chemosensitivity of MCF-7 cells to doxorubicin and tamoxifen, but had no effect on MCF-10A cells. These findings suggested that pharmacological inhibition of CRY by KS15 exerts an anti-proliferative effect and increases sensitivity to anti-tumor drugs in a specific type of breast cancer. - Highlights: • Cryptochrome inhibitor (KS15) has anti-tumor activity to human breast cancer cells. • KS15 induces differential changes in cell cycle regulators and pro-apoptotic genes. • KS15 inhibits MCF-7 cell growth and enhances susceptibility to anti-tumor drugs.

  5. Myricetin is a novel inhibitor of human inosine 5′-monophosphate dehydrogenase with anti-leukemia activity

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Huiling; Hu, Qian; Wang, Jingyuan; Liu, Zehui; Wu, Dang [Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237 (China); Lu, Weiqiang, E-mail: wqlu@bio.ecnu.edu.cn [Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241 (China); Huang, Jin, E-mail: huangjin@ecust.edu.cn [Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237 (China)

    2016-09-02

    Human inosine 5′-monophosphate dehydrogenase (hIMPDH) is a rate-limiting enzyme in the de novo biosynthetic pathway of purine nucleotides, playing crucial roles in cellular proliferation, differentiation, and transformation. Dysregulation of hIMPDH expression and activity have been found in a variety of human cancers including leukemia. In this study, we found that myricetin, a naturally occurring phytochemical existed in berries, wine and tea, was a novel inhibitor of human type 1 and type 2 IMPDH (hIMPDH1/2) with IC{sub 50} values of 6.98 ± 0.22 μM and 4.10 ± 0.14 μM, respectively. Enzyme kinetic analysis using Lineweaver-Burk plot revealed that myricetin is a mix-type inhibitor for hIMPDH1/2. Differential scanning fluorimetry and molecular docking simulation data demonstrate that myricetin is capable of binding with hIMPDH1/2. Myricetin treatment exerts potent anti-proliferative and pro-apoptotic effects on K562 human leukemia cells in a dose-dependent manner. Importantly, cytotoxicity of myricetin on K562 cells were markedly attenuated by exogenous addition of guanosine, a salvage pathway of maintaining intracellular pool of guanine nucleotides. Taking together, these results indicate that natural product myricetin exhibits potent anti-leukemia activity by interfering with purine nucleotides biosynthetic pathway through the suppression of hIMPDH1/2 catalytic activity. - Highlights: • Myricetin, a common dietary flavonoid, is a novel inhibitor of hIMPDH1/2. • Myricetin directly binds with hIMPDH1/2 and induces cell cycle arrest and apoptosis of leukemia cells. • The cytotoxicity of myricetin on K562 cells is markedly attenuated by exogenous addition of guanosine.

  6. The age-dependent effects of selective serotonin reuptake inhibitors in humans and rodents: A review.

    NARCIS (Netherlands)

    Olivier, J.D.A.; Blom, T.; Arentsen, T.; Homberg, J.R.

    2011-01-01

    The selective serotonin reuptake inhibitor (SSRI) Prozac(R) (fluoxetine) is widely prescribed for the treatment of depression and anxiety-related disorders. While extensive research has established that fluoxetine is safe for adults, safety is not guaranteed for (unborn) children and adolescents.

  7. Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines

    DEFF Research Database (Denmark)

    Mohammed, M Z; Vyjayanti, V N; Laughton, C A

    2011-01-01

    Modulation of DNA base excision repair (BER) has the potential to enhance response to chemotherapy and improve outcomes in tumours such as melanoma and glioma. APE1, a critical protein in BER that processes potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer....... In the current study, we aimed to develop small molecule inhibitors of APE1 for cancer therapy....

  8. An Ixodes ricinus Tick Salivary Lectin Pathway Inhibitor Protects Borrelia burgdorferi sensu lato from Human Complement

    NARCIS (Netherlands)

    Wagemakers, Alex; Coumou, Jeroen; Schuijt, Tim J.; Oei, Anneke; Nijhof, Ard M.; van 't Veer, Cornelis; van der Poll, Tom; Bins, Adriaan D.; Hovius, Joppe W. R.

    2016-01-01

    We previously identified tick salivary lectin pathway inhibitor (TSLPI) in Ixodes scapularis, a vector for Borrelia burgdorferi sensu stricto (s.s.) in North America. TSLPI is a salivary protein facilitating B. burgdorferi s.s. transmission and acquisition by inhibiting the host lectin complement

  9. Comparison of responses of human melanoma cell lines to MEK and BRAF inhibitors

    Directory of Open Access Journals (Sweden)

    Clare Judith Stones

    2013-05-01

    Full Text Available The NRAS and BRAF genes are frequently mutated in melanoma, suggesting that the NRAS-BRAF-MEK-ERK signalling pathway is an important target for therapy. Two classes of drugs, one targeting activated BRAF and one targeting MEK, are currently undergoing clinical evaluation. We have analysed the NRAS and BRAF mutational status of a series of 44 early passage lines developed from New Zealand patients with metastatic melanoma. 41% of the lines analysed had BRAF mutations, 23% had NRAS mutations and 36% had neither. We then determined IC50 values (drug concentrations for 50% growth inhibition for CI-1040, a commonly used inhibitor of MEK kinase; trametinib, a clinical agent targeting MEK kinase; and vemurafenib, an inhibitor of mutant BRAF kinase. Cell lines with activating BRAF mutations were significantly more sensitive to vemurafenib than lines with NRAS mutations or lines lacking either mutation (p < 0.001. IC50 values for CI-1040 and trametinib were strongly correlated (r = 0.98 with trametinib showing ~100-fold greater potency. Cell lines sensitive to vemurafenib were also sensitive to CI-1040 and trametinib, but there was no relationship between IC50 values and NRAS mutation status. A small number of lines lacking a BRAF mutation were sensitive to CI-1040 but resistant to vemurafenib. We used western blotting to investigate the effect on ERK phosphorylation of CI-1040 in four lines, of vemurafenib in two lines and of trametinib in two lines. The results support the view that MEK inhibitors might be combined with BRAF inhibitors in the treatment melanomas of with activated BRAF. The high sensitivity to trametinib of some lines with wild-type BRAF status also suggests that MEK inhibitors could have a therapeutic effect against some melanomas as single agents.

  10. Effects of GSK3 inhibitors on in vitro expansion and differentiation of human adipose-derived stem cells into adipocytes

    Directory of Open Access Journals (Sweden)

    Peraldi Pascal

    2008-02-01

    Full Text Available Abstract Background Multipotent stem cells exist within adipose tissue throughout life. An abnormal recruitment of these adipose precursor cells could participate to hyperplasia of adipose tissue observed in severe obesity or to hypoplasia of adipose tissue observed in lipodystrophy. Therefore, pharmacological molecules that control the pool of stem cells in adipose tissue are of great interest. Glycogen Synthase Kinase (GSK 3 has been previously described as involved in differentiation of preadipose cells and might be a potential therapeutic target to modulate proliferation and differentiation of adipocyte precursors. However, the impact of GSK3 inhibition on human adipose-derived stem cells remained to be investigated. The aim of this study was to investigate GSK3 as a possible target for pharmacological inhibition of stem cell adipogenesis. To reach this goal, we studied the effects of pharmacological inhibitors of GSK3, i.e. lithium chloride (LiCl and BIO on proliferation and adipocyte differentiation of multipotent stem cells derived from human adipose tissue. Results Our results showed that GSK3 inhibitors inhibited proliferation and clonogenicity of human stem cells, strongly suggesting that GSK3 inhibitors could be potent regulators of the pool of adipocyte precursors in adipose tissue. The impact of GSK3 inhibition on differentiation of hMADS cells was also investigated. Adipogenic and osteogenic differentiations were inhibited upon hMADS treatment with BIO. Whereas a chronic treatment was required to inhibit osteogenesis, a treatment that was strictly restricted to the early step of differentiation was sufficient to inhibit adipogenesis. Conclusion These results demonstrated the feasibility of a pharmacological approach to regulate adipose-derived stem cell function and that GSK3 could represent a potential target for controlling adipocyte precursor pool under conditions where fat tissue formation is impaired.

  11. Treatment of human pre-B acute lymphoblastic leukemia with the Aurora kinase inhibitor PHA-739358 (Danusertib

    Directory of Open Access Journals (Sweden)

    Fei Fei

    2012-06-01

    Full Text Available Abstract Background Treatment of Philadelphia chromosome-positive acute lymphoblastic leukemias (Ph-positive ALL with clinically approved inhibitors of the Bcr/Abl tyrosine kinase frequently results in the emergence of a leukemic clone carrying the T315I mutation in Bcr/Abl, which confers resistance to these drugs. PHA-739358, an Aurora kinase inhibitor, was reported to inhibit the Bcr/Abl T315I mutant in CML cells but no preclinical studies have examined this in detail in human ALL. Results We compared the sensitivity of human Bcr/Abl T315I, Bcr/Abl wild type and non-Bcr/Abl ALL cells to this drug. PHA-739358 inhibited proliferation and induced apoptosis independently of Bcr/Abl, the T315I mutation, or presence of the tumor suppressor p53, but the degree of effectiveness varied between different ALL samples. Since short-term treatment with a single dose of drug only transiently inhibited proliferation, we tested combination treatments of PHA-739358 with the farnesyltransferase inhibitor Lonafarnib, with vincristine and with dasatinib. All combinations reduced viability and cell numbers compared to treatment with a single drug. Clonogenic assays showed that 25 nM PHA-739358 significantly reduced the colony growth potential of Ph-positive ALL cells, and combined treatment with a second drug abrogated colony growth in this assay. PHA-739358 further effectively blocked Bcr/Abl tyrosine kinase activity and Aurora kinase B in vivo, and mice transplanted with human Bcr/Abl T315I ALL cells treated with a 3x 7-day cycle of PHA-739358 as mono-treatment had significantly longer survival. Conclusions PHA-739358 represents an alternative drug for the treatment of both Ph-positive and negative ALL, although combined treatment with a second drug may be needed to eradicate the leukemic cells.

  12. The effects of metal ion PCR inhibitors on results obtained with the Quantifiler(®) Human DNA Quantification Kit.

    Science.gov (United States)

    Combs, Laura Gaydosh; Warren, Joseph E; Huynh, Vivian; Castaneda, Joanna; Golden, Teresa D; Roby, Rhonda K

    2015-11-01

    Forensic DNA samples may include the presence of PCR inhibitors, even after extraction and purification. Studies have demonstrated that metal ions, co-purified at specific concentrations, inhibit DNA amplifications. Metal ions are endogenous to sample types, such as bone, and can be introduced from environmental sources. In order to examine the effect of metal ions as PCR inhibitors during quantitative real-time PCR, 2800 M DNA was treated with 0.0025-18.750 mM concentrations of aluminum, calcium, copper, iron, nickel, and lead. DNA samples, both untreated and metal-treated, were quantified using the Quantifiler(®) Human DNA Quantification Kit. Quantification cycle (Cq) values for the Quantifiler(®) Human DNA and internal PCR control (IPC) assays were measured and the estimated concentrations of human DNA were obtained. Comparisons were conducted between metal-treated and control DNA samples to determine the accuracy of the quantification estimates and to test the efficacy of the IPC inhibition detection. This kit is most resistant to the presence of calcium as compared to all metals tested; the maximum concentration tested does not affect the amplification of the IPC or quantification of the sample. This kit is most sensitive to the presence of aluminum; concentrations greater than 0.0750 mM negatively affected the quantification, although the IPC assay accurately assessed the presence of PCR inhibition. The Quantifiler(®) Human DNA Quantification Kit accurately quantifies human DNA in the presence of 0.5000 mM copper, iron, nickel, and lead; however, the IPC does not indicate the presence of PCR inhibition at this concentration of these metals. Unexpectedly, estimates of DNA quantity in samples treated with 18.750 mM copper yielded values in excess of the actual concentration of DNA in the samples; fluorescence spectroscopy experiments indicated this increase was not a direct interaction between the copper metal and 6-FAM dye used to label the probe that

  13. Docking to Explicate Interface between Plant-Originated Inhibitors and E6 Oncogenic Protein of Highly Threatening Human Papillomavirus 18

    Directory of Open Access Journals (Sweden)

    Satish Kumar

    2015-06-01

    Full Text Available The leading cause of cancer mortality globally amongst the women is due to human papillomavirus (HPV infection. There is need to explore anti-cancerous drugs against this life-threatening infection. Traditionally, different natural compounds such as withaferin A, artemisinin, ursolic acid, ferulic acid, (--epigallocatechin-3-gallate, berberin, resveratrol, jaceosidin, curcumin, gingerol, indol-3-carbinol, and silymarin have been used as hopeful source of cancer treatment. These natural inhibitors have been shown to block HPV infection by different researchers. In the present study, we explored these natural compounds against E6 oncoprotein of high risk HPV18, which is known to inactivate tumor suppressor p53 protein. E6, a high throughput protein model of HPV18, was predicted to anticipate the interaction mechanism of E6 oncoprotein with these natural inhibitors using structure-based drug designing approach. Docking analysis showed the interaction of these natural inhibitors with p53 binding site of E6 protein residues 108-117 (CQKPLNPAEK and help reinstatement of normal p53 functioning. Further, docking analysis besides helping in silico validations of natural compounds also helped elucidating the molecular mechanism of inhibition of HPV oncoproteins.

  14. Several Human Cyclin-Dependent Kinase Inhibitors, Structurally Related to Roscovitine, As New Anti-Malarial Agents

    Directory of Open Access Journals (Sweden)

    Sandrine Houzé

    2014-09-01

    Full Text Available In Africa, malaria kills one child each minute. It is also responsible for about one million deaths worldwide each year. Plasmodium falciparum, is the protozoan responsible for the most lethal form of the disease, with resistance developing against the available anti-malarial drugs. Among newly proposed anti-malaria targets, are the P. falciparum cyclin-dependent kinases (PfCDKs. There are involved in different stages of the protozoan growth and development but share high sequence homology with human cyclin-dependent kinases (CDKs. We previously reported the synthesis of CDKs inhibitors that are structurally-related to (R-roscovitine, a 2,6,9-trisubstituted purine, and they showed activity against neuronal diseases and cancers. In this report, we describe the synthesis and the characterization of new CDK inhibitors, active in reducing the in vitro growth of P. falciparum (3D7 and 7G8 strains. Six compounds are more potent inhibitors than roscovitine, and three exhibited IC50 values close to 1 µM for both 3D7 and 7G8 strains. Although, such molecules do inhibit P. falciparum growth, they require further studies to improve their selectivity for PfCDKs.

  15. Designing Second Generation Anti-Alzheimer Compounds as Inhibitors of Human Acetylcholinesterase: Computational Screening of Synthetic Molecules and Dietary Phytochemicals.

    Directory of Open Access Journals (Sweden)

    Hafsa Amat-Ur-Rasool

    Full Text Available Alzheimer's disease (AD, a big cause of memory loss, is a progressive neurodegenerative disorder. The disease leads to irreversible loss of neurons that result in reduced level of acetylcholine neurotransmitter (ACh. The reduction of ACh level impairs brain functioning. One aspect of AD therapy is to maintain ACh level up to a safe limit, by blocking acetylcholinesterase (AChE, an enzyme that is naturally responsible for its degradation. This research presents an in-silico screening and designing of hAChE inhibitors as potential anti-Alzheimer drugs. Molecular docking results of the database retrieved (synthetic chemicals and dietary phytochemicals and self-drawn ligands were compared with Food and Drug Administration (FDA approved drugs against AD as controls. Furthermore, computational ADME studies were performed on the hits to assess their safety. Human AChE was found to be most approptiate target site as compared to commonly used Torpedo AChE. Among the tested dietry phytochemicals, berberastine, berberine, yohimbine, sanguinarine, elemol and naringenin are the worth mentioning phytochemicals as potential anti-Alzheimer drugs The synthetic leads were mostly dual binding site inhibitors with two binding subunits linked by a carbon chain i.e. second generation AD drugs. Fifteen new heterodimers were designed that were computationally more efficient inhibitors than previously reported compounds. Using computational methods, compounds present in online chemical databases can be screened to design more efficient and safer drugs against cognitive symptoms of AD.

  16. Canagliflozin, an SGLT2 inhibitor, attenuates the development of hepatocellular carcinoma in a mouse model of human NASH.

    Science.gov (United States)

    Shiba, Kumiko; Tsuchiya, Kyoichiro; Komiya, Chikara; Miyachi, Yasutaka; Mori, Kentaro; Shimazu, Noriko; Yamaguchi, Shinobu; Ogasawara, Naomi; Katoh, Makoto; Itoh, Michiko; Suganami, Takayoshi; Ogawa, Yoshihiro

    2018-02-05

    Sodium glucose cotransporter 2 (SGLT2) inhibitors, an antidiabetic drug, promotes urinary excretion of glucose by blocking its reabsorption in the renal proximal tubules. It is unclear whether SGLT2 inhibition could attenuate nonalcoholic steatohepatitis (NASH) and NASH-associated hepatocellular carcinoma. We examined the preventive effects of an SGLT2 inhibitor canagliflozin (CANA) in Western diet (WD)-fed melanocortin 4 receptor-deficient (MC4R-KO) mice, a mouse model of human NASH. An eight-week CANA treatment attenuated hepatic steatosis in WD-fed MC4R-KO mice, with increased epididymal fat mass without inflammatory changes. CANA treatment for 20 weeks inhibited the development of hepatic fibrosis in WD-fed MC4R-KO mice. After one year of CANA treatment, the number of liver tumors was significantly reduced in WD-fed MC4R-KO mice. In adipose tissue, CANA suppressed the ratio of oxidative to reduced forms of glutathiones (GSSG/GSH) in WD-fed MC4R-KO mice. Treatment with GSH significantly attenuated the H 2 O 2 -induced upregulation of genes related to NADPH oxidase in 3T3-L1 adipocytes, and that of Il6, Tgfb, and Pdgfb in RAW264.7 cells. This study provides evidence that SGLT2 inhibitors represent the unique class of drugs that can attenuate or delay the onset of NASH and eventually hepatocellular carcinoma, at least partly, through "healthy adipose expansion".

  17. Monoamine oxidase inhibitors l-deprenyl and clorgyline protect nonmalignant human cells from ionising radiation and chemotherapy toxicity.

    LENUS (Irish Health Repository)

    Seymour, C B

    2003-11-17

    l-Deprenyl (R-(-)-deprenyl, selegiline) is an inhibitor of monoamine oxidase-B (MAO-B) that is known to protect nerve cells from a variety of chemical and physical insults. As apoptosis is a common mechanism of radiation-induced cell death, the effect of l-deprenyl on the survival of cultured cells and tissue explants was studied following exposure to gamma radiation. The results obtained were compared with the effects of the less-selective MAO-B inhibitor pargyline and the MAO-A inhibitor clorgyline. l-Deprenyl at a concentration of 10(-9) M protected the nontumorigenic cell line (HaCaT) and normal human urothelial explants from the effects of cobalt-60 gamma radiation, but did not protect tumorigenic human cell lines HaCaT-ras, HPV-transfected human keratinocytes (HPV-G cells), or PC3. Human bladder carcinoma explants were not protected. Clorgyline showed a smaller protective effect of normal cells, whereas pargyline had no effect. Radiation-induced delayed effects (genomic instability measured as delayed cell death) were prevented in normal cells by l-deprenyl but, interestingly, deprenyl appeared to increase the amount of delayed death in the tumorigenic cell lines. Studies using l-deprenyl prior to the exposure of nonmalignant cells to cisplatin showed that cell death due to this agent was also reduced. Treatment of cultures of nontumorigenic cells with l-deprenyl or clorgyline significantly increased the levels of the protein Bcl-2 following irradiation, but there was no such effect on the already-elevated levels of this protein in the tumour samples. Since the Bcl-2 has been shown to be an inhibitor of apoptosis or programmed cell death, this would imply that the protective effects of l-deprenyl and clorgyline involve activation of antiapoptotic pathways within the normal cell. This hypothesis is supported by data showing reduced levels of apoptosis in HaCAT cells and in normal bladder explant cultures following treatment with l-deprenyl.

  18. Liposomal-delivery of phosphodiesterase 5 inhibitors augments UT-15C-stimulated ATP release from human erythrocytes

    Directory of Open Access Journals (Sweden)

    Elizabeth A. Bowles

    2017-12-01

    Here we investigate the hypothesis that targeted delivery of PDE5 inhibitors to human erythrocytes, using a liposomal delivery system, potentiates prostacyclin analog- induced ATP release. The findings are consistent with the hypothesis that directed delivery of this class of drugs to erythrocytes could be a new and important method to augment prostacyclin analog-induced ATP release from these cells. Such an approach could significantly limit side effects of both classes of drugs without compromising their therapeutic effectiveness in diseases such as PAH.

  19. Large-scale prediction of human kinase-inhibitor interactions using protein sequences and molecular topological structures.

    Science.gov (United States)

    Cao, Dong-Sheng; Zhou, Guang-Hua; Liu, Shao; Zhang, Liu-Xia; Xu, Qing-Song; He, Min; Liang, Yi-Zeng

    2013-08-20

    The kinase family is one of the largest target families in the human genome. The family's key function in signal transduction for all organisms makes it a very attractive target class for the therapeutic interventions in many diseases states such as cancer, diabetes, inflammation and arthritis. A first step toward accelerating kinase drug discovery process is to fast identify whether a chemical and a kinase interact or not. Experimentally, these interactions can be identified by in vitro binding assay - an expensive and laborious procedure that is not applicable on a large scale. Therefore, there is an urgent need to develop statistically efficient approaches for identifying kinase-inhibitor interactions. For the first time, the quantitative binding affinities of kinase-inhibitor pairs are differentiated as a measurement to define if an inhibitor interacts with a kinase, and then a chemogenomics framework using an unbiased set of general integrated features (drug descriptors and protein descriptors) and random forest (RF) is employed to construct a predictive model which can accurately classify kinase-inhibitor pairs. Our results show that RF with integrated features gave prediction accuracy of 93.76%, sensitivity of 92.26%, and specificity of 95.27%, respectively. The results are superior to those by only considering two separated spaces (chemical space and protein space), demonstrating that these integrated features contribute cooperatively. Based on the constructed model, we provided a high confidence list of drug-target associations for subsequent experimental investigation guidance at a low false discovery rate. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Structure-activity study of new inhibitors of human betaine-homocysteine S-methyltransferase

    Czech Academy of Sciences Publication Activity Database

    Vaněk, Václav; Buděšínský, Miloš; Kabeleová, Petra; Šanda, Miloslav; Kožíšek, Milan; Hančlová, Ivona; Mládková, Jana; Brynda, Jiří; Rosenberg, Ivan; Koutmos, M.; Garrow, T. A.; Jiráček, Jiří

    2009-01-01

    Roč. 52, č. 12 (2009), s. 3652-3665 ISSN 0022-2623 R&D Projects: GA MŠk 1M0508 Grant - others:GA MŠk(CZ) LC06077; NIH(US) R01TW0052501 Program:LC Institutional research plan: CEZ:AV0Z40550506 Keywords : BHMT * betain * homocysteine * methionine * inhibitor Subject RIV: CE - Biochemistry Impact factor: 4.802, year: 2009

  1. S-alkylated homocysteine derivatives: New inhibitors of human betaine-homocysteine S-methyltransferase

    Czech Academy of Sciences Publication Activity Database

    Jiráček, Jiří; Collinsová, Michaela; Rosenberg, Ivan; Buděšínský, Miloš; Protivínská, Eva; Netušilová, Hana; Garrow, T. A.

    2006-01-01

    Roč. 49, č. 13 (2006), s. 3982-3989 ISSN 0022-2623 R&D Projects: GA AV ČR(CZ) IAA4055302 Grant - others:NIH(US) DK52501; NIH(US) R01TW0052501; IARS(US) 50-352 Institutional research plan: CEZ:AV0Z40550506 Keywords : BHMT * S-alkylated homocysteine * inhibitor Subject RIV: CE - Biochemistry Impact factor: 5.115, year: 2006

  2. In vitro effect of inhibitors on the activity of glucosyltransferase, isolated from human dental plaque.

    Science.gov (United States)

    Pinheiro, C E; Poletto, M I; Pinheiro, C R; Negrato, M L

    1989-01-01

    The enzyme glucosyltransferase plays an important role in plaque formation and growth. Therefore, chemical inhibition of glucosyltransferase may become an effective method for plaque control. In this investigation we have evaluated the effects of some antiplaque substances (chlorhexidine, cetylpiridinium chloride, iodine, sodium fluoride and sodium dodecyl sulfate) on glucosyltransferase activity. Our results revealed that iodine was the most effective inhibitor. Based on in vitro glucosyltransferase inhibition we may suggest that topical iodine could be an auxiliary method for plaque control.

  3. Molecular Characterization of Clinical Isolates of Human Immunodeficiency Virus Resistant to the Protease Inhibitor Darunavir

    Czech Academy of Sciences Publication Activity Database

    Grantz Šašková, Klára; Kožíšek, Milan; Řezáčová, Pavlína; Brynda, Jiří; Yashina, T.; Kagan, R. M.; Konvalinka, Jan

    2009-01-01

    Roč. 83, č. 17 (2009), s. 8810-8818 ISSN 0022-538X R&D Projects: GA MŠk 1M0508 EU Projects: European Commission(XE) 37693 - HIV PI RESISTANCE Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : HIV -1 protease * darunavir * X-ray structural analysis * protease inhibitor * mutations Subject RIV: CE - Biochemistry Impact factor: 5.150, year: 2009

  4. Novel Substrate-Based Inhibitors of Human Glutamate Carboxypeptidase II with Enhanced Lipophilicity

    Czech Academy of Sciences Publication Activity Database

    Plechanovová, Anna; Byun, Y.; Alquicer, Glenda; Škultétyová, Ĺubica; Mlčochová, Petra; Němcová, Adriana; Kim, H.-J.; Navrátil, Michal; Mease, R.; Lubkowski, J.; Pomper, M.; Konvalinka, Jan; Rulíšek, Lubomír; Bařinka, Cyril

    2011-01-01

    Roč. 54, č. 21 (2011), s. 7535-7546 ISSN 0022-2623 R&D Projects: GA MŠk(CZ) ME10031; GA MŠk LC512 Grant - others:EMBO(DE) 1978 Institutional research plan: CEZ:AV0Z50520701; CEZ:AV0Z40550506 Keywords : Glutamate carboxypeptidase II. * QM/MM calculations * X-ray crystallography * lipophilicity * inhibitors Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.248, year: 2011

  5. Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia

    International Nuclear Information System (INIS)

    Shimada, Hidenori; Hashimoto, Yoshiya; Nakada, Akira; Shigeno, Keiji; Nakamura, Tatsuo

    2012-01-01

    Highlights: ► Very rapid generation of human iPS cells under optimized conditions. ► Five chemical inhibitors under hypoxia boosted reprogramming. ► We performed genome-wide DNA methylation analysis. -- Abstract: Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1 month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly generating bona fide human iPS cells and facilitates the application of i

  6. Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Shimada, Hidenori [Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan); Hashimoto, Yoshiya [Department of Biomaterials, Osaka Dental University, 8-1, Hanazonocho, Kuzuha, Hirakatashi, Osaka 573-1121 (Japan); Nakada, Akira; Shigeno, Keiji [Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan); Nakamura, Tatsuo, E-mail: nakamura@frontier.kyoto-u.ac.jp [Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan)

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer Very rapid generation of human iPS cells under optimized conditions. Black-Right-Pointing-Pointer Five chemical inhibitors under hypoxia boosted reprogramming. Black-Right-Pointing-Pointer We performed genome-wide DNA methylation analysis. -- Abstract: Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1 month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly

  7. Structural optimization of a retrograde trafficking inhibitor that protects cells from infections by human polyoma- and papillomaviruses.

    Science.gov (United States)

    Carney, Daniel W; Nelson, Christian D S; Ferris, Bennett D; Stevens, Julia P; Lipovsky, Alex; Kazakov, Teymur; DiMaio, Daniel; Atwood, Walter J; Sello, Jason K

    2014-09-01

    Human polyoma- and papillomaviruses are non-enveloped DNA viruses that cause severe pathologies and mortalities. Under circumstances of immunosuppression, JC polyomavirus causes a fatal demyelinating disease called progressive multifocal leukoencephalopathy (PML) and the BK polyomavirus is the etiological agent of polyomavirus-induced nephropathy and hemorrhagic cystitis. Human papillomavirus type 16, another non-enveloped DNA virus, is associated with the development of cancers in tissues like the uterine cervix and oropharynx. Currently, there are no approved drugs or vaccines to treat or prevent polyomavirus infections. We recently discovered that the small molecule Retro-2(cycl), an inhibitor of host retrograde trafficking, blocked infection by several human and monkey polyomaviruses. Here, we report diversity-oriented syntheses of Retro-2(cycl) and evaluation of the resulting analogs using an assay of human cell infections by JC polyomavirus. We defined structure-activity relationships and also discovered analogs with significantly improved potency as suppressors of human polyoma- and papillomavirus infection in vitro. Our findings represent an advance in the development of drug candidates that can broadly protect humans from non-enveloped DNA viruses and toxins that exploit retrograde trafficking as a means for cell entry. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Fem1b, a proapoptotic protein, mediates proteasome inhibitor-induced apoptosis of human colon cancer cells.

    Science.gov (United States)

    Subauste, M Cecilia; Sansom, Owen J; Porecha, Nehal; Raich, Natacha; Du, Liqin; Maher, Joseph F

    2010-02-01

    In the treatment of colon cancer, the development of resistance to apoptosis is a major factor in resistance to therapy. New molecular approaches to overcome apoptosis resistance, such as selectively upregulating proapoptotic proteins, are needed in colon cancer therapy. In a mouse model with inactivation of the adenomatous polyposis coli (Apc) tumor suppressor gene, reflecting the pathogenesis of most human colon cancers, the gene encoding feminization-1 homolog b (Fem1b) is upregulated in intestinal epithelium following Apc inactivation. Fem1b is a proapoptotic protein that interacts with apoptosis-inducing proteins Fas, tumor necrosis factor receptor-1 (TNFR1), and apoptotic protease activating factor-1 (Apaf-1). Increasing Fem1b expression induces apoptosis of cancer cells, but effects on colon cancer cells have not been reported. Fem1b is a homolog of feminization-1 (FEM-1), a protein in Caenorhabditis elegans that is regulated by proteasomal degradation, but whether Fem1b is likewise regulated by proteasomal degradation is unknown. Herein, we found that Fem1b protein is expressed in primary human colon cancer specimens, and in malignant SW620, HCT-116, and DLD-1 colon cancer cells. Increasing Fem1b expression, by transfection of a Fem1b expression construct, induced apoptosis of these cells. We found that proteasome inhibitor treatment of SW620, HCT-116, and DLD-1 cells caused upregulation of Fem1b protein levels, associated with induction of apoptosis. Blockade of Fem1b upregulation with morpholino antisense oligonucleotide suppressed the proteasome inhibitor-induced apoptosis of these cells. In conclusion, the proapoptotic protein Fem1b is downregulated by the proteasome in malignant colon cancer cells and mediates proteasome inhibitor-induced apoptosis of these cells. Therefore, Fem1b could represent a novel molecular target to overcome apoptosis resistance in therapy of colon cancer.

  9. SNX-25a, a novel Hsp90 inhibitor, inhibited human cancer growth more potently than 17-AAG.

    Science.gov (United States)

    Wang, Shaoxiang; Wang, Xiao; Du, Zhan; Liu, Yuting; Huang, Dane; Zheng, Kai; Liu, Kaisheng; Zhang, Yi; Zhong, Xueyun; Wang, Yifei

    2014-07-18

    17-Allylamino-17-demethoxygeldanamycin (17-AAG), a typical Hsp90 inhibitor derived from geldanamycin (GA), has entered Phase III clinical trials for cancer therapy. However, it has several significant limitations such as poor solubility, limited bioavailability and unacceptable hepatotoxicity. In this study, the anticancer activity and mechanism of SNX-25a, a novel Hsp90 inhibitor, was investigated comparing with that of 17-AAG. We showed that SNX-25a triggered growth inhibition more sensitively than 17-AAG against many human cancer cells, including K562, SW-620, A375, Hep-2, MCF-7, HepG2, HeLa, and A549 cell lines, especially at low concentrations (AAG, SNX-25a was more potent in arresting the cell cycle at G2 phase, and displayed more potent effects on human cancer cell apoptosis and Hsp90 client proteins. It also exhibited a stronger binding affinity to Hsp90 than 17-AAG using molecular docking. Considering the superiority effects on Hsp90 affinity, cell growth, cell cycle, apoptosis, and Hsp90 client proteins, SNX-25a is supposed as a potential anticancer agent that needs to be explored in detail. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Lersivirine, a Nonnucleoside Reverse Transcriptase Inhibitor with Activity against Drug-Resistant Human Immunodeficiency Virus Type 1▿ ‡

    Science.gov (United States)

    Corbau, Romuald; Mori, Julie; Phillips, Chris; Fishburn, Lesley; Martin, Alex; Mowbray, Charles; Panton, Wendy; Smith-Burchnell, Caroline; Thornberry, Adele; Ringrose, Heather; Knöchel, Thorsten; Irving, Steve; Westby, Mike; Wood, Anthony; Perros, Manos

    2010-01-01

    The nonnucleoside reverse transcriptase inhibitors (NNRTIs) are key components of highly active antiretroviral therapy (HAART) for the treatment of human immunodeficiency virus type 1 (HIV-1). A major problem with the first approved NNRTIs was the emergence of mutations in the HIV-1 reverse transcriptase (RT), in particular K103N and Y181C, which led to resistance to the entire class. We adopted an iterative strategy to synthesize and test small molecule inhibitors from a chemical series of pyrazoles against wild-type (wt) RT and the most prevalent NNRTI-resistant mutants. The emerging candidate, lersivirine (UK-453,061), binds the RT enzyme in a novel way (resulting in a unique resistance profile), inhibits over 60% of viruses bearing key RT mutations, with 50% effective concentrations (EC50s) within 10-fold of those for wt viruses, and has excellent selectivity against a range of human targets. Altogether lersivirine is a highly potent and selective NNRTI, with excellent efficacy against NNRTI-resistant viruses. PMID:20660667

  11. Lersivirine, a nonnucleoside reverse transcriptase inhibitor with activity against drug-resistant human immunodeficiency virus type 1.

    Science.gov (United States)

    Corbau, Romuald; Mori, Julie; Phillips, Chris; Fishburn, Lesley; Martin, Alex; Mowbray, Charles; Panton, Wendy; Smith-Burchnell, Caroline; Thornberry, Adele; Ringrose, Heather; Knöchel, Thorsten; Irving, Steve; Westby, Mike; Wood, Anthony; Perros, Manos

    2010-10-01

    The nonnucleoside reverse transcriptase inhibitors (NNRTIs) are key components of highly active antiretroviral therapy (HAART) for the treatment of human immunodeficiency virus type 1 (HIV-1). A major problem with the first approved NNRTIs was the emergence of mutations in the HIV-1 reverse transcriptase (RT), in particular K103N and Y181C, which led to resistance to the entire class. We adopted an iterative strategy to synthesize and test small molecule inhibitors from a chemical series of pyrazoles against wild-type (wt) RT and the most prevalent NNRTI-resistant mutants. The emerging candidate, lersivirine (UK-453,061), binds the RT enzyme in a novel way (resulting in a unique resistance profile), inhibits over 60% of viruses bearing key RT mutations, with 50% effective concentrations (EC(50)s) within 10-fold of those for wt viruses, and has excellent selectivity against a range of human targets. Altogether lersivirine is a highly potent and selective NNRTI, with excellent efficacy against NNRTI-resistant viruses.

  12. Gas chromatography-mass spectrometric study of 19-oxygenation of the aromatase inhibitor 19-methylandrostenedione with human placental microsomes.

    Science.gov (United States)

    Numazawa, Mitsuteru; Nagaoka, Masao; Handa, Wakako; Yamada, Akane

    2006-06-01

    To gain insight into the catalytic function of aromatase, we studied 19-oxygenation of 19-methyl-substituted derivative of the natural substrate androstenedione (AD), compound 1, with human placental aromatase by use of gas chromatography-mass spectrometry (GC-MS). Incubation of the 19-methyl derivative 1 with human placental microsomes in the presence of NADPH under an aerobic condition did not yield a detectable amount of [19S]19-hydroxy product 2 or its [19R]-isomer 3 when the product was analyzed as the bis-methoxime-trimethylsilyl (TMS) derivative by GC-MS; moreover, the production of estrogen was not detected as the bis-TMS derivative of estradiol (detection limit: about 3 ng and 10 pg per injection for the 19-ol and estradiol, respectively). The results reveal that the 19-methyl steroid 1 does not serve as a substrate of aromatase, although it does serve as a powerful inhibitor of the enzyme.

  13. Molecular modeling of human acidic mammalian chitinase in complex with the natural-product cyclopentapeptide chitinase inhibitor argifin.

    Science.gov (United States)

    Gouda, Hiroaki; Terashima, Shinichi; Iguchi, Kanami; Sugawara, Akihiro; Saito, Yoshifumi; Yamamoto, Tsuyoshi; Hirose, Tomoyasu; Shiomi, Kazuro; Sunazuka, Toshiaki; Omura, Satoshi; Hirono, Shuichi

    2009-09-01

    Human acidic mammalian chitinase (hAMCase) is an attractive target for developing anti-asthma medications. We used a variety of computational methods to investigate the interaction between hAMCase and the natural-product cyclopentapeptide chitinase inhibitor argifin. The three-dimensional structure of hAMCase was first constructed using homology modeling. The interaction mode and binding free energy between argifin and hAMCase were then examined by the molecular-docking calculation and the molecular mechanics Poisson-Boltzmann surface area method combined with molecular dynamics simulation, respectively. The results suggested that argifin binds to hAMCase in a similar fashion to the interaction mode observed in the crystal structure of argifin-human chitotriosidase complex, and possesses inhibitory activity against hAMCase in the micromolar range. We further designed argifin derivatives expected to be selective for hAMCase.

  14. Single-molecule supercoil-relaxation assay as a screening tool to determine the mechanism and efficacy of human topoisomerase IB inhibitors

    Science.gov (United States)

    Seol, Yeonee; Zhang, Hongliang; Agama, Keli; Lorence, Nicholas; Pommier, Yves; Neuman, Keir C.

    2015-01-01

    Human nuclear type IB topoisomerase (Top1) inhibitors are widely used and powerful anti-cancer agents. In this study, we introduce and validate a single-molecule supercoil relaxation assay as a molecular pharmacology tool for characterizing therapeutically relevant Top1 inhibitors. Using this assay, we determined the effects on Top1 supercoil relaxation activity of four Top1 inhibitors; three clinically relevant: camptothecin, LMP-400, LMP-776 (both indenoisoquinoline derivatives), and one natural product in preclinical development, lamellarin-D. Our results demonstrate that Top1 inhibitors have two distinct effects on Top1 activity: a decrease in supercoil relaxation rate and an increase in religation inhibition. The type and magnitude of the inhibition mode depend both on the specific inhibitor and on the topology of the DNA substrate. In general, the efficacy of inhibition is significantly higher with supercoiled than with relaxed DNA substrates. Comparing single-molecule inhibition with cell growth inhibition (IC50) measurements showed a correlation between the binding time of the Top1 inhibitors and their cytotoxic efficacy, independent of the mode of inhibition. This study demonstrates that the single-molecule supercoil relaxation assay is a sensitive method to elucidate the detailed mechanisms of Top1 inhibitors and is relevant for the cellular efficacy of Top1 inhibitors. PMID:26351326

  15. Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors

    DEFF Research Database (Denmark)

    Ermert, David; Shaughnessy, Jutamas; Joeris, Thorsten

    2015-01-01

    Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement...

  16. Disulfiram promotes the conversion of carcinogenic cadmium to a proteasome inhibitor with pro-apoptotic activity in human cancer cells

    International Nuclear Information System (INIS)

    Li Lihua; Yang Huanjie; Chen Di; Cui, Cindy; Ping Dou, Q.

    2008-01-01

    The ubiquitin-proteasome system is involved in various cellular processes, including transcription, apoptosis, and cell cycle. In vitro, in vivo, and clinical studies suggest the potential use of proteasome inhibitors as anticancer drugs. Cadmium (Cd) is a widespread environmental pollutant that has been classified as a human carcinogen. Recent study in our laboratory suggested that the clinically used anti-alcoholism drug disulfiram (DSF) could form a complex with tumor cellular copper, resulting in inhibition of the proteasomal chymotrypsin-like activity and induction of cancer cell apoptosis. In the current study, we report, for the first time, that DSF is able to convert the carcinogen Cd to a proteasome-inhibitor and cancer cell apoptosis inducer. Although the DSF-Cd complex inhibited the chymotrypsin-like activity of a purified 20S proteasome with an IC 50 value of 32 μmol/L, this complex was much more potent in inhibiting the chymotrypsin-like activity of prostate cancer cellular 26S proteasome. Inhibition of cellular proteasome activity by the DSF-Cd complex resulted in the accumulation of ubiquitinated proteins and the natural proteasome substrate p27, which was followed by activation of calpain and induction of apoptosis. Importantly, human breast cancer MCF10DCIS cells were much more sensitive to the DSF-Cd treatment than immortalized but non-tumorigenic human breast MCF-10A cells, demonstrating that the DSF-Cd complex could selectively induce proteasome inhibition and apoptosis in human tumor cells. Our work suggests the potential use of DSF for treatment of cells with accumulated levels of carcinogen Cd

  17. Liposomal-delivery of phosphodiesterase 5 inhibitors augments UT-15C-stimulated ATP release from human erythrocytes.

    Science.gov (United States)

    Bowles, Elizabeth A; Feys, Dimitri; Ercal, Nuran; Sprague, Randy S

    2017-12-01

    The use of liposomes to affect targeted delivery of pharmaceutical agents to specific sites may result in the reduction of side effects and an increase in drug efficacy. Since liposomes are delivered intravascularly, erythrocytes, which constitute almost half of the volume of blood, are ideal targets for liposomal drug delivery. In vivo, erythrocytes serve not only in the role of oxygen transport but also as participants in the regulation of vascular diameter through the regulated release of the potent vasodilator, adenosine triphosphate (ATP). Unfortunately, erythrocytes of humans with pulmonary arterial hypertension (PAH) do not release ATP in response to the physiological stimulus of exposure to increases in mechanical deformation as would occur when these cells traverse the pulmonary circulation. This defect in erythrocyte physiology has been suggested to contribute to pulmonary hypertension in these individuals. In contrast to deformation, both healthy human and PAH erythrocytes do release ATP in response to incubation with prostacyclin analogs via a well-characterized signaling pathway. Importantly, inhibitors of phosphodiesterase 5 (PDE5) have been shown to significantly increase prostacyclin analog-induced ATP release from human erythrocytes. Here we investigate the hypothesis that targeted delivery of PDE5 inhibitors to human erythrocytes, using a liposomal delivery system, potentiates prostacyclin analog- induced ATP release. The findings are consistent with the hypothesis that directed delivery of this class of drugs to erythrocytes could be a new and important method to augment prostacyclin analog-induced ATP release from these cells. Such an approach could significantly limit side effects of both classes of drugs without compromising their therapeutic effectiveness in diseases such as PAH.

  18. Development of an on-line high performance liquid chromatography detection system for human cytochrome P450 1A2 inhibitors in extracts of natural products

    NARCIS (Netherlands)

    Jeurissen, S.M.F.; Claassen, F.W.; Havlik, J.; Bouwmans, E.E.; Cnubben, N.H.P.; Sudhölter, E.J.R.; Rietjens, I.M.C.M.; Beek, T.A. van

    2007-01-01

    An on-line HPLC screening method for detection of inhibitors of human cytochrome P450 1A2 in extracts was developed. HPLC separation of extracts is connected to a continuous methoxyresorufin-O-demethylation (MROD) assay in which recombinant human P450 1A2 converts methoxyresorufin to its fluorescent

  19. Flavonoids Are Inhibitors of Human Organic Anion Transporter 1 (OAT1)–Mediated Transport

    Science.gov (United States)

    An, Guohua; Wang, Xiaodong

    2014-01-01

    Organic anion transporter 1 (OAT1) has been reported to be involved in the nephrotoxicity of many anionic xenobiotics. As current clinically used OAT1 inhibitors are often associated with safety issues, identifying potent OAT1 inhibitors with little toxicity is of great value in reducing OAT1-mediated drug nephrotoxicity. Flavonoids are a class of polyphenolic compounds with exceptional safety records. Our objective was to evaluate the effects of 18 naturally occurring flavonoids, and some of their glycosides, on the uptake of para-aminohippuric acid (PAH) in both OAT1-expressing and OAT1-negative LLC-PK1 cells. Most flavonoid aglycones produced substantial decreases in PAH uptake in OAT1-expressing cells. Among the flavonoids screened, fisetin, luteolin, morin, and quercetin exhibited the strongest effect and produced complete inhibition of OAT1-mediated PAH uptake at a concentration of 50 μM. Further concentration-dependent studies revealed that both morin and luteolin are potent OAT1 inhibitors, with IC50 values of flavonoid aglycones, all flavonoid glycosides had negligible or small effects on OAT1. In addition, the role of OAT1 in the uptake of fisetin, luteolin, morin, and quercetin was investigated and fisetin was found to be a substrate of OAT1. Taken together, our results indicate that flavonoids are a novel class of OAT1 modulators. Considering the high consumption of flavonoids in the diet and in herbal products, OAT1-mediated flavonoid-drug interactions may be clinically relevant. Further investigation is warranted to evaluate the nephroprotective role of flavonoids in relation to drug-induced nephrotoxicity mediated by the OAT1 pathway. PMID:25002746

  20. Immune checkpoint inhibitors enhance cytotoxicity of cytokine-induced killer cells against human myeloid leukaemic blasts.

    Science.gov (United States)

    Poh, Su Li; Linn, Yeh Ching

    2016-05-01

    We studied whether blockade of inhibitory receptors on cytokine-induced killer (CIK) cells by immune checkpoint inhibitors could increase its anti-tumour potency against haematological malignancies. CIK cultures were generated from seven normal donors and nine patients with acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL) or multiple myeloma (MM). The inhibitory receptors B and T lymphocyte attenuator, CD200 receptor, lymphocyte activation gene-3 (LAG-3) and T cell immunoglobulin and mucin-domain-containing-3 (TIM-3) were present at variable percentages in most CIK cultures, while cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed death-1 (PD-1) and killer cell immunoglobulin-like receptors (KIR2DL1/2/3) were expressed at low level in most cultures. Without blockade, myeloid leukaemia cells were susceptible to autologous and allogeneic CIK-mediated cytotoxicity. Blockade of KIR, LAG-3, PD-1 and TIM-3 but not CTLA-4 resulted in remarkable increase in killing against these targets, even in those with poor baseline cytotoxicity. ALL and MM targets were resistant to CIK-mediated cytotoxicity, and blockade of receptors did not increase cytotoxicity to a meaningful extent. Combination of inhibitors against two receptors did not further increase cytotoxicity. Interestingly, potentiation of CIK killing by blocking antibodies was not predicted by expression of receptors on CIK and their respective ligands on the targets. Compared to un-activated T and NK cells, blockade potentiated the cytotoxicity of CIK cells to a greater degree and at a lower E:T ratio, but without significant increase in cytotoxicity against normal white cell. Our findings provide the basis for clinical trial combining autologous CIK cells with checkpoint inhibitors for patients with AML.

  1. Synthesis and Evaluation of Novel RSK Inhibitors in a Living Human Breast Model

    Science.gov (United States)

    2015-09-01

    SL0101 inhibits RSK in both the breast cancer cell line MCF7 and the normal breast cell line MCF-10A, but only inhibits the proliferation of the...but a much less potent inhibitor of the proliferation of MCF7 breast cancer cells (EC50 = 50 µM), suggesting that it does not readily pass through the...cell membrane (3). In order to develop a drug, the potency against MCF7 cells must be improved. The scope of this project is to design and chemically

  2. Altered Expression of Matrix Metalloproteinases and Their Endogenous Inhibitors in a Human Isogenic Stem Cell Model of Huntington's Disease

    Directory of Open Access Journals (Sweden)

    Swati Naphade

    2018-02-01

    Full Text Available Huntington's disease (HD is an autosomal dominant neurodegenerative disorder characterized by a progressive movement disorder, psychiatric symptoms, and cognitive impairments. HD is caused by a CAG repeat expansion encoding a stretch of polyglutamine residues in the N-terminus of mutant huntingtin (mHTT protein. Proteolytic processing of mHTT yields toxic fragments, which cause neurotoxicity and massive neuronal cell death predominantly in the striatum and cortex. Inhibition of mHTT cleavage reduces neuronal toxicity suggesting mHTT proteolysis contributes to HD pathogenesis. A previously conducted unbiased siRNA screen in our lab for known human proteases identified matrix metalloproteinases (MMPs as modifiers of mHTT proteolysis and toxicity. To further study MMP activation in HD, isogenic HD, and control corrected (C116 neural stem cells (NSCs prepared from HD patient-derived induced pluripotent stem cells were used to examine the role of MMPs and their endogenous inhibitors in this highly relevant model system. We found altered expression of MMP-2 and MMP-9 (gelatinases, MMP-3/10, and MMP-14, activity in HD-NSCs when compared to control C116-NSCs. Dysregulation in MMP activity was accompanied with concomitant changes in levels of endogenous inhibitors of MMPs, called tissue inhibitors of matrix metalloproteinases (TIMPs. Specifically, we observed decreased levels of TIMP-1 and TIMP-2 in HD-NSCs, suggesting part of the altered expression and activity of MMPs is due to lower abundance of these endogenous inhibitors. Immunofluorescence analysis revealed increased MMP/TIMP localization in the nucleus or aggregates of HD-NSCs, suggesting potential interaction with mHTT. TIMP-1 was found to associate with mHTT aggregates in discrete punctate structures in HD-NSCs. These events collectively contribute to increased neurotoxicity in HD. Previous characterization of these NSCs revealed transforming growth factor beta (TGF-β pathway as the top

  3. In Silico Design of Human IMPDH Inhibitors Using Pharmacophore Mapping and Molecular Docking Approaches

    Directory of Open Access Journals (Sweden)

    Rui-Juan Li

    2015-01-01

    Full Text Available Inosine 5′-monophosphate dehydrogenase (IMPDH is one of the crucial enzymes in the de novo biosynthesis of guanosine nucleotides. It has served as an attractive target in immunosuppressive, anticancer, antiviral, and antiparasitic therapeutic strategies. In this study, pharmacophore mapping and molecular docking approaches were employed to discover novel Homo sapiens IMPDH (hIMPDH inhibitors. The Güner-Henry (GH scoring method was used to evaluate the quality of generated pharmacophore hypotheses. One of the generated pharmacophore hypotheses was found to possess a GH score of 0.67. Ten potential compounds were selected from the ZINC database using a pharmacophore mapping approach and docked into the IMPDH active site. We find two hits (i.e., ZINC02090792 and ZINC00048033 that match well the optimal pharmacophore features used in this investigation, and it is found that they form interactions with key residues of IMPDH. We propose that these two hits are lead compounds for the development of novel hIMPDH inhibitors.

  4. Identification and characterization of inhibitors of human apurinic/apyrimidinic endonuclease APE1.

    Directory of Open Access Journals (Sweden)

    Anton Simeonov

    2009-06-01

    Full Text Available APE1 is the major nuclease for excising abasic (AP sites and particular 3'-obstructive termini from DNA, and is an integral participant in the base excision repair (BER pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5-fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC(1280, a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays -- a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen -- and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals.

  5. Zoniporide: a potent and selective inhibitor of the human sodium-hydrogen exchanger isoform 1 (NHE-1).

    Science.gov (United States)

    Tracey, W Ross; Allen, Mary C; Frazier, Donald E; Fossa, Anthony A; Johnson, Celeste G; Marala, Ravi B; Knight, Delvin R; Guzman-Perez, Angel

    2003-01-01

    The sodium-hydrogen exchanger isoform-1 (NHE-1) plays an important role in the myocardial response to ischemia-reperfusion; inhibition of this exchanger protects against ischemic injury, including reduction in infarct size. Herein we describe a novel, potent, and highly selective NHE-1 inhibitor, zoniporide (CP-597,396; [1-(quinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl] guanidine). Zoniporide inhibits human NHE-1 with an IC(50) of 14 nM, has >150-fold selectivity vs. other NHE isoforms, and potently inhibits ex vivo NHE-1-dependent swelling of human platelets. This compound is well tolerated in preclinical animal models, exhibits moderate plasma protein binding, has a t(1/2) of 1.5 h in monkeys, and has one major active metabolite. In both in vitro and in vivo rabbit models of myocardial ischemia-reperfusion injury, zoniporide markedly reduced infarct size without adversely affecting hemodynamics or cardiac function. In the isolated heart (Langendorff), zoniporide elicited a concentration-dependent reduction in infarct size (EC(50) = 0.25 nM). At 50 nM it reduced infarct size by 83%. This compound was 2.5-20-fold more potent than either eniporide or cariporide (EC(50)s of 0.69 and 5.11 nM, respectively), and reduced infarct size to a greater extent than eniporide. In open chest, anesthetized rabbits, zoniporide also elicited a dose-dependent reduction in infarct size (ED(50) = 0.45 mg/kg/h) and inhibited NHE-1-mediated platelet swelling (93% inhibition at 4 mg/kg/h). Furthermore, zoniporide attenuated postischemic cardiac contractile dysfunction in conscious primates, and reduced both the incidence and duration of ischemia-reperfusion-induced ventricular fibrillation in rats. Zoniporide represents a novel class of potent and selective human NHE-1 inhibitors with potential utility for providing cardioprotection in a clinical setting.

  6. SERPINE2, an inhibitor of plasminogen activators, is highly expressed in the human endometrium during the secretory phase

    Directory of Open Access Journals (Sweden)

    Hwu Yuh-Ming

    2011-03-01

    Full Text Available Abstract Background SERPINE2, also known as protease nexin-1, belongs to the serine protease inhibitor (SERPIN superfamily. It is one of the potent SERPINs that modulates the activity of plasminogen activators (PAs. PAs and their SERPIN inhibitors, such as SERPINB2 and SERPINE1, were expressed in the human endometrium and were implicated in implantation. However, expression data about SERPINE2 in the human endometrium is still unknown. Thus, we conducted an investigation to reveal the spatiotemporal and cellular expression of SERPINE2 in the human uterus during the menstrual cycle. Methods Seven patients who underwent a hysterectomy and samples of 120 archived patients' endometrial curettage or parts of the uterus that were formalin-fixed and embedded in paraffin. Western blotting was performed to evaluate the specificity and sensitivity of the antibody. Immunohistochemistry was conducted to localize the SERPINE2 expression site. Quantitative analysis was conducted to evaluate expression levels of SERPINE2 in various sub-phases of the menstrual cycle. Results The SERPINE2 protein was primarily detected in the uterine fluid during the mid- and late-secretory phases of the menstrual cycle. It was predominantly expressed in the luminal and glandular epithelium, less in the myometrium, and only dispersedly in certain stromal cells throughout the menstrual cycle. A quantitative analysis of expression levels of SERPINE2 in the glandular epithelium revealed that it was highly expressed in the endometrium during the secretory phase compared to the proliferative phase. Conclusions The SERPINE2 protein is highly expressed in the endometrium during the secretory phase, indicating that it may participate in tissue remodeling involved in implantation.

  7. The Novel HSP90 inhibitor, IPI-493, is highly effective in human gastrostrointestinal stromal tumor xenografts carrying heterogeneous KIT mutations.

    Science.gov (United States)

    Floris, Giuseppe; Sciot, Raf; Wozniak, Agnieszka; Van Looy, Thomas; Wellens, Jasmien; Faa, Gavino; Normant, Emmanuel; Debiec-Rychter, Maria; Schöffski, Patrick

    2011-09-01

    KIT activity is crucial for gastrointestinal stromal tumors (GIST). Imatinib (IMA) and sunitinib (SUN) are very effective KIT-inhibitors in patients with advanced GIST but have no curative potential. We evaluated the efficacy of the novel HSP90 inhibitor IPI-493 alone, or in combination with IMA or SUN in GIST xenografts with KIT mutations. Nude mice (n = 98) were grafted bilaterally with human GIST carrying KIT exon 11 (GIST-PSW), KIT exon 9 (GIST-BOE), or double, KIT imatinib-sensitive exon 11 and imatinib-resistant exon 17 mutations (GIST-48). Mice were divided into six treatment groups and dosed orally for 15 days as follows: (i) control group, sterile water; (ii) IMA alone; (iii) SUN alone; (iv) IPI-493 alone; (v) IPI-493+IMA; and (vi) IPI-493+SUN. Treatment with IPI-493 resulted in tumor growth stabilization, variable proliferation arrest, induction of apoptosis and necrosis, and downregulation of KIT and its signaling cascade, especially in the GIST-BOE model. Significant reduction of vessel density was observed with IPI-493 treatment, and was equal to SUN treatment in GIST-PSW and GIST-BOE xenografts. IPI-493 treatment effects were enhanced in combination with TKIs, especially with IPI-493+SUN. In our hands, IPI-493 showed dose-dependent liver damages. When administered as a single agent in a xenograft model, the HSP90 inhibitor IPI-493 has consistent antitumor activity and induces KIT downregulation in GISTs with heterogeneous KIT mutations. IPI-493 synergizes with TKIs that are commonly used for the treatment of advanced or IMA-resistant GIST. The antitumor response of IPI-493 is particularly enhanced in combination with SUN. ©2011 AACR.

  8. Peroxisome Proliferator-Activated Receptorα Agonists Differentially Regulate Inhibitor of DNA Binding Expression in Rodents and Human Cells

    Directory of Open Access Journals (Sweden)

    María del Carmen González

    2012-01-01

    Full Text Available Inhibitor of DNA binding (Id2 is a helix-loop-helix (HLH transcription factor that participates in cell differentiation and proliferation. Id2 has been linked to the development of cardiovascular diseases since thiazolidinediones, antidiabetic agents and peroxisome proliferator-activated receptor (PPAR gamma agonists, have been reported to diminish Id2 expression in human cells. We hypothesized that PPARα activators may also alter Id2 expression. Fenofibrate diminished hepatic Id2 expression in both late pregnant and unmated rats. In 24 hour fasted rats, Id2 expression was decreased under conditions known to activate PPARα. In order to determine whether the fibrate effects were mediated by PPARα, wild-type mice and PPARα-null mice were treated with Wy-14,643 (WY. WY reduced Id2 expression in wild-type mice without an effect in PPARα-null mice. In contrast, fenofibrate induced Id2 expression after 24 hours of treatment in human hepatocarcinoma cells (HepG2. MK-886, a PPARα antagonist, did not block fenofibrate-induced activation of Id2 expression, suggesting a PPARα-independent effect was involved. These findings confirm that Id2 is a gene responsive to PPARα agonists. Like other genes (apolipoprotein A-I, apolipoprotein A-V, the opposite directional transcriptional effect in rodents and a human cell line further emphasizes that PPARα agonists have different effects in rodents and humans.

  9. Safety and Efficacy of the Complement Inhibitor AMY-101 in a Natural Model of Periodontitis in Non-human Primates

    Directory of Open Access Journals (Sweden)

    Tetsuhiro Kajikawa

    2017-09-01

    Full Text Available Periodontitis is a chronic inflammatory disease associated with overactivation of the complement system. Recent preclinical studies suggest that host-modulation therapies may contribute to effective treatment of human periodontitis, which may lead to loss of teeth and function if untreated. We previously showed that locally administered AMY-101 (Cp40, a peptidic inhibitor of the central complement component C3, can inhibit naturally occurring periodontitis in non-human primates (NHPs when given once a week. This study was undertaken to determine the local safety of increasing doses of the drug as well as its efficacy when given at a reduced frequency or after systemic administration. Our findings have determined a local dose of AMY-101 (0.1 mg/site that is free of local irritation and effective when given once every 3 weeks. Moreover, a daily subcutaneous dose of AMY-101 (4 mg/kg bodyweight was protective against NHP periodontitis, suggesting that patients treated for systemic disorders (e.g., paroxysmal nocturnal hemoglobinuria can additionally benefit in terms of improved periodontal condition. In summary, AMY-101 appears to be a promising candidate drug for the adjunctive treatment of human periodontitis, a notion that merits investigation in human clinical trials.

  10. Structural basis for the immunomodulatory function of cysteine protease inhibitor from human roundworm Ascaris lumbricoides.

    Science.gov (United States)

    Mei, Guoqiang; Dong, Jianmei; Li, Zhaotao; Liu, Sanling; Liu, Yunfeng; Sun, Mingze; Liu, Guiyun; Su, Zhong; Liu, Jinsong

    2014-01-01

    Immunosuppression associated with infections of nematode parasites has been documented. Cysteine protease inhibitor (CPI) released by the nematode parasites is identified as one of the major modulators of host immune response. In this report, we demonstrated that the recombinant CPI protein of Ascaris lumbricoides (Al-CPI) strongly inhibited the activities of cathepsin L, C, S, and showed weaker effect to cathepsin B. Crystal structure of Al-CPI was determined to 2.1 Å resolution. Two segments of Al-CPI, loop 1 and loop 2, were proposed as the key structure motifs responsible for Al-CPI binding with proteases and its inhibitory activity. Mutations at loop 1 and loop 2 abrogated the protease inhibition activity to various extents. These results provide the molecular insight into the interaction between the nematode parasite and its host and will facilitate the development of anthelmintic agents or design of anti-autoimmune disease drugs.

  11. Localization of Tissue Inhibitor of Metalloproteinases 1 (TIMP-1) in Human Colorectal Adenoma and Adenocarcinoma

    DEFF Research Database (Denmark)

    Holten-Andersen, Mads N.; Hansen, Ulla; Brünner, Nils

    2005-01-01

    Tissue inhibitor of matrix metalloproteases 1 (TIMP-1) inhibits the proteolytic activity of matrix metalloproteases and hereby prevents cancer invasion. However, TIMP-1 also possesses other functions such as inhibition of apoptosis, induction of malignant transformation and stimulation of cell......-growth. We have previously demonstrated that TIMP-1 is elevated in blood from colorectal cancer patients and that high TIMP-1 levels predict poor prognosis. To clarify the role of TIMP-1 in colorectal tumorigenesis, the expression pattern of TIMP-1 in benign and malignant colorectal tumors was studied....... In all of 24 cases of colorectal adenocarcinoma TIMP-1 mRNA was detected by in situ hybridization. In all cases TIMP-1 expression was found in fibroblast-like cells located at the invasive front but was seen only sporadically in normal mucosa. No TIMP-1 mRNA was seen in any of the cases in benign...

  12. 6-Thioguanine is a noncompetitive and slow binding inhibitor of human deubiquitinating protease USP2.

    Science.gov (United States)

    Chuang, Shang-Ju; Cheng, Shu-Chun; Tang, Hui-Chi; Sun, Chiao-Yin; Chou, Chi-Yuan

    2018-02-15

    Ubiquitin-specific protease 2 (USP2) belongs to the family of deubiquitinases that can rescue protein targets from proteasomal degradation by reversing their ubiquitination. In various cancers, including prostate cancer and ovarian carcinoma, upregulation of USP2 leads to an increase in the levels of deubiquitinated substrates such as fatty acid synthase, MDM2, cyclin D1 and Aurora-A. USP2 thus plays a critical role in tumor cells' survival and therefore represents a therapeutic target. Here a leukemia drug, 6-thioguanine, was found to be a potent inhibitor of USP2. Enzyme-kinetic and X-ray crystallographic data suggest that 6-thioguanine displays a noncompetitive and slow-binding inhibitory mechanism against USP2. Our study provides a clear rationale for the clinical evaluation of 6-thioguanine for USP2-upregulated cancers.

  13. Synthesis and Screening of Human Monoamine Oxidase-A Inhibitor Effect of New 2-Pyrazoline and Hydrazone Derivatives.

    Science.gov (United States)

    Evranos-Aksöz, Begüm; Baysal, İpek; Yabanoğlu-Çiftçi, Samiye; Djikic, Teodora; Yelekçi, Kemal; Uçar, Gülberk; Ertan, Rahmiye

    2015-10-01

    A group of 3,5-diaryl-2-pyrazoline and hydrazone derivatives was prepared via the reaction of various chalcones with hydrazide compounds in ethanol. Twenty original compounds were synthesized. Ten of these original compounds have a pyrazoline structure, nine of these original compounds have a hydrazone structure, and one of these original compounds has a chalcone structure. Structural elucidation of the compounds was performed by IR, (1)H NMR, (13)C NMR, mass spectral data, and elemental analyses. These compounds were tested for their inhibitory activities toward the A and B isoforms of human monoamine oxidase (MAO). Except for 3k and 6c, all compounds were found to be competitive, reversible, and selective inhibitors for either one of the isoforms (hMAO-A or MAO-B). Compounds 3k and 6c were found to be competitive, reversible, but non-selective MAO inhibitors. Compound 6h showed hMAO-B inhibitory activity whereas the others potently inhibited hMAO-A. Compound 5c showed higher selectivity than the standard drug moclobemide. According to the experimental K(i) values, compounds 6i, 6d, and 6a exhibited the highest inhibitory activity toward hMAO-A. The AutoDock 4.2 program was employed to perform automated molecular docking. The calculated results obtained computationally were in good agreement with the experimental values. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Phosphonic and arsonic acids as inhibitors of human red cell acid phosphatase and their use in affinity chromatography.

    Science.gov (United States)

    Dissing, J; Dahl, O; Svensmark, O

    1979-08-15

    1. In order to obtain an effective ligand for affinity chromatography of the low molecular weight acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) from human red cells nine phosphonic and two arsonic acid substrate analogues were investigated as potential inhibitors. The two forms of acid phosphatase type B (b1 and b2) were isolated and partially purified using conventional methods and the inhibitory action of the substrate analogs investigated. 2. Four of the phosphonic acids were relatively effective competitive inhibitors. It appears that certain structural and electronic requirements have to be fulfilled by the phosphonic acids in order to exhibit significant affinity for the enzyme. A high affinity appears to require the presence of a bulky, hydrophobic moiety which has to be separated from the phosphorus atom by the distance of one atom. 3. p-Aminobenzylphosphonic acid exerted the highest affinity for acid phosphatase with a pH optimum at 6.5. Ki values of 4 . 10(-4) and 6 . 10(-4) M were found for the b1 and b2 forms, respectively. 4. Coupling of p-aminobenzylphosphonic acid to Agarose yielded an effective and specific affinity medium. By means of affinity chromatography using this medium, acid phosphatase was purified 500-fold in a single step.

  15. Comparison of the Binding of Reversible Inhibitors to Human Butyrylcholinesterase and Acetylcholinesterase: A Crystallographic, Kinetic and Calorimetric Study

    Directory of Open Access Journals (Sweden)

    Terrone L. Rosenberry

    2017-11-01

    Full Text Available Acetylcholinesterase (AChE and butyrylcholinesterase (BChE hydrolyze the neurotransmitter acetylcholine and, thereby, function as coregulators of cholinergic neurotransmission. Although closely related, these enzymes display very different substrate specificities that only partially overlap. This disparity is largely due to differences in the number of aromatic residues lining the active site gorge, which leads to large differences in the shape of the gorge and potentially to distinct interactions with an individual ligand. Considerable structural information is available for the binding of a wide diversity of ligands to AChE. In contrast, structural data on the binding of reversible ligands to BChE are lacking. In a recent effort, an inhibitor competition approach was used to probe the overlap of ligand binding sites in BChE. Here, we extend this study by solving the crystal structures of human BChE in complex with five reversible ligands, namely, decamethonium, thioflavin T, propidium, huprine, and ethopropazine. We compare these structures to equivalent AChE complexes when available in the protein data bank and supplement this comparison with kinetic data and observations from isothermal titration calorimetry. This new information now allows us to define the binding mode of various ligand families and will be of importance in designing specific reversible ligands of BChE that behave as inhibitors or reactivators.

  16. Human SHMT inhibitors reveal defective glycine import as a targetable metabolic vulnerability of diffuse large B-cell lymphoma.

    Science.gov (United States)

    Ducker, Gregory S; Ghergurovich, Jonathan M; Mainolfi, Nello; Suri, Vipin; Jeong, Stephanie K; Hsin-Jung Li, Sophia; Friedman, Adam; Manfredi, Mark G; Gitai, Zemer; Kim, Hahn; Rabinowitz, Joshua D

    2017-10-24

    The enzyme serine hydroxymethyltransferse (SHMT) converts serine into glycine and a tetrahydrofolate-bound one-carbon unit. Folate one-carbon units support purine and thymidine synthesis, and thus cell growth. Mammals have both cytosolic SHMT1 and mitochondrial SHMT2, with the mitochondrial isozyme strongly up-regulated in cancer. Here we show genetically that dual SHMT1/2 knockout blocks HCT-116 colon cancer tumor xenograft formation. Building from a pyrazolopyran scaffold that inhibits plant SHMT, we identify small-molecule dual inhibitors of human SHMT1/2 (biochemical IC 50 ∼ 10 nM). Metabolomics and isotope tracer studies demonstrate effective cellular target engagement. A cancer cell-line screen revealed that B-cell lines are particularly sensitive to SHMT inhibition. The one-carbon donor formate generally rescues cells from SHMT inhibition, but paradoxically increases the inhibitor's cytotoxicity in diffuse large B-cell lymphoma (DLBCL). We show that this effect is rooted in defective glycine uptake in DLBCL cell lines, rendering them uniquely dependent upon SHMT enzymatic activity to meet glycine demand. Thus, defective glycine import is a targetable metabolic deficiency of DLBCL.

  17. Isomeric mono-, di-, and tri-bromobenzo-1H-triazoles as inhibitors of human protein kinase CK2α.

    Directory of Open Access Journals (Sweden)

    Romualda Wąsik

    Full Text Available To further clarify the role of the individual bromine atoms of 4,5,6,7-tetrabromotriazole (TBBt, a relatively selective inhibitor of protein kinase CK2, we have examined the inhibition (IC(50 of human CK2α by the two mono-, the four di-, and the two tri- bromobenzotriazoles relative to that of TBBt. Halogenation of the central vicinal C(5/C(6 atoms proved to be a key factor in enhancing inhibitory activity, in that 5,6-di-Br(2Bt and 4,5,6-Br(3Bt were almost as effective inhibitors as TBBt, notwithstanding their marked differences in pK(a for dissociation of the triazole proton. The decrease in pK(a on halogenation of the peripheral C(4/C(7 atoms virtually nullifies the gain due to hydrophobic interactions, and does not lead to a decrease in IC(50. Molecular modeling of structures of complexes of the ligands with the enzyme, as well as QSAR analysis, pointed to a balance of hydrophobic and electrostatic interactions as a discriminator of inhibitory activity. The role of halogen bonding remains debatable, as originally noted for the crystal structure of TBBt with CK2α (pdb1j91. Finally we direct attention to the promising applicability of our series of well-defined halogenated benzotriazoles to studies on inhibition of kinases other than CK2.

  18. Exploration of natural product ingredients as inhibitors of human HMG-CoA reductase through structure-based virtual screening.

    Science.gov (United States)

    Lin, Shih-Hung; Huang, Kao-Jean; Weng, Ching-Feng; Shiuan, David

    2015-01-01

    Cholesterol plays an important role in living cells. However, a very high level of cholesterol may lead to atherosclerosis. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase is the key enzyme in the cholesterol biosynthesis pathway, and the statin-like drugs are inhibitors of human HMG-CoA reductase (hHMGR). The present study aimed to virtually screen for potential hHMGR inhibitors from natural product to discover hypolipidemic drug candidates with fewer side effects and lesser toxicities. We used the 3D structure 1HWK from the PDB (Protein Data Bank) database of hHMGR as the target to screen for the strongly bound compounds from the traditional Chinese medicine database. Many interesting molecules including polyphenolic compounds, polisubstituted heterocyclics, and linear lipophilic alcohols were identified and their ADMET (absorption, disrtibution, metabolism, excretion, toxicity) properties were predicted. Finally, four compounds were obtained for the in vitro validation experiments. The results indicated that curcumin and salvianolic acid C can effectively inhibit hHMGR, with IC50 (half maximal inhibitory concentration) values of 4.3 µM and 8 µM, respectively. The present study also demonstrated the feasibility of discovering new drug candidates through structure-based virtual screening.

  19. Molecular docking and 3D-QSAR studies on inhibitors of DNA damage signaling enzyme human PARP-1.

    Science.gov (United States)

    Fatima, Sabiha; Bathini, Raju; Sivan, Sree Kanth; Manga, Vijjulatha

    2012-08-01

    Poly (ADP-ribose) polymerase-1 (PARP-1) operates in a DNA damage signaling network. Molecular docking and three dimensional-quantitative structure activity relationship (3D-QSAR) studies were performed on human PARP-1 inhibitors. Docked conformation obtained for each molecule was used as such for 3D-QSAR analysis. Molecules were divided into a training set and a test set randomly in four different ways, partial least square analysis was performed to obtain QSAR models using the comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). Derived models showed good statistical reliability that is evident from their r², q²(loo) and r²(pred) values. To obtain a consensus for predictive ability from all the models, average regression coefficient r²(avg) was calculated. CoMFA and CoMSIA models showed a value of 0.930 and 0.936, respectively. Information obtained from the best 3D-QSAR model was applied for optimization of lead molecule and design of novel potential inhibitors.

  20. Phosphatase Inhibitors Function as Novel, Broad Spectrum Botulinum Neurotoxin Antagonists in Mouse and Human Embryonic Stem Cell-Derived Motor Neuron-Based Assays.

    Science.gov (United States)

    Kiris, Erkan; Nuss, Jonathan E; Stanford, Stephanie M; Wanner, Laura M; Cazares, Lisa; Maestre, Michael F; Du, Hao T; Gomba, Glenn Y; Burnett, James C; Gussio, Rick; Bottini, Nunzio; Panchal, Rekha G; Kane, Christopher D; Tessarollo, Lino; Bavari, Sina

    2015-01-01

    There is an urgent need to develop novel treatments to counter Botulinum neurotoxin (BoNT) poisoning. Currently, the majority of BoNT drug development efforts focus on directly inhibiting the proteolytic components of BoNT, i.e. light chains (LC). Although this is a rational approach, previous research has shown that LCs are extremely difficult drug targets and that inhibiting multi-serotype BoNTs with a single LC inhibitor may not be feasible. An alternative approach would target neuronal pathways involved in intoxication/recovery, rather than the LC itself. Phosphorylation-related mechanisms have been implicated in the intoxication pathway(s) of BoNTs. However, the effects of phosphatase inhibitors upon BoNT activity in the physiological target of BoNTs, i.e. motor neurons, have not been investigated. In this study, a small library of phosphatase inhibitors was screened for BoNT antagonism in the context of mouse embryonic stem cell-derived motor neurons (ES-MNs). Four inhibitors were found to function as BoNT/A antagonists. Subsequently, we confirmed that these inhibitors protect against BoNT/A in a dose-dependent manner in human ES-MNs. Additionally, these compounds provide protection when administered in post-intoxication scenario. Importantly, the inhibitors were also effective against BoNT serotypes B and E. To the best of our knowledge, this is the first study showing phosphatase inhibitors as broad-spectrum BoNT antagonists.

  1. Application of cultured human mast cells (CHMC) for the design and structure-activity relationship of IgE-mediated mast cell activation inhibitors.

    Science.gov (United States)

    Argade, Ankush; Bhamidipati, Somasekhar; Li, Hui; Carroll, David; Clough, Jeffrey; Keim, Holger; Sylvain, Catherine; Rossi, Alexander B; Coquilla, Christina; Issakani, Sarkiz D; Masuda, Esteban S; Payan, Donald G; Singh, Rajinder

    2015-01-01

    Here we report the optimization of small molecule inhibitors of human mast cell degranulation via anti-IgE-mediated tryptase release following cross-linking and activation of IgE-loaded FcεR1 receptors. The compounds are selective upstream inhibitors of FcεR1-dependent human mast cell degranulation and proved to be devoid of activity in downstream ionomycin mediated degranulation. Structure-activity relationship (SAR) leading to compound 26 is outlined. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. 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...... count increased mainly due to increases in numbers of CD4+ CD28+ and CD4+ CD45RO+ cells, whereas increases in numbers of CD4+ CD45RA+ cells contributed little to the increase in CD4+ cell count. The total cytotoxic T-cell (CTL) killing of autologous B cells infected with HIV-encoding recombinant...

  3. Small molecule inhibitors of the LEDGF site of human immunodeficiency virus integrase identified by fragment screening and structure based design.

    Directory of Open Access Journals (Sweden)

    Thomas S Peat

    Full Text Available A fragment-based screen against human immunodeficiency virus type 1 (HIV integrase led to a number of compounds that bound to the lens epithelium derived growth factor (LEDGF binding site of the integrase catalytic core domain. We determined the crystallographic structures of complexes of the HIV integrase catalytic core domain for 10 of these compounds and quantitated the binding by surface plasmon resonance. We demonstrate that the compounds inhibit the interaction of LEDGF with HIV integrase in a proximity AlphaScreen assay, an assay for the LEDGF enhancement of HIV integrase strand transfer and in a cell based assay. The compounds identified represent a potential framework for the development of a new series of HIV integrase inhibitors that do not bind to the catalytic site of the enzyme.

  4. 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...... count increased mainly due to increases in numbers of CD4+ CD28+ and CD4+ CD45RO+ cells, whereas increases in numbers of CD4+ CD45RA+ cells contributed little to the increase in CD4+ cell count. The total cytotoxic T-cell (CTL) killing of autologous B cells infected with HIV-encoding recombinant...... Vaccinia virus was increased after 3-6 months, whereas the specific HIV-directed CTL activity and the concentration and lytic activity of natural killer (NK) cells were unchanged during follow-up. These results demonstrate that the initiation of a treatment including an HIV protease inhibitor is followed...

  5. 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...... Vaccinia virus was increased after 3-6 months, whereas the specific HIV-directed CTL activity and the concentration and lytic activity of natural killer (NK) cells were unchanged during follow-up. These results demonstrate that the initiation of a treatment including an HIV protease inhibitor is followed...... count increased mainly due to increases in numbers of CD4+ CD28+ and CD4+ CD45RO+ cells, whereas increases in numbers of CD4+ CD45RA+ cells contributed little to the increase in CD4+ cell count. The total cytotoxic T-cell (CTL) killing of autologous B cells infected with HIV-encoding recombinant...

  6. Pectinesterase Inhibitor from Jelly Fig (Ficus awkeotsang Makino Achene Inhibits Surface Antigen Expression by Human Hepatitis B Virus

    Directory of Open Access Journals (Sweden)

    Yu-Chuen Huang

    2013-01-01

    Full Text Available Pectinesterase inhibitor (PEI isolated from jelly fig (Ficus awkeotsang Makino is an edible component of a popular drink consumed in Asia. Hepatitis B virus (HBV infection is prevalent in Asia, and current treatments for HBV infection need improvement. This study aimed to evaluate the effect of PEI on the surface antigen expression by HBV (HBsAg. Human hepatoma cell lines Hep3B and Huh7 served as in vitro models for assessing the cytotoxicity and HBsAg expression. A culture of primary hepatocytes cultured from mice served as the normal counterpart. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT colorimetric assay. HBsAg expression was evaluated by measuring HBsAg secretion into the culture medium using an enzyme-linked immunosorbent assay. The results showed that PEI did not affect the viability of the human hepatoma cell lines or primary mouse hepatocytes. PEI inhibited the expression of HBsAg in hepatoma cell lines harboring endogenous (Hep3B and integrated (Huh7 HBV genomes in a concentration- and time-dependent manner, thus implicating a universal activity against HBV gene expression. In conclusion, it suggests that PEI from jelly fig inhibits the expression of human HBsAg in host cells without toxic effects on normal primary hepatocytes.

  7. Pectinesterase Inhibitor from Jelly Fig (Ficus awkeotsang Makino) Achene Inhibits Surface Antigen Expression by Human Hepatitis B Virus.

    Science.gov (United States)

    Huang, Yu-Chuen; Jiang, Chii-Ming; Chen, Yu-Jen; Chen, Yu-Yawn

    2013-01-01

    Pectinesterase inhibitor (PEI) isolated from jelly fig (Ficus awkeotsang Makino) is an edible component of a popular drink consumed in Asia. Hepatitis B virus (HBV) infection is prevalent in Asia, and current treatments for HBV infection need improvement. This study aimed to evaluate the effect of PEI on the surface antigen expression by HBV (HBsAg). Human hepatoma cell lines Hep3B and Huh7 served as in vitro models for assessing the cytotoxicity and HBsAg expression. A culture of primary hepatocytes cultured from mice served as the normal counterpart. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. HBsAg expression was evaluated by measuring HBsAg secretion into the culture medium using an enzyme-linked immunosorbent assay. The results showed that PEI did not affect the viability of the human hepatoma cell lines or primary mouse hepatocytes. PEI inhibited the expression of HBsAg in hepatoma cell lines harboring endogenous (Hep3B) and integrated (Huh7) HBV genomes in a concentration- and time-dependent manner, thus implicating a universal activity against HBV gene expression. In conclusion, it suggests that PEI from jelly fig inhibits the expression of human HBsAg in host cells without toxic effects on normal primary hepatocytes.

  8. Histone Deacetylase Inhibitor Induced Radiation Sensitization Effects on Human Cancer Cells after Photon and Hadron Radiation Exposure

    Directory of Open Access Journals (Sweden)

    Ariungerel Gerelchuluun

    2018-02-01

    Full Text Available Suberoylanilide hydroxamic acid (SAHA is a histone deacetylase inhibitor, which has been widely utilized throughout the cancer research field. SAHA-induced radiosensitization in normal human fibroblasts AG1522 and lung carcinoma cells A549 were evaluated with a combination of γ-rays, proton, and carbon ion exposure. Growth delay was observed in both cell lines during SAHA treatment; 2 μM SAHA treatment decreased clonogenicity and induced cell cycle block in G1 phase but 0.2 μM SAHA treatment did not show either of them. Low LET (Linear Energy Transfer irradiated A549 cells showed radiosensitization effects on cell killing in cycling and G1 phase with 0.2 or 2 μM SAHA pretreatment. In contrast, minimal sensitization was observed in normal human cells after low and high LET radiation exposure. The potentially lethal damage repair was not affected by SAHA treatment. SAHA treatment reduced the rate of γ-H2AX foci disappearance and suppressed RAD51 and RPA (Replication Protein A focus formation. Suppression of DNA double strand break repair by SAHA did not result in the differences of SAHA-induced radiosensitization between human cancer cells and normal cells. In conclusion, our results suggest SAHA treatment will sensitize cancer cells to low and high LET radiation with minimum effects to normal cells.

  9. Metabolism and excretion of anacetrapib, a novel inhibitor of the cholesteryl ester transfer protein, in humans.

    Science.gov (United States)

    Kumar, Sanjeev; Tan, Eugene Y; Hartmann, Georgy; Biddle, Zachary; Bergman, Arthur J; Dru, James; Ho, Jonathan Z; Jones, Allen N; Staskiewicz, Steve J; Braun, Matthew P; Karanam, Bindhu; Dean, Dennis C; Gendrano, Isaias Noel; Graves, Mark W; Wagner, John A; Krishna, Rajesh

    2010-03-01

    Anacetrapib is a novel cholesteryl ester transfer protein inhibitor being developed for the treatment of primary hypercholesterolemia and mixed dyslipidemia. The absorption, distribution, metabolism, and excretion of anacetrapib were investigated in an open-label study in which six healthy male subjects received a single oral dose of 150 mg and 165 microCi of [(14)C]anacetrapib. Plasma, urine, and fecal samples were collected at predetermined times for up to 14 days postdose and were analyzed for total radioactivity, the parent compound, and metabolites. The majority of the administered radioactivity (87%) was eliminated by fecal excretion, with negligible amounts present in urine (0.1%). The peak level of radioactivity in plasma (approximately 2 microM equivalents of [(14)C]anacetrapib) was achieved approximately 4 h postdose. The parent compound was the major radioactive component (79-94% of total radioactivity) in both plasma and feces. Three oxidative metabolites, M1, M2, and M3, were detected in plasma and feces and were identified as the O-demethylated species (M1) and two secondary hydroxylated derivatives of M1 (M2 and M3). Each metabolite was detected at low levels, representing excretion of metabolites by the biliary-fecal route.

  10. Evaluation of Effective MMP Inhibitors from Eight Different Brown Algae in Human Fibrosarcoma HT1080 Cells.

    Science.gov (United States)

    Bae, Min Joo; Karadeniz, Fatih; Ahn, Byul-Nim; Kong, Chang-Suk

    2015-09-01

    Matrix metalloproteinases (MMPs) are crucial extracellular matrices degrading enzymes that have important roles in metastasis of cancer progression as well as other significant conditions such as oxidative stress and hepatic fibrosis. Marine plants are on the rise for their potential to provide natural products that exhibit remarkable health benefits. In this context, brown algae species have been of much interest in the pharmaceutical field with reported instances of isolation of bioactive compounds against tumor growth and MMP activity. In this study, eight different brown algae species were harvested, and their extracts were compared in regard to their anti-MMP effects. According to gelatin zymography results, Ecklonia cava, Ecklonia bicyclis, and Ishige okamurae showed higher inhibitory effects than the other samples on MMP-2 and -9 activity at the concentrations of 10, 50, and 100 μg/mL. However, only I. okamurae was able to regulate the MMP activity through the expression of MMP and tissue inhibitor of MMP observed by mRNA levels. Overall, brown algae species showed to be good sources for anti-MMP agents, while I. okamurae needs to be further studied for its potential to yield pharmaceutical molecules that can regulate MMP-activity through cellular pathways as well as enzymatic inhibition.

  11. Small molecule ice recrystallization inhibitors enable freezing of human red blood cells with reduced glycerol concentrations.

    Science.gov (United States)

    Capicciotti, Chantelle J; Kurach, Jayme D R; Turner, Tracey R; Mancini, Ross S; Acker, Jason P; Ben, Robert N

    2015-04-08

    In North America, red blood cells (RBCs) are cryopreserved in a clinical setting using high glycerol concentrations (40% w/v) with slow cooling rates (~1°C/min) prior to storage at -80°C, while European protocols use reduced glycerol concentrations with rapid freezing rates. After thawing and prior to transfusion, glycerol must be removed to avoid intravascular hemolysis. This is a time consuming process requiring specialized equipment. Small molecule ice recrystallization inhibitors (IRIs) such as β-PMP-Glc and β-pBrPh-Glc have the ability to prevent ice recrystallization, a process that contributes to cellular injury and decreased cell viability after cryopreservation. Herein, we report that addition of 110 mM β-PMP-Glc or 30 mM β-pBrPh-Glc to a 15% glycerol solution increases post-thaw RBC integrity by 30-50% using slow cooling rates and emphasize the potential of small molecule IRIs for the preservation of cells.

  12. Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

    Directory of Open Access Journals (Sweden)

    Yi-Hsuan Wu

    Full Text Available ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

  13. Zinc porphyrins: Potent inhibitors of hematopoieses in animal and human bone marrow

    OpenAIRE

    Lutton, John D.; Abraham, Nader G.; Drummond, George S.; Levere, Richard D.; Kappas, Attallah

    1997-01-01

    The effects of selected heme analogues on heme oxygenase activity in tissues and on human and rabbit bone marrow hematopoietic colony growth were examined. Zinc protoporphyrin (ZnPP) and zinc mesoporphyrin (ZnMP), at concentrations ranging between 1 and 20 μM, produced significant inhibition of human and rabbit bone marrow erythroid (CFU-E, BFU-E) and myeloid (CFU-GM) colony growth. The growth inhibition produced by ZnPP or ZnMP was not overcome with exposure of cultures to elevated levels of...

  14. Identification of a macromolecular crystal growth inhibitor in human urine as osteopontin

    DEFF Research Database (Denmark)

    Sørensen, Steen; Justesen, S J; Johnsen, A H

    1995-01-01

    Macromolecules occurring in human urine inhibit the growth and/or aggregation of calcium oxalate crystals and may prevent the formation of kidney stones. Attention has focused particularly on proteins, as these seem to be most responsible for the inhibitory activity; three proteins, nephrocalcin...

  15. Proteomic profiling of human colon cancer cells treated with the histone deacetylase inhibitor belinostat

    DEFF Research Database (Denmark)

    Beck, Hans Christian; Petersen, Jørgen; Nielsen, Søren Jensby

    2010-01-01

    in the human colon cancer cell line HCT116. Protein extracts from untreated HCT116 cells, and cells grown for 24 h in the presence of 1 and 10 muM belinostat were analysed by 2-D gel electrophoresis. Proteins were visualized by colloidal Coomassie blue staining and quantitative analysis of gel images revealed...

  16. Differential effects of rapalogues, dual kinase inhibitors on human ovarian carcinoma cells in vitro.

    Science.gov (United States)

    Rogers-Broadway, Karly-Rai; Chudasama, Dimple; Pados, George; Tsolakidis, Dimitris; Goumenou, Anastasia; Hall, Marcia; Karteris, Emmanouil

    2016-07-01

    Ovarian cancer is the second most common gynaecological malignancy and was diagnosed in over 7,000 women in 2011 in the UK. There are currently no reliable biomarkers available for use in a regular screening assay for ovarian cancer and due to characteristic late presentation (78% in stages III and IV) ovarian cancer has a low survival rate (35% after 10 years). The mTOR pathway is a central regulator of growth, proliferation, apoptosis and angiogenesis; providing balance between available resources such as amino acids and growth factors, and stresses such as hypoxia, to control cellular behaviour accordingly. Emerging data links mTOR with the aetiopathogenesis of ovarian cancer. We hypothesised that mTOR inhibitors could play a therapeutic role in ovarian cancer treatment. In this study we began by validating the expression of four main mTOR pathway components, mTOR, DEPTOR, rictor and raptor, at gene and protein level in in vitro models of endometrioid (MDAH‑2774) and clear cell (SKOV3) ovarian cancer using qPCR and ImageStream technology. Using a wound healing assay we show that inhibition of the mTOR pathway using rapamycin, rapalogues, resveratrol and NVP BEZ-235 induces a cytostatic and not cytotoxic response up to 18 h in these cell lines. We extended these findings up to 72 h with a proliferation assay and show that the effects of inhibition of the mTOR pathway are primarily mediated by the dephosphorylation of p70S6 kinase. We show that mTOR inhibition does not involve alteration of mTOR pathway components or induce caspase 9 cleavage. Preclinical studies including ovarian tissue of ovarian cancer patients, unaffected controls and patients with unrelated gynaecological conditions show that DEPTOR is reliably upregulated in ovarian cancer.

  17. An Ixodes ricinus Tick Salivary Lectin Pathway Inhibitor Protects Borrelia burgdorferi sensu lato from Human Complement.

    Science.gov (United States)

    Wagemakers, Alex; Coumou, Jeroen; Schuijt, Tim J; Oei, Anneke; Nijhof, Ard M; van 't Veer, Cornelis; van der Poll, Tom; Bins, Adriaan D; Hovius, Joppe W R

    2016-04-01

    We previously identified tick salivary lectin pathway inhibitor (TSLPI) in Ixodes scapularis, a vector for Borrelia burgdorferi sensu stricto (s.s.) in North America. TSLPI is a salivary protein facilitating B. burgdorferi s.s. transmission and acquisition by inhibiting the host lectin complement pathway through interference with mannose binding lectin (MBL) activity. Since Ixodes ricinus is the predominant vector for Lyme borreliosis in Europe and transmits several complement sensitive B. burgdorferi sensu lato (s.l.) strains, we aimed to identify, describe, and characterize the I. ricinus ortholog of TSLPI. We performed (q)PCRs on I. ricinus salivary gland cDNA to identify a TSLPI ortholog. Next, we generated recombinant (r)TSLPI in a Drosophila expression system and examined inhibition of the MBL complement pathway and complement-mediated killing of B. burgdorferi s.l. in vitro. We identified a TSLPI ortholog in I. ricinus salivary glands with 93% homology at the RNA and 89% at the protein level compared to I. scapularis TSLPI, which was upregulated during tick feeding. In silico analysis revealed that TSLPI appears to be part of a larger family of Ixodes salivary proteins among which I. persulcatus basic tail salivary proteins and I. scapularis TSLPI and Salp14. I. ricinus rTSLPI inhibited the MBL complement pathway and protected B. burgdorferi s.s. and Borrelia garinii from complement-mediated killing. We have identified a TSLPI ortholog, which protects B. burgdorferi s.l. from complement-mediated killing in I. ricinus, the major vector for tick-borne diseases in Europe.

  18. p53 modulates the AMPK inhibitor compound C induced apoptosis in human skin cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Shi-Wei [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Wu, Chun-Ying [Division of Gastroenterology and Hepatology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Wang, Yen-Ting [Department of Medical Research and Education, Cheng Hsin General Hospital, Taipei, Taiwan (China); Kao, Jun-Kai [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Department of Pediatrics, Children' s Hospital, Changhua Christian Hospital, Changhua, Taiwan (China); Lin, Chi-Chen; Chang, Chia-Che; Mu, Szu-Wei; Chen, Yu-Yu [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Chiu, Husan-Wen [Institute of Biotechnology, National Cheng-Kung University, Tainan, Taiwan (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan (China); Chang, Chuan-Hsun [Department of Surgical Oncology, Cheng Hsin General Hospital, Taipei, Taiwan (China); Department of Nutrition Therapy, Cheng Hsin General Hospital, Taipei, Taiwan (China); School of Nutrition and Health Sciences, Taipei Medical University, Taipei, Taiwan (China); Liang, Shu-Mei [Institute of Biotechnology, National Cheng-Kung University, Tainan, Taiwan (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan (China); Chen, Yi-Ju [Department of Dermatology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Huang, Jau-Ling [Department of Bioscience Technology, Chang Jung Christian University, Tainan, Taiwan (China); Shieh, Jeng-Jer, E-mail: shiehjj@vghtc.gov.tw [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan (China)

    2013-02-15

    Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53 status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cell's p53 status. - Highlights: ► Compound C caused more apoptosis in p53 wildtype than p53-mutant skin cancer cells. ► Compound C can upregulate p53 expression and induce p53 activation. ► Compound C induced p53 effects were dependent on ROS induced DNA damage pathway. ► p53-knockdown attenuated compound C-induced apoptosis but not autophagy. ► Compound C-induced apoptosis in skin cancer cells was dependent on p53 status.

  19. Neuraminidase Inhibitor Susceptibility Testing in Human Influenza Viruses: A Laboratory Surveillance Perspective

    Science.gov (United States)

    Okomo-Adhiambo, Margaret; Sleeman, Katrina; Ballenger, Kristina; Nguyen, Ha T.; Mishin, Vasiliy P.; Sheu, Tiffany G.; Smagala, James; Li, Yan; Klimov, Alexander I.; Gubareva, Larisa V.

    2010-01-01

    Neuraminidase inhibitors (NAIs) are vital in managing seasonal and pandemic influenza infections. NAI susceptibilities of virus isolates (n = 5540) collected during the 2008–2009 influenza season were assessed in the chemiluminescent neuraminidase inhibition (NI) assay. Box-and-whisker plot analyses of log-transformed IC50s were performed for each virus type/subtype and NAI to identify outliers which were characterized based on a statistical cutoff of IC50 >3 interquartile ranges (IQR) from the 75th percentile. Among 1533 seasonal H1N1 viruses tested, 1431 (93.3%) were outliers for oseltamivir; they all harbored the H275Y mutation in the neuraminidase (NA) and were reported as oseltamivir-resistant. Only 15 (0.7%) of pandemic 2009 H1N1 viruses tested (n = 2259) were resistant to oseltamivir. All influenza A(H3N2) (n = 834) and B (n = 914) viruses were sensitive to oseltamivir, except for one A(H3N2) and one B virus, with D151V and D197E (D198E in N2 numbering) mutations in the NA, respectively. All viruses tested were sensitive to zanamivir, except for six seasonal A(H1N1) and several A(H3N2) outliers (n = 22) which exhibited cell culture induced mutations at residue D151 of the NA. A subset of viruses (n = 1058) tested for peramivir were sensitive to the drug, with exception of H275Y variants that exhibited reduced susceptibility to this NAI. This study summarizes baseline susceptibility patterns of seasonal and pandemic influenza viruses, and seeks to contribute towards criteria for defining NAI resistance. PMID:21994620

  20. Specificity of Protein Covalent Modification by the Electrophilic Proteasome Inhibitor Carfilzomib in Human Cells*

    Science.gov (United States)

    Federspiel, Joel D.; Codreanu, Simona G.; Goyal, Sandeep; Albertolle, Matthew E.; Lowe, Eric; Teague, Juli; Wong, Hansen; Guengerich, F. Peter; Liebler, Daniel C.

    2016-01-01

    Carfilzomib (CFZ) is a second-generation proteasome inhibitor that is Food and Drug Administration and European Commission approved for the treatment of relapsed or refractory multiple myeloma. CFZ is an epoxomicin derivative with an epoxyketone electrophilic warhead that irreversibly adducts the catalytic threonine residue of the β5 subunit of the proteasome. Although CFZ produces a highly potent, sustained inactivation of the proteasome, the electrophilic nature of the drug could potentially produce off-target protein adduction. To address this possibility, we synthesized an alkynyl analog of CFZ and investigated protein adduction by this analog in HepG2 cells. Using click chemistry coupled with streptavidin based IP and shotgun tandem mass spectrometry (MS/MS), we identified two off-target proteins, cytochrome P450 27A1 (CYP27A1) and glutathione S-transferase omega 1 (GSTO1), as targets of the alkynyl CFZ probe. We confirmed the adduction of CYP27A1 and GSTO1 by streptavidin capture and immunoblotting methodology and then site-specifically mapped the adducts with targeted MS/MS methods. Although CFZ adduction of CYP27A1 and GSTO1 in vitro decreased the activities of these enzymes, the small fraction of these proteins modified by CFZ in intact cells should limit the impact of these off-target modifications. The data support the high selectivity of CFZ for covalent modification of its therapeutic targets, despite the presence of a reactive electrophile. The approach we describe offers a generalizable method to evaluate the safety profile of covalent protein-modifying therapeutics. PMID:27503896

  1. The neuropeptide galanin is a novel inhibitor of human hair growth.

    Science.gov (United States)

    Holub, B S; Kloepper, J E; Tóth, B I; Bíro, T; Kofler, B; Paus, R

    2012-07-01

    Galanin is a trophic factor of the central and peripheral nervous system that shows widespread distribution in human skin. However, the exact localization and the role of galanin in the hair follicle (HF) remain to be clarified. To characterize galanin expression in human scalp HFs and to examine the effects of galanin on normal human scalp HF growth in organ culture. Immunohistochemistry was performed on cryosections of human female scalp skin. Anagen HFs were microdissected and cultured up to 9 days and treated with 100 nmol L(-1) galanin. Staining for Ki-67, TUNEL and Masson-Fontana were used to analyse proliferation, apoptosis and hair cycle staging of the HFs. Functional effects of galanin were tested in serum-free HF organ culture. Galanin-like immunoreactivity was detected in the outer root sheath (ORS) and inner root sheath. Additionally, galanin mRNA was detected in ORS keratinocytes and all HF samples tested. Galanin receptor transcripts (GalR2, GalR3) were also detected in selected samples. Galanin reduced proliferation of hair matrix keratinocytes in situ compared with vehicle-treated controls, shortened the hair growth phase (anagen) in vitro and reduced hair shaft elongation. This was accompanied by the premature development of a catagen-like morphology of galanin-treated HFs. We present the first evidence that human HFs are both a source and a functionally relevant target of galanin. Due to its hair growth-inhibitory properties in vitro, galanin application deserves further exploration as a potential new treatment strategy for unwanted hair growth (hirsutism, hypertrichosis). © 2012 The Authors. BJD © 2012 British Association of Dermatologists.

  2. Characterization of SNARE proteins in human pituitary adenomas: targeted secretion inhibitors as a new strategy for the treatment of acromegaly?

    Science.gov (United States)

    Garcia, Edwin A; Trivellin, Giampaolo; Aflorei, Elena D; Powell, Michael; Grieve, Joana; Alusi, Ghassan; Pobereskin, Luis; Shariati, Babak; Cudlip, Simon; Roncaroli, Federico; Mendoza, Nigel; Grossman, Ashley B; Harper, Elaine A; Korbonits, Márta

    2013-12-01

    Targeted secretion inhibitors (TSIs), a new class of recombinant biotherapeutic proteins engineered from botulinum toxin, represent a novel approach for treating diseases with excess secretion. They inhibit hormone secretion from targeted cell types through cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor-activating protein receptor) proteins. qGHRH-LH(N)/D is a TSI targeting pituitary somatotroph through binding to the GHRH-receptor and cleavage of the vesicle-associated membrane protein (VAMP) family of SNARE proteins. Our objective was to study SNARE protein expression in pituitary adenomas and to inhibit GH secretion from somatotropinomas using qGHRH-LH(N)/D. We analyzed human pituitary adenoma analysis for SNARE expression and response to qGHRH-LH(N)/D treatment. The study was conducted in University Hospitals. We used pituitary adenoma samples from 25 acromegaly and 47 nonfunctioning pituitary adenoma patients. Vesicle-SNARE (VAMP1-3), target-SNARE (syntaxin1, SNAP-23, and SNAP-25), and GHRH-receptor detection with RT-qPCR, immunocytochemistry, and immunoblotting. Assessment of TSI catalytic activity on VAMPs and release of GH from adenoma cells. SNARE proteins were variably expressed in pituitary samples. In vitro evidence using recombinant GFP-VAMP2&3 or pituitary adenoma lysates suggested sufficient catalytic activity of qGHRH-LH(N)/D to degrade VAMPs, but was unable to inhibit GH secretion in somatotropinoma cell cultures. SNARE proteins are present in human pituitary somatotroph adenomas that can be targeted by TSIs to inhibit GH secretion. qGHRH-LH(N)/D was unable to inhibit GH secretion from human somatotroph adenoma cells. Further studies are required to understand how the SNARE proteins drive GH secretion in human somatotrophs to allow the development of novel TSIs with a potential therapeutic benefit.

  3. PATTERNS OF CYTOKINES, PLASMA ENDOTOXIN, PLASMINOGEN-ACTIVATOR INHIBITOR, AND ACUTE-PHASE PROTEINS DURING THE TREATMENT OF SEVERE SEPSIS IN HUMANS

    NARCIS (Netherlands)

    DOFFERHOFF, ASM; BOM, VJJ; DEVRIESHOSPERS, HG; VANINGEN, J; VANDERMEER, J; HAZENBERG, BPC; MULDER, POM; WEITS, J

    Objective: To study the patterns of plasma concentrations of endotoxin, tumor necrosis factor-alpha (TNF), interleukin-6 (IL-6), plasminogen activator inhibitor-1, C-reactive protein, and serum amyloid A during the treatment of human sepsis. Design: A prospective case series study. Setting: ICU of

  4. Liquid chromatography-tandem mass spectrometric assay for the T790M mutant EGFR inhibitor osimertinib (AZD9291) in human plasma

    NARCIS (Netherlands)

    Rood, Johannes J M; van Bussel, Mark T J; Schellens, Jan H M; Beijnen, Jos H; Sparidans, Rolf W

    2016-01-01

    A method for the quantitative analysis by ultra-performance liquid chromatography-tandem mass spectrometry of the highly selective irreversible covalent inhibitor of EGFR-TK, osimertinib in human plasma was developed and validated, using pazopanib as an internal standard. The validation was

  5. Preclinical evaluation of sunitinib, a multi-tyrosine kinase inhibitor, as a radiosensitizer for human prostate cancer

    International Nuclear Information System (INIS)

    Brooks, Colin; Sheu, Tommy; Bridges, Kathleen; Mason, Kathy; Kuban, Deborah; Mathew, Paul; Meyn, Raymond

    2012-01-01

    Many prostate cancers demonstrate an increased expression of growth factor receptors such as vascular endothelial growth factor receptor (VEGFR) and platelet derived growth factor receptor (PDGFR) which have been correlated with increased resistance to radiotherapy and poor prognosis in other tumors. Therefore, response to radiation could potentially be improved by using inhibitors of these abnormally activated pathways. We have investigated the radiosensitizing effects of sunitinib, a potent, multi-tyrosine kinase inhibitor of the VEGFR and PDGFR receptors, on human prostate cancer cells. The radiosensitizing effects of sunitinib were assessed on human prostate cancer cell lines DU145, PC3 and LNCaP by clonogenic assay. Sunitinib’s ability to inhibit the activities of its key targets was determined by immunoblot analysis. The radiosensitizing effects of sunitinib in vivo were tested on human tumor xenografts growing in nude mice where response was assessed by tumor growth delay. Clonogenic survival curve assays for both DU145 and PC3 cells showed that the surviving fraction at 2 Gy was reduced from 0.70 and 0.52 in controls to 0.44 and 0.38, respectively, by a 24 hr pretreatment with 100 nM sunitinib. LNCaP cells were not radiosensitized by sunitinib. Dose dependent decreases in VEGFR and PDGFR activation were also observed following sunitinib in both DU145 and PC3 cells. We assessed the ability of sunitinib to radiosensitize PC3 xenograft tumors growing in the hind limb of nude mice. Sunitinib given concurrently with radiation did not prolong tumor growth delay. However, when animals were treated with sunitinib commencing the day after fractionated radiation was complete, tumor growth delay was enhanced compared to radiation alone. We conclude, based on the in vivo results, that sunitinib and radiation do not interact directly to radiosensitize the PC3 tumor cells in vivo as they did in vitro. The fact that tumor growth delay was enhanced when sunitinib was

  6. Preclinical evaluation of sunitinib, a multi-tyrosine kinase inhibitor, as a radiosensitizer for human prostate cancer

    Directory of Open Access Journals (Sweden)

    Brooks Colin

    2012-09-01

    Full Text Available Abstract Background Many prostate cancers demonstrate an increased expression of growth factor receptors such as vascular endothelial growth factor receptor (VEGFR and platelet derived growth factor receptor (PDGFR which have been correlated with increased resistance to radiotherapy and poor prognosis in other tumors. Therefore, response to radiation could potentially be improved by using inhibitors of these abnormally activated pathways. We have investigated the radiosensitizing effects of sunitinib, a potent, multi-tyrosine kinase inhibitor of the VEGFR and PDGFR receptors, on human prostate cancer cells. Methods The radiosensitizing effects of sunitinib were assessed on human prostate cancer cell lines DU145, PC3 and LNCaP by clonogenic assay. Sunitinib’s ability to inhibit the activities of its key targets was determined by immunoblot analysis. The radiosensitizing effects of sunitinib in vivo were tested on human tumor xenografts growing in nude mice where response was assessed by tumor growth delay. Results Clonogenic survival curve assays for both DU145 and PC3 cells showed that the surviving fraction at 2 Gy was reduced from 0.70 and 0.52 in controls to 0.44 and 0.38, respectively, by a 24 hr pretreatment with 100 nM sunitinib. LNCaP cells were not radiosensitized by sunitinib. Dose dependent decreases in VEGFR and PDGFR activation were also observed following sunitinib in both DU145 and PC3 cells. We assessed the ability of sunitinib to radiosensitize PC3 xenograft tumors growing in the hind limb of nude mice. Sunitinib given concurrently with radiation did not prolong tumor growth delay. However, when animals were treated with sunitinib commencing the day after fractionated radiation was complete, tumor growth delay was enhanced compared to radiation alone. Conclusions We conclude, based on the in vivo results, that sunitinib and radiation do not interact directly to radiosensitize the PC3 tumor cells in vivo as they did in vitro

  7. Crystal structures of human HMG-CoA synthase isoforms provide insights into inherited ketogenesis disorders and inhibitor design.

    Science.gov (United States)

    Shafqat, Naeem; Turnbull, Andrew; Zschocke, Johannes; Oppermann, Udo; Yue, Wyatt W

    2010-05-14

    3-Hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (HMGCS) catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl CoA. It is ubiquitous across the phylogenetic tree and is broadly classified into three classes. The prokaryotic isoform is essential in Gram-positive bacteria for isoprenoid synthesis via the mevalonate pathway. The eukaryotic cytosolic isoform also participates in the mevalonate pathway but its end product is cholesterol. Mammals also contain a mitochondrial isoform; its deficiency results in an inherited disorder of ketone body formation. Here, we report high-resolution crystal structures of the human cytosolic (hHMGCS1) and mitochondrial (hHMGCS2) isoforms in binary product complexes. Our data represent the first structures solved for human HMGCS and the mitochondrial isoform, allowing for the first time structural comparison among the three isoforms. This serves as a starting point for the development of isoform-specific inhibitors that have potential cholesterol-reducing and antibiotic applications. In addition, missense mutations that cause mitochondrial HMGCS deficiency have been mapped onto the hHMGCS2 structure to rationalize the structural basis for the disease pathology. (c) 2010 Elsevier Ltd. All rights reserved.

  8. Peroxisome Proliferator-Activated Receptor γ Induces the Expression of Tissue Factor Pathway Inhibitor-1 (TFPI-1 in Human Macrophages

    Directory of Open Access Journals (Sweden)

    G. Chinetti-Gbaguidi

    2016-01-01

    Full Text Available Tissue factor (TF is the initiator of the blood coagulation cascade after interaction with the activated factor VII (FVIIa. Moreover, the TF/FVIIa complex also activates intracellular signalling pathways leading to the production of inflammatory cytokines. The TF/FVIIa complex is inhibited by the tissue factor pathway inhibitor-1 (TFPI-1. Peroxisome proliferator-activated receptor gamma (PPARγ is a transcription factor that, together with PPARα and PPARβ/δ, controls macrophage functions. However, whether PPARγ activation modulates the expression of TFP1-1 in human macrophages is not known. Here we report that PPARγ activation increases the expression of TFPI-1 in human macrophages in vitro as well as in vivo in circulating peripheral blood mononuclear cells. The induction of TFPI-1 expression by PPARγ ligands, an effect shared by the activation of PPARα and PPARβ/δ, occurs also in proinflammatory M1 and in anti-inflammatory M2 polarized macrophages. As a functional consequence, treatment with PPARγ ligands significantly reduces the inflammatory response induced by FVIIa, as measured by variations in the IL-8, MMP-2, and MCP-1 expression. These data identify a novel role for PPARγ in the control of TF the pathway.

  9. The heat shock protein 90 inhibitor 17-AAG suppresses growth and induces apoptosis in human cholangiocarcinoma cells.

    Science.gov (United States)

    Zhang, Jianjun; Zheng, Zhichao; Zhao, Yan; Zhang, Tao; Gu, Xiaohu; Yang, Wei

    2013-11-01

    The aim of this study was to investigate the effects of 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (HSP90) inhibitor, on the proliferation, cell cycle, and apoptosis of human cholangiocarcinoma (CCA) cells. Cell proliferation and cell cycle distribution were measured by the MTT assay and flow cytometry analysis, respectively. Induction of apoptosis was determined by flow cytometry and Hoechst staining. The expressions of cleaved poly ADP-ribose polymerase (PARP), Bcl-2, Survivin, and Cyclin B1 were detected by Western blot analysis. The activity of caspase-3 was also examined. We found that 17-AAG inhibited cell growth and induced G2/M cell cycle arrest and apoptosis in CCA cells together with the down-regulation of Bcl-2, Survivin and Cyclin B1, and the up-regulation of cleaved PARP. Moreover, increased caspase-3 activity was also observed in CCA cells treated with 17-AAG. In conclusion, our data suggest that the inhibition of HSP90 function by 17-AAG may provide a promising therapeutic strategy for the treatment of human CCA.

  10. Pectinesterase inhibitor from jelly fig (Ficus awkeotsang Makino) achene induces apoptosis of human leukemic U937 cells.

    Science.gov (United States)

    Chang, Jia-Huei; Wang, Yuh-Tai; Chang, Hung-Min

    2005-05-01

    The antitumor activity of pectinesterase inhibitor (PEI), a group of cationic polypeptides, from jelly fig (Ficus awkeotsang Makino) achene was first examined as a treatment for leukemia in this study. PEI displayed strong growth inhibition against human leukemic U937 cells via induction of apoptosis in a dose- and time-dependent manner. At a level of 50 microg/mL, PEI inhibited 90% of cell growth, and the concentration of PEI required to induce 50% of cell viability (LC50) was about 180 microg/mL. Meanwhile, cell cycle arrest at G2/M phase was observed when cells were incubated with 100 microg PEI/mL for 24 h. PEI displayed a dose-dependent influence on mitochondria transmembrane potential (MTP, delta psi m) of cells when detected by a flow cytometry. MTP of more than 50% cells was reduced when cells were incubated with PEI at levels higher than 50 microg PEI/mL for 24 h. In addition, PEI upregulated caspase-3 activity. Taken together, PEI potently inhibited the proliferation of human leukemic U937 cells via cell cycle arrest and apoptosis in association with MTP reduction and caspase-3 activation, respectively, and showed therapy potential for U937 cells.

  11. Complement C3 inhibitor Cp40 attenuates xenoreactions in pig hearts perfused with human blood.

    Science.gov (United States)

    Abicht, Jan-Michael; Kourtzelis, Ioannis; Reichart, Bruno; Koutsogiannaki, Sophia; Primikyri, Alexandra; Lambris, John D; Chavakis, Triantafyllos; Holdt, Lesca; Kind, Alexander; Guethoff, Sonja; Mayr, Tanja

    2017-01-01

    The complement system plays a crucial role in acute xenogeneic reactions after cardiac transplantation. We used an ex vivo perfusion model to investigate the effect of Cp40, a compstatin analog and potent inhibitor of complement at the level of C3. Fifteen wild-type pig hearts were explanted, cardiopleged, and reperfused ex vivo after 150 minutes of cold ischemia. Hearts were challenged in a biventricular working heart mode to evaluate cardiac perfusion and function. In the treatment group (n=5), the complement cascade was blocked at the level of C3 using Cp40, using diluted human blood. Untreated human and porcine blood was used for controls. Throughout the perfusion, C3 activation was inhibited when Cp40 was used (mean of all time points: 1.11 ± 0.34% vs 3.12 ± 0.48% control activation; Phearts xenoperfused ex vivo. We suggest that this compstatin analog, which blocks all main pathways of complement activation, could be a beneficial perioperative treatment in preclinical and in future clinical xenotransplantation. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Antioxidants cause rapid expansion of human adipose-derived mesenchymal stem cells via CDK and CDK inhibitor regulation

    Science.gov (United States)

    2013-01-01

    Background Antioxidants have been shown to enhance the proliferation of adipose-derived mesenchymal stem cells (ADMSCs) in vitro, although the detailed mechanism(s) and potential side effects are not fully understood. In this study, human ADMSCs cultured in ImF-A medium supplemented with antioxidants (N-acetyl-l-cysteine and ascorbic acid-2-phosphate) and fibroblast growth factor 2 (FGF-2) were compared with ADMSCs cultured with FGF-2 alone (ImF) or with FGF-2 under 5% pO2 conditions (ImF-H). Results During log-phase growth, exposure to ImF-A resulted in a higher percentage of ADMSCs in the S phase of the cell cycle and a smaller percentage in G0/G1 phase. This resulted in a significantly reduced cell-doubling time and increased number of cells in the antioxidant-supplemented cultures compared with those supplemented with FGF-2 alone, an approximately 225% higher cell density after 7 days. Western blotting showed that the levels of the CDK inhibitors p21 and p27 decreased after ImF-A treatment, whereas CDK2, CDK4, and CDC2 levels clearly increased. In addition, ImF-A resulted in significant reduction in the expression of CD29, CD90, and CD105, whereas relative telomere length, osteogenesis, adipogenesis, and chondrogenesis were enhanced. The results were similar for ADMSCs treated with antioxidants and those under hypoxic conditions. Conclusion Antioxidant treatment promotes entry of ADMSCs into the S phase by suppressing cyclin-dependent kinase inhibitors and results in rapid cell proliferation similar to that observed under hypoxic conditions. PMID:23915242

  13. Pharmacovirological Impact of an Integrase Inhibitor on Human Immunodeficiency Virus Type 1 cDNA Species In Vivo ▿

    Science.gov (United States)

    Goffinet, Christine; Allespach, Ina; Oberbremer, Lena; Golden, Pamela L.; Foster, Scott A.; Johns, Brian A.; Weatherhead, Jason G.; Novick, Steven J.; Chiswell, Karen E.; Garvey, Edward P.; Keppler, Oliver T.

    2009-01-01

    Clinical trials of the first approved integrase inhibitor (INI), raltegravir, have demonstrated a drop in the human immunodeficiency virus type 1 (HIV-1) RNA loads of infected patients that was unexpectedly more rapid than that with a potent reverse transcriptase inhibitor, and apparently dose independent. These clinical outcomes are not understood. In tissue culture, although their inhibition of integration is well documented, the effects of INIs on levels of unintegrated HIV-1 cDNAs have been variable. Furthermore, there has been no report to date on an INI's effect on these episomal species in vivo. Here, we show that prophylactic treatment of transgenic rats with the strand transfer INI GSK501015 reduced levels of viral integrants in the spleen by up to 99.7%. Episomal two-long-terminal-repeat (LTR) circles accumulated up to sevenfold in this secondary lymphoid organ, and this inversely correlated with the impact on the proviral burden. Contrasting raltegravir's dose-ranging study with HIV patients, titration of GSK501015 in HIV-infected animals demonstrated dependence of the INI's antiviral effect on its serum concentration. Furthermore, the in vivo 50% effective concentration calculated from these data best matched GSK501015's in vitro potency when serum protein binding was accounted for. Collectively, this study demonstrates a titratable, antipodal impact of an INI on integrated and episomal HIV-1 cDNAs in vivo. Based on these findings and known biological characteristics of viral episomes, we discuss how integrase inhibition may result in additional indirect antiviral effects that contribute to more rapid HIV-1 decay in HIV/AIDS patients. PMID:19458008

  14. MG132, a proteasome inhibitor, induces human pulmonary fibroblast cell death via increasing ROS levels and GSH depletion.

    Science.gov (United States)

    Park, Woo Hyun; Kim, Suhn Hee

    2012-04-01

    MG132 as a proteasome inhibitor can induce apoptotic cell death in lung cancer cells. However, little is known about the toxicological cellular effects of MG132 on normal primary lung cells. Here, we investigated the effects of N-acetyl cysteine (NAC) and vitamin C (well known antioxidants) or L-buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) on MG132-treated human pulmonary fibroblast (HPF) cells in relation to cell death, reactive oxygen species (ROS) and glutathione (GSH). MG132 induced growth inhibition and death in HPF cells, accompanied by the loss of mitochondrial membrane potential (MMP; ∆ψm). MG132 increased ROS levels and GSH-depleted cell numbers in HPF cells. Both antioxidants, NAC and vitamin C, prevented growth inhibition, death and MMP (∆ψm) loss in MG132-treated HPF cells and also attenuated ROS levels in these cells. BSO showed a strong increase in ROS levels in MG132-treated HPF cells and slightly enhanced the growth inhibition, cell death, MMP (∆ψm) loss and GSH depletion. In addition, NAC decreased anonymous ubiquitinated protein levels in MG132-treated HPF cells. Furthermore, superoxide dismutase (SOD) 2, catalase (CTX) and GSH peroxidase (GPX) siRNAs enhanced HPF cell death by MG132, which was not correlated with ROS and GSH level changes. In conclusion, MG132 induced the growth inhibition and death of HPF cells, which were accompanied by increasing ROS levels and GSH depletion. Both NAC and vitamin C attenuated HPF cell death by MG132, whereas BSO slightly enhanced the death.

  15. Efficacy of the HSP90 inhibitor 17-AAG in human glioma cell lines and tumorigenic glioma stem cells.

    Science.gov (United States)

    Sauvageot, Claire Marie-Elisabeth; Weatherbee, Jessica Leigh; Kesari, Santosh; Winters, Susan Elizabeth; Barnes, Jessica; Dellagatta, Jamie; Ramakrishna, Naren Raj; Stiles, Charles Dean; Kung, Andrew Li-Jen; Kieran, Mark W; Wen, Patrick Yung Chih

    2009-04-01

    Glioblastoma multiforme (GBM) arises from genetic and signaling abnormalities in components of signal transduction pathways involved in proliferation, survival, and the cell cycle axis. Studies to date with single-agent targeted molecular therapy have revealed only modest effects in attenuating the growth of these tumors, suggesting that targeting multiple aberrant pathways may be more beneficial. Heat-shock protein 90 (HSP90) is a molecular chaperone that is involved in the conformational maturation of a defined group of client proteins, many of which are deregulated in GBM. 17-allylamino-17-demethoxygeldanamycin (17-AAG) is a well-characterized HSP90 inhibitor that should be able to target many of the aberrant signal transduction pathways in GBM. We assessed the ability of 17-AAG to inhibit the growth of glioma cell lines and glioma stem cells both in vitro and in vivo and assessed its ability to synergize with radiation and/or temozolomide, the standard therapies for GBM. Our results reveal that 17-AAG is able to inhibit the growth of both human glioma cell lines and glioma stem cells in vitro and is able to target the appropriate proteins within these cells. In addition, 17-AAG can inhibit the growth of intracranial tumors and can synergize with radiation both in tissue culture and in intracranial tumors. This compound was not found to synergize with temozolomide in any of our models of gliomas. Our results suggest that HSP90 inhibitors like 17-AAG may have therapeutic potential in GBM, either as a single agent or in combination with radiation.

  16. Exploration of natural product ingredients as inhibitors of human HMG-CoA reductase through structure-based virtual screening

    Directory of Open Access Journals (Sweden)

    Lin SH

    2015-06-01

    Full Text Available Shih-Hung Lin,1 Kao-Jean Huang,1,2 Ching-Feng Weng,1 David Shiuan1 1Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan, Republic of China; 2Development Center of Biotechnology, Taipei, Taiwan, Republic of China Abstract: Cholesterol plays an important role in living cells. However, a very high level of cholesterol may lead to atherosclerosis. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A reductase is the key enzyme in the cholesterol biosynthesis pathway, and the statin-like drugs are inhibitors of human HMG-CoA reductase (hHMGR. The present study aimed to virtually screen for potential hHMGR inhibitors from natural product to discover hypolipidemic drug candidates with fewer side effects and lesser toxicities. We used the 3D structure 1HWK from the PDB (Protein Data Bank database of hHMGR as the target to screen for the strongly bound compounds from the traditional Chinese medicine database. Many interesting molecules including polyphenolic compounds, polisubstituted heterocyclics, and linear lipophilic alcohols were identified and their ADMET (absorption, disrtibution, metabolism, excretion, toxicity properties were predicted. Finally, four compounds were obtained for the in vitro validation experiments. The results indicated that curcumin and salvianolic acid C can effectively inhibit hHMGR, with IC50 (half maximal inhibitory concentration values of 4.3 µM and 8 µM, respectively. The present study also demonstrated the feasibility of discovering new drug candidates through structure-based virtual screening. Keywords: HMG-CoA reductase, virtual screening, curcumin, salvianolic acid C

  17. Lysosomal degradation of receptor-bound urokinase-type plasminogen activator is enhanced by its inhibitors in human trophoblastic choriocarcinoma cells

    DEFF Research Database (Denmark)

    Jensen, Poul Henning; Christensen, Erik Ilsø; Ebbesen, P.

    1990-01-01

    We have studied the effect of plasminogen activator inhibitors PAI-1 and PAI-2 on the binding of urokinase-type plasminogen activator (u-PA) to its receptor in the human choriocarcinoma cell line JAR. With 125I-labeled ligands in whole-cell binding assays, both uncomplexed u-PA and u-PA-inhibitor......We have studied the effect of plasminogen activator inhibitors PAI-1 and PAI-2 on the binding of urokinase-type plasminogen activator (u-PA) to its receptor in the human choriocarcinoma cell line JAR. With 125I-labeled ligands in whole-cell binding assays, both uncomplexed u-PA and u...... in an apparently intact form in the medium or was still cell associated. The degradation could be inhibited by inhibitors of vesicle transport and lysosomal hydrolases. By electron microscopic autoradiography, both 125I-u-PA and 125I-u-PA-inhibitor complexes were located over the cell membrane at 4 degrees C...

  18. Novel human ZAKI-4 isoforms: hormonal and tissue-specific regulation and function as calcineurin inhibitors.

    Science.gov (United States)

    Cao, Xia; Kambe, Fukushi; Miyazaki, Takashi; Sarkar, Devanand; Ohmori, Sachiko; Seo, Hisao

    2002-01-01

    We identified a thyroid hormone [3,5,3'-tri-iodothyronine (T(3))]-responsive gene, ZAKI-4, in cultured human skin fibroblasts. It belongs to a family of genes that encode proteins containing a conserved motif. The motif binds to calcineurin and inhibits its phosphatase activity. In the present study, we have demonstrated three different ZAKI-4 transcripts, alpha, beta1 and beta2, in human brain by 5'- and 3'-RACE (rapid amplification of cDNA ends). The alpha transcript was identical with the one that we originally cloned from human fibroblasts and the other two are novel. The three transcripts are generated by alternative initiation and splicing from a single gene on the short arm of chromosome 6. It is predicted that beta1 and beta2 encode an identical protein product, beta, which differs from alpha in its N-terminus. Since alpha and beta contain an identical C-terminal region harbouring the conserved motif, both isoforms are suggested to inhibit calcineurin activity. Indeed, each isoform associates with calcineurin A and inhibits its activity in a similar manner, suggesting that the difference in N-terminus of each isoform does not affect the inhibitory function on calcineurin. An examination of the expression profile of the three transcripts in 12 human tissues revealed that the alpha transcript is expressed exclusively in the brain, whereas beta transcripts are expressed ubiquitously, most abundantly in brain, heart, skeletal muscle and kidney. It was also demonstrated that human skin fibroblasts express both alpha and beta transcripts, raising the question of which transcript is up-regulated by T(3). It was revealed that T(3) markedly induced the expression of alpha isoform but not of beta. This T(3)-mediated increase in the alpha isoform was associated with a significant decrease in endogenous calcineurin activity. These results suggest that the expression of ZAKI-4 isoforms is subjected to distinct hormonal as well as tissue-specific regulation, constituting

  19. MicroRNA-21 inhibitor sensitizes human glioblastoma cells U251 (PTEN-mutant) and LN229 (PTEN-wild type) to taxol

    International Nuclear Information System (INIS)

    Ren, Yu; Kang, Chun-Sheng; Zhou, Xuan; Mei, Mei; Yuan, Xu-Bo; Han, Lei; Wang, Guang-Xiu; Jia, Zhi-Fan; Xu, Peng; Pu, Pei-Yu

    2010-01-01

    Substantial data indicate that the oncogene microRNA 21 (miR-21) is significantly elevated in glioblastoma multiforme (GBM) and regulates multiple genes associated with cancer cell proliferation, apoptosis, and invasiveness. Thus, miR-21 can theoretically become a target to enhance the chemotherapeutic effect in cancer therapy. So far, the effect of downregulating miR-21 to enhance the chemotherapeutic effect to taxol has not been studied in human GBM. Human glioblastoma U251 (PTEN-mutant) and LN229 (PTEN wild-type) cells were treated with taxol and the miR-21 inhibitor (in a poly (amidoamine) (PAMAM) dendrimer), alone or in combination. The 50% inhibitory concentration and cell viability were determined by the MTT assay. The mechanism between the miR-21 inhibitor and the anticancer drug taxol was analyzed using the Zheng-Jun Jin method. Annexin V/PI staining was performed, and apoptosis and the cell cycle were evaluated by flow cytometry analysis. Expression of miR-21 was investigated by RT-PCR, and western blotting was performed to evaluate malignancy related protein alteration. IC(50) values were dramatically decreased in cells treated with miR-21 inhibitor combine with taxol, to a greater extent than those treated with taxol alone. Furthermore, the miR-21 inhibitor significantly enhanced apoptosis in both U251 cells and LN229 cells, and cell invasiveness was obviously weakened. Interestingly, the above data suggested that in both the PTEN mutant and the wild-type GBM cells, miR-21 blockage increased the chemosensitivity to taxol. It is worth noting that the miR-21 inhibitor additively interacted with taxol on U251cells and synergistically on LN229 cells. Thus, the miR-21 inhibitor might interrupt the activity of EGFR pathways, independently of PTEN status. Meanwhile, the expression of STAT3 and p-STAT3 decreased to relatively low levels after miR-21 inhibitor and taxol treatment. The data strongly suggested that a regulatory loop between miR-21 and STAT3 might

  20. Prediction of Human Pharmacokinetics of Ulixertinib, a Novel ERK1/2 Inhibitor from Mice, Rats, and Dogs Pharmacokinetics.

    Science.gov (United States)

    Suresh, Ponnayyan Sulochana; Jairam, Ravi Kumar; Chandrasekhar, Devaraj V; Vinod, Anera Balakrishna; Hiremath, Rakesh A; Raj, Anusha; Zainuddin, Mohd; Bhamidipati, Ravi Kanth; Mullangi, Ramesh

    2018-02-22

    Ulixertinib (BVD-523) is a novel and selective reversible inhibitor of ERK1/ERK2. The primary objectives of the study were to evaluate the pharmacokinetics of ulixertinib in mice, rats, and dogs followed by prediction of human pharmacokinetic profile by allometric equations with/without correction factors. Oral and intravenous pharmacokinetic profiles of ulixertinib were generated in mice, rats, and dogs. The human intravenous pharmacokinetics profiles [volume of distribution (V ss ) and clearance (CL)] were predicted employing simple allometry and using correction factors [maximum life span potential (MLP) and brain weight (BW)]. Pharmacokinetic data obtained from dogs were used to simulate human oral profile [area under the curve (AUC) and maximum plasma concentrations (C max )]. Post-intravenous administration the CL was moderate in dogs (15.5 mL/min/kg) and low in mice (6.24 mL/min/kg) and rats (1.67 mL/min/kg). V ss was 0.56, 0.36, and 1.61 L/kg in mice, rats, and dogs, respectively. The half-life (t ½ ) of ulixertinib ranged between 1.0 and 2.5 h across the animal species. Following oral administration ulixertinib attained maximum concentration in plasma (T max ) within 0.50-0.75 h in mice and rats, indicating that absorption was rapid; however, in dogs, T max attained at 2 h. Absolute oral bioavailability in mice and rats was > 92%; however, in dogs, it was 34%. By different allometric approaches, simple method and brain weight correction factor shown clear improvement in the prediction efficiency of allometric scaling for V ss (1.34-1.70 L/kg) and CL (4.18-6.09 mL/min/kg), respectively, comparing with the MLP method and simple method for CL. Similarly, simulation of oral human profile was attained from scaled values and dog data to predict reported human profile (AUC and C max ). The derived pharmacokinetic parameters (AUC and C max at 600 mg dose) and simulated plasma concentration-time profiles of ulixertinib in humans were predicted with good

  1. Repeated administration of an acetylcholinesterase inhibitor attenuates nicotine taking in rats and smoking behavior in human smokers

    Science.gov (United States)

    Ashare, R L; Kimmey, B A; Rupprecht, L E; Bowers, M E; Hayes, M R; Schmidt, H D

    2016-01-01

    Tobacco smoking remains the leading cause of preventable death worldwide and current smoking cessation medications have limited efficacy. Thus, there is a clear need for translational research focused on identifying novel pharmacotherapies for nicotine addiction. Our previous studies demonstrated that acute administration of an acetylcholinesterase inhibitor (AChEI) attenuates nicotine taking and seeking in rats and suggest that AChEIs could be repurposed for smoking cessation. Here, we expand upon these findings with experiments designed to determine the effects of repeated AChEI administration on voluntary nicotine taking in rats as well as smoking behavior in human smokers. Rats were trained to self-administer intravenous infusions of nicotine (0.03 mg kg−1 per 0.59 ml) on a fixed-ratio-5 schedule of reinforcement. Once rats maintained stable nicotine taking, galantamine or donepezil was administered before 10 consecutive daily nicotine self-administration sessions. Repeated administration of 5.0 mg kg−1 galantamine and 3.0 mg kg−1 donepezil attenuated nicotine self-administration in rats. These effects were reinforcer-specific and not due to adverse malaise-like effects of drug treatment as repeated galantamine and donepezil administration had no effects on sucrose self-administration, ad libitum food intake and pica. The effects of repeated galantamine (versus placebo) on cigarette smoking were also tested in human treatment-seeking smokers. Two weeks of daily galantamine treatment (8.0 mg (week 1) and 16.0 mg (week 2)) significantly reduced smoking rate as well as smoking satisfaction and reward compared with placebo. This translational study indicates that repeated AChEI administration reduces nicotine reinforcement in rats and smoking behavior in humans at doses not associated with tolerance and/or adverse effects. PMID:26784967

  2. Novel PI3K/Akt inhibitors screened by the cytoprotective function of human immunodeficiency virus type 1 Tat.

    Directory of Open Access Journals (Sweden)

    Yuri Kim

    Full Text Available The PI3K/Akt pathway regulates various stress-related cellular responses such as cell survival, cell proliferation, metabolism and protein synthesis. Many cancer cell types display the activation of this pathway, and compounds inhibiting this cell survival pathway have been extensively evaluated as anti-cancer agents. In addition to cancers, several human viruses, such as HTLV, HPV, HCV and HIV-1, also modulate this pathway, presumably in order to extend the life span of the infected target cells for productive viral replication. The expression of HIV-1 Tat protein exhibited the cytoprotective effect in macrophages and a human microglial cell line by inhibiting the negative regulator of this pathway, PTEN. This cytoprotective effect of HIV-1 appears to contribute to the long-term survival and persistent HIV-1 production in human macrophage reservoirs. In this study we exploited the PI3K/Akt dependent cytoprotective effect of Tat-expressing CHME5 cells. We screened a collection of compounds known to modulate inflammation, and identified three novel compounds: Lancemaside A, Compound K and Arctigenin that abolished the cytoprotective phenotype of Tat-expressing CHME5 cells. All three compounds antagonized the kinase activity of Akt. Further detailed signaling studies revealed that each of these three compounds targeted different steps of the PI3K/Akt pathway. Arctigenin regulates the upstream PI3K enzyme from converting PIP2 to PIP3. Lancemaside A1 inhibited the movement of Akt to the plasma membrane, a critical step for Akt activation. Compound K inhibited Akt phosphorylation. This study supports that Tat-expressing CHME5 cells are an effective model system for screening novel PI3K/Akt inhibitors.

  3. Novel PI3K/Akt Inhibitors Screened by the Cytoprotective Function of Human Immunodeficiency Virus Type 1 Tat

    Science.gov (United States)

    Kim, Dong-Hyun; Kim, Baek

    2011-01-01

    The PI3K/Akt pathway regulates various stress-related cellular responses such as cell survival, cell proliferation, metabolism and protein synthesis. Many cancer cell types display the activation of this pathway, and compounds inhibiting this cell survival pathway have been extensively evaluated as anti-cancer agents. In addition to cancers, several human viruses, such as HTLV, HPV, HCV and HIV-1, also modulate this pathway, presumably in order to extend the life span of the infected target cells for productive viral replication. The expression of HIV-1 Tat protein exhibited the cytoprotective effect in macrophages and a human microglial cell line by inhibiting the negative regulator of this pathway, PTEN. This cytoprotective effect of HIV-1 appears to contribute to the long-term survival and persistent HIV-1 production in human macrophage reservoirs. In this study we exploited the PI3K/Akt dependent cytoprotective effect of Tat-expressing CHME5 cells. We screened a collection of compounds known to modulate inflammation, and identified three novel compounds: Lancemaside A, Compound K and Arctigenin that abolished the cytoprotective phenotype of Tat-expressing CHME5 cells. All three compounds antagonized the kinase activity of Akt. Further detailed signaling studies revealed that each of these three compounds targeted different steps of the PI3K/Akt pathway. Arctigenin regulates the upstream PI3K enzyme from converting PIP2 to PIP3. Lancemaside A1 inhibited the movement of Akt to the plasma membrane, a critical step for Akt activation. Compound K inhibited Akt phosphorylation. This study supports that Tat-expressing CHME5 cells are an effective model system for screening novel PI3K/Akt inhibitors. PMID:21765914

  4. The Anti-Inflammatory Effects of Lipoxygenase and Cyclo-Oxygenase Inhibitors in Inflammation-Induced Human Fetal Glia Cells and the Aβ Degradation Capacity of Human Fetal Astrocytes in an Ex vivo Assay

    Directory of Open Access Journals (Sweden)

    Rea Pihlaja

    2017-05-01

    Full Text Available Chronic inflammation is a common phenomenon present in the background of multiple neurodegenerative diseases, including Alzheimer's disease (AD. The arachidonic acid pathway overproduces proinflammatory eicosanoids during these states and glial cells in the brain gradually lose their vital functions of protecting and supporting neurons. In this study, the role of different key enzymes of the eicosanoid pathway mediating inflammatory responses was examined in vitro and ex vivo using human fetal glial cells. Astrocytes and microglia were exposed to proinflammatory agents i.e., cytokines interleukin 1-β (IL-1β and tumor necrosis factor (TNF-α. ELISA assays were used to examine the effects of inhibitors of key enzymes in the eicosanoid pathway. Inhibitors for 5-lipoxygenase (5-LOX and cyclo-oxygenase 2 (COX-2 in both cell types and 5-, 12-, and 15-LOX-inhibitor in astrocytes reduced significantly IL-6 secretion, compared to exposed glial cells without inhibitors. The cytokine antibody array showed that especially treatments with 5, -12, and -15 LOX inhibitor in astrocytes, 5-LOX inhibitor in microglia and COX-2 inhibitor in both glial cell types significantly reduced the expression of multiple proinflammatory cytokines. Furthermore, human fetal astrocytes and microglia were cultured on top of AD-affected and control human brain sections for 30 h. According to the immunochemical evaluation of the level of total Aβ, astrocytes were very efficient at degrading Aβ from AD-affected brain sections ex vivo; simultaneously added enzyme inhibitors did not increase their Aβ degradation capabilities. Microglia were not able to reduce the level of total Aβ during the 30 h incubation time.

  5. Mechanisms underlying the growth inhibitory effects of the cyclo-oxygenase-2 inhibitor celecoxib in human breast cancer cells

    International Nuclear Information System (INIS)

    Basu, Gargi D; Pathangey, Latha B; Tinder, Teresa L; Gendler, Sandra J; Mukherjee, Pinku

    2005-01-01

    Inhibitors of cyclo-oxygenase (COX)-2 are being extensively studied as anticancer agents. In the present study we evaluated the mechanisms by which a highly selective COX-2 inhibitor, celecoxib, affects tumor growth of two differentially invasive human breast cancer cell lines. MDA-MB-231 (highly invasive) and MDA-MB-468 (moderately invasive) cell lines were treated with varying concentrations of celecoxib in vitro, and the effects of this agent on cell growth and angiogenesis were monitored by evaluating cell proliferation, apoptosis, cell cycle arrest, and vasculogenic mimicry. The in vitro results of MDA-MB-231 cell line were further confirmed in vivo in a mouse xenograft model. The highly invasive MDA-MB-231 cells express higher levels of COX-2 than do the less invasive MDA-MB-468 cells. Celecoxib treatment inhibited COX-2 activity, indicated by prostaglandin E 2 secretion, and caused significant growth arrest in both breast cancer cell lines. In the highly invasive MDA-MB-231 cells, the mechanism of celecoxib-induced growth arrest was by induction of apoptosis, associated with reduced activation of protein kinase B/Akt, and subsequent activation of caspases 3 and 7. In the less invasive MDA-MB-468 cells, growth arrest was a consequence of cell cycle arrest at the G 0 /G 1 checkpoint. Celecoxib-induced growth inhibition was reversed by addition of exogenous prostaglandin E 2 in MDA-MB-468 cells but not in MDA-MB-231 cells. Furthermore, MDA-MB-468 cells formed significantly fewer extracellular matrix associated microvascular channels in vitro than did the high COX-2 expressing MDA-MB-231 cells. Celecoxib treatment not only inhibited cell growth and vascular channel formation but also reduced vascular endothelial growth factor levels. The in vitro findings corroborated in vivo data from a mouse xenograft model in which daily administration of celecoxib significantly reduced tumor growth of MDA-MB-231 cells, which was associated with reduced vascularization and

  6. Mechanisms underlying the growth inhibitory effects of the cyclo-oxygenase-2 inhibitor celecoxib in human breast cancer cells.

    Science.gov (United States)

    Basu, Gargi D; Pathangey, Latha B; Tinder, Teresa L; Gendler, Sandra J; Mukherjee, Pinku

    2005-01-01

    Inhibitors of cyclo-oxygenase (COX)-2 are being extensively studied as anticancer agents. In the present study we evaluated the mechanisms by which a highly selective COX-2 inhibitor, celecoxib, affects tumor growth of two differentially invasive human breast cancer cell lines. MDA-MB-231 (highly invasive) and MDA-MB-468 (moderately invasive) cell lines were treated with varying concentrations of celecoxib in vitro, and the effects of this agent on cell growth and angiogenesis were monitored by evaluating cell proliferation, apoptosis, cell cycle arrest, and vasculogenic mimicry. The in vitro results of MDA-MB-231 cell line were further confirmed in vivo in a mouse xenograft model. The highly invasive MDA-MB-231 cells express higher levels of COX-2 than do the less invasive MDA-MB-468 cells. Celecoxib treatment inhibited COX-2 activity, indicated by prostaglandin E2 secretion, and caused significant growth arrest in both breast cancer cell lines. In the highly invasive MDA-MB-231 cells, the mechanism of celecoxib-induced growth arrest was by induction of apoptosis, associated with reduced activation of protein kinase B/Akt, and subsequent activation of caspases 3 and 7. In the less invasive MDA-MB-468 cells, growth arrest was a consequence of cell cycle arrest at the G0/G1 checkpoint. Celecoxib-induced growth inhibition was reversed by addition of exogenous prostaglandin E2 in MDA-MB-468 cells but not in MDA-MB-231 cells. Furthermore, MDA-MB-468 cells formed significantly fewer extracellular matrix associated microvascular channels in vitro than did the high COX-2 expressing MDA-MB-231 cells. Celecoxib treatment not only inhibited cell growth and vascular channel formation but also reduced vascular endothelial growth factor levels. The in vitro findings corroborated in vivo data from a mouse xenograft model in which daily administration of celecoxib significantly reduced tumor growth of MDA-MB-231 cells, which was associated with reduced vascularization and

  7. Calcineurin inhibitors acutely improve insulin sensitivity without affecting insulin secretion in healthy human volunteers

    DEFF Research Database (Denmark)

    Øzbay, Aygen; Møller, Niels; Juhl, Claus

    2012-01-01

    WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: New onset diabetes after transplantation is related to treatment with immunosuppressive medications. Clinical studies have shown that risk of new onset diabetes is greater with tacrolimus compared with ciclosporin. The diabetogenicity of ciclosporin...... of NODAT remains unclear. We sought to compare the impact of CsA and Tac on glucose metabolism in human subjects. METHODS: Ten healthy men underwent 5 h infusions of CsA, Tac and saline in a randomized, double-blind, crossover study. During infusion glucose metabolism was investigated using following.......047), whereas first phase and pulsatile insulin secretion were unaffected. Coinciding with the CNI induced improved insulin sensitivity, glucose oxidation rates increased, while insulin clearance rates decreased, only non-significantly. Tac singularly lowered hsCRP concentrations, otherwise no changes were...

  8. Improving virtual screening predictive accuracy of Human kallikrein 5 inhibitors using machine learning models.

    Science.gov (United States)

    Fang, Xingang; Bagui, Sikha; Bagui, Subhash

    2017-08-01

    The readily available high throughput screening (HTS) data from the PubChem database provides an opportunity for mining of small molecules in a variety of biological systems using machine learning techniques. From the thousands of available molecular descriptors developed to encode useful chemical information representing the characteristics of molecules, descriptor selection is an essential step in building an optimal quantitative structural-activity relationship (QSAR) model. For the development of a systematic descriptor selection strategy, we need the understanding of the relationship between: (i) the descriptor selection; (ii) the choice of the machine learning model; and (iii) the characteristics of the target bio-molecule. In this work, we employed the Signature descriptor to generate a dataset on the Human kallikrein 5 (hK 5) inhibition confirmatory assay data and compared multiple classification models including logistic regression, support vector machine, random forest and k-nearest neighbor. Under optimal conditions, the logistic regression model provided extremely high overall accuracy (98%) and precision (90%), with good sensitivity (65%) in the cross validation test. In testing the primary HTS screening data with more than 200K molecular structures, the logistic regression model exhibited the capability of eliminating more than 99.9% of the inactive structures. As part of our exploration of the descriptor-model-target relationship, the excellent predictive performance of the combination of the Signature descriptor and the logistic regression model on the assay data of the Human kallikrein 5 (hK 5) target suggested a feasible descriptor/model selection strategy on similar targets. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. TAS-116, a novel Hsp90 inhibitor, selectively enhances radio-sensitivity of human cancer cells to X-rays and carbon ion radiation

    Science.gov (United States)

    Lee, Younghyun; Sunada, Shigeaki; Hirakawa, Hirokazu; Fujimori, Akira; Nickoloff, Jac A.; Okayasu, Ryuichi

    2016-01-01

    Hsp90 inhibitors have been investigated as cancer therapeutics in mono-therapy and to augment radiotherapy, however serious adverse effects of early generation Hsp90 inhibitors limited their development. TAS-116 is a novel Hsp90 inhibitor with lower adverse effects than other Hsp90 inhibitors, and here we investigated the radio-sensitizing effects of TAS-116 in low LET X-ray, and high LET carbon ion irradiated human cancer cells and mouse tumor xenografts. TAS-116 decreased cell survival of both X-ray and carbon ion-irradiated human cancer cell lines (HeLa and H1299 cells), and similar to other Hsp90 inhibitors, it did not affect radiosensitivity of non-cancerous human fibroblasts. TAS-116 increased the number of radiation-induced γ-H2AX foci, and delayed the repair of DNA double-strand breaks (DSBs). TAS-116 reduced the expression of proteins that mediate repair of DSBs by homologous recombination (RAD51) and non-homologous end joining (Ku, DNA-PKcs), and suppressed formation of RAD51 foci and phosphorylation/activation of DNA-PKcs. TAS-116 also decreased expression of the cdc25 cell cycle progression marker, markedly increasing G2/M arrest. Combined treatment of mouse tumor xenografts with carbon ions and TAS-116 showed promising delay in tumor growth compared to either individual treatment. These results demonstrate that TAS-116 radio-sensitizes human cancer cells to both X rays and carbon ions by inhibiting the two major DSB repair pathways, and these effects were accompanied by marked cell cycle arrest. The promising results of combination TAS-116 + carbon ion radiation therapy of tumor xenografts justify further exploration of TAS-116 as an adjunct to radiotherapy using low or high LET radiation. PMID:28062703

  10. 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,

  11. Recovery of human lymphocytes damaged with. gamma. -radiation or enzymatically produced oxygen radicals: different effects of poly(ADP-ribosyl)polymerase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Marini, M.; Zunica, G. (Ist. di Istologia ed Embriologia Generale, Bologna (Italy)); Tamba, M. (Consiglio Nazionale delle Ricerche, Bologna (Italy). Lab. di Fotochimica e Radiazioni d' Alta Energia); Cossarizza, A.; Monti, D.; Franceschi, C. (Ist. di Patologia Generale, Modena (Italy))

    1990-08-01

    Quiescent human lymphocytes were damaged in two different ways, both producing toxic oxygen radicals: xanthine oxidase plus hypoxanthine (XOD/HYP), or {gamma}-rays. Under conditions where DNA synthesis was reduced to 10-20% of control, inhibitors of poly(ADP-ribosyl)polymerase (ADPRP, an enzyme that becomes activated in the presence of DNA strand breaks) allowed lymphocytes to recover completely when the damage was caused by XOD/HYP, but they did not affect DNA synthesis of irradiated cells. However, a protective effect of ADPRP inhibitors was observed with irradiated lymphocytes receiving doses {ge}50Gy. Unscheduled DNA synthesis was significantly increased when lymphocytes were damaged by high radiation doses in the presence of ADPRP inhibitors. (author).

  12. Engineered aggregation inhibitor fusion for production of highly amyloidogenic human islet amyloid polypeptide.

    Science.gov (United States)

    Mirecka, Ewa Agnieszka; Gremer, Lothar; Schiefer, Stephanie; Oesterhelt, Filipp; Stoldt, Matthias; Willbold, Dieter; Hoyer, Wolfgang

    2014-12-10

    Human islet amyloid polypeptide (IAPP) is the major component of pancreatic amyloid deposits in type 2 diabetes. The structural conversion of IAPP from a monomeric state into amyloid assemblies is the subject of intense research. Recombinant production of IAPP is, however, difficult due to its extreme aggregation propensity. Here we describe a novel strategy for expression of IAPP in Escherichia coli, based on an engineered protein tag, which sequesters IAPP monomers and prevents IAPP aggregation. The IAPP-binding protein HI18 was selected by phage display from a β-wrapin library. Fusion of HI18 to IAPP enabled the soluble expression of the construct. IAPP was cleaved from the fusion construct and purified to homogeneity with a yield of 3mg of isotopically labeled peptide per liter of culture. In the monomeric state, IAPP was largely disordered as evidenced by far-UV CD and liquid-state NMR spectroscopy but competent to form amyloid fibrils according to atomic force microscopy. These results demonstrate the ability of the engineered β-wrapin HI18 for shielding the hydrophobic sequence of IAPP during expression and purification. Fusion of aggregation-inhibiting β-wrapins is a suitable approach for the recombinant production of aggregation-prone proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. ZnO nanoparticles and their acarbose-capped nanohybrids as inhibitors for human salivary amylase.

    Science.gov (United States)

    Shaik, Firdoz; Kumar, Anil

    2017-04-01

    The authors report a controlled synthesis of biocompatible ZnO and acarbose-capped nanohybrids, and examined the inhibition activities of these nanosystems with human salivary α -amylase (HSA) activity. XRD measurements reveal ZnO present in wurtzite phase with hexagonal structure. The average size of ZnO particles for the two studied nanosystems was estimated to lie between 10 to 12 nm using Scherrer equation. These particles depict the onset of absorption at about 320 nm and the band-gap emission at about 370 nm, which are fairly blue shifted as compared with the bulk ZnO and have been understood due to the size quantisation effect. The inhibitory action of thioglycerol capped ZnO nanoparticles (SP1) and acarbose drug (used for diabetes type II) capped ZnO (SP2) for HSA was observed to 61 and72%, respectively. The inhibition activity of the SP1 alone was found to be very similar to that of acarbose and the coating of these particles with drug (SP2) demonstrated an enhancement in inhibition activity of the enzyme by about 30%. From the inhibition studies, it is confirmed that these nanosystems showed better inhibition activity at physiological temperature and pH. These nanosystems are projected to have potential applications in diabetes type II control.

  14. The efficacy of viral capsid inhibitors in human enterovirus infection and associated diseases.

    Science.gov (United States)

    Li, Chin; Wang, Hongtao; Shih, Shin-Ru; Chen, Tzu-Chun; Li, Mei-Ling

    2007-01-01

    Enteroviruses are members of picornavirus family which causes diverse and severe diseases in humans and animals. Clinical manifestations of enterovirus infections include fever, hand, foot, and mouth disease, and herpangina. Enteroviruses also cause potentially severe and life-threatening infections such as meningitis, encephalitis, myocarditis, polio-like syndrome, and neonatal sepsis. With the emergence of enterovirus all over the world as the major causative agent of HFMD fatalities in recent years and in the absence of any effective anti-enteroviral therapy, there is clearly a need to find a specific antiviral therapy. Steps such as viral attachment, uncoating, viral RNA replication, and protein synthesis in the replication cycle can serve as potential targets for antiviral agents. Agents targeted at viral protein 1 (VP1), a relatively conserved capsid structure mediating viral adsorption and uncoating process, is of great potential to be anti-enterovirus drugs. Recently, considerable efforts have been made in the development of antiviral compounds targeting the capsid protein of enterovirus. This review summarizes the development of small molecules targeting enteroviral capsid protein as effective antiviral therapy.

  15. Phosphatase Inhibitors Function as Novel, Broad Spectrum Botulinum Neurotoxin Antagonists in Mouse and Human Embryonic Stem Cell-Derived Motor Neuron-Based Assays.

    Directory of Open Access Journals (Sweden)

    Erkan Kiris

    Full Text Available There is an urgent need to develop novel treatments to counter Botulinum neurotoxin (BoNT poisoning. Currently, the majority of BoNT drug development efforts focus on directly inhibiting the proteolytic components of BoNT, i.e. light chains (LC. Although this is a rational approach, previous research has shown that LCs are extremely difficult drug targets and that inhibiting multi-serotype BoNTs with a single LC inhibitor may not be feasible. An alternative approach would target neuronal pathways involved in intoxication/recovery, rather than the LC itself. Phosphorylation-related mechanisms have been implicated in the intoxication pathway(s of BoNTs. However, the effects of phosphatase inhibitors upon BoNT activity in the physiological target of BoNTs, i.e. motor neurons, have not been investigated. In this study, a small library of phosphatase inhibitors was screened for BoNT antagonism in the context of mouse embryonic stem cell-derived motor neurons (ES-MNs. Four inhibitors were found to function as BoNT/A antagonists. Subsequently, we confirmed that these inhibitors protect against BoNT/A in a dose-dependent manner in human ES-MNs. Additionally, these compounds provide protection when administered in post-intoxication scenario. Importantly, the inhibitors were also effective against BoNT serotypes B and E. To the best of our knowledge, this is the first study showing phosphatase inhibitors as broad-spectrum BoNT antagonists.

  16. Combined 5-FU and ChoKα inhibitors as a new alternative therapy of colorectal cancer: evidence in human tumor-derived cell lines and mouse xenografts.

    Directory of Open Access Journals (Sweden)

    Ana de la Cueva

    Full Text Available Colorectal cancer (CRC is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα, an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy.ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS and thymidine kinase (TK1 levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action.Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.

  17. An innovative strategy for dual inhibitor design and its application in dual inhibition of human thymidylate synthase and dihydrofolate reductase enzymes.

    Directory of Open Access Journals (Sweden)

    Mahreen Arooj

    Full Text Available Due to the diligence of inherent redundancy and robustness in many biological networks and pathways, multitarget inhibitors present a new prospect in the pharmaceutical industry for treatment of complex diseases. Nevertheless, to design multitarget inhibitors is concurrently a great challenge for medicinal chemists. We have developed a novel computational approach by integrating the affinity predictions from structure-based virtual screening with dual ligand-based pharmacophore to discover potential dual inhibitors of human Thymidylate synthase (hTS and human dihydrofolate reductase (hDHFR. These are the key enzymes in folate metabolic pathway that is necessary for the biosynthesis of RNA, DNA, and protein. Their inhibition has found clinical utility as antitumor, antimicrobial, and antiprotozoal agents. A druglike database was utilized to perform dual-target docking studies. Hits identified through docking experiments were mapped over a dual pharmacophore which was developed from experimentally known dual inhibitors of hTS and hDHFR. Pharmacophore mapping procedure helped us in eliminating the compounds which do not possess basic chemical features necessary for dual inhibition. Finally, three structurally diverse hit compounds that showed key interactions at both active sites, mapped well upon the dual pharmacophore, and exhibited lowest binding energies were regarded as possible dual inhibitors of hTS and hDHFR. Furthermore, optimization studies were performed for final dual hit compound and eight optimized dual hits demonstrating excellent binding features at target systems were also regarded as possible dual inhibitors of hTS and hDHFR. In general, the strategy used in the current study could be a promising computational approach and may be generally applicable to other dual target drug designs.

  18. Isolation and identification of intestinal CYP3A inhibitors from cranberry (Vaccinium macrocarpon) using human intestinal microsomes.

    Science.gov (United States)

    Kim, Eunkyung; Sy-Cordero, Arlene; Graf, Tyler N; Brantley, Scott J; Paine, Mary F; Oberlies, Nicholas H

    2011-02-01

    Cranberry juice is used routinely, especially among women and the elderly, to prevent and treat urinary tract infections. These individuals are likely to be taking medications concomitantly with cranberry juice, leading to concern about potential drug-dietary substance interactions, particularly in the intestine, which, along with the liver, is rich in expression of the prominent drug metabolizing enzyme, cytochrome P450 3A (CYP3A). Using a systematic in vitro-in vivo approach, a cranberry juice product was identified recently that elicited a pharmacokinetic interaction with the CYP3A probe substrate midazolam in 16 healthy volunteers. Relative to water, cranberry juice inhibited intestinal first-pass midazolam metabolism. In vitro studies were initiated to identify potential enteric CYP3A inhibitors from cranberry via a bioactivity-directed fractionation approach involving dried whole cranberry [Vaccinium macrocarpon Ait. (Ericaceae)], midazolam, and human intestinal microsomes (HIM). Three triterpenes (maslinic acid, corosolic acid, and ursolic acid) were isolated. The inhibitory potency (IC(50)) of maslinic acid, corosolic acid, and ursolic acid was 7.4, 8.8, and cranberry juice interaction observed in the clinical study. © Georg Thieme Verlag KG Stuttgart · New York.

  19. Effect of Known Inhibitors of Ion Transport on Pendrin (SLC26A4) Activity in a Human Kidney Cell Line.

    Science.gov (United States)

    Bernardinelli, Emanuele; Costa, Roberta; Nofziger, Charity; Paulmichl, Markus; Dossena, Silvia

    2016-01-01

    Pendrin is a Cl-/I-/HCO3- exchanger playing a fundamental role in controlling blood pressure and airway function, therefore representing an attractive target for the treatment of hypertensive states and respiratory distresses. A review of the literature regarding the ability of some compounds (namely several known inhibitors of ion transport) to block pendrin activity revealed discordant findings. These incongruous findings may be due, in part, to the concentration of compound and/or the nature of the model system used in the study. Pendrin activity was evaluated by measuring pendrin-dependent iodide influx following overexpression of the transporter in a human kidney cell line, in the presence of selected test compounds or the respective vehicles. Pendrin activity was significantly hampered by 0.1 mM 5-nitro-2-[(3-phenylpropyl)amino]benzoic acid (NPPB), niflumic acid and tenidap, but was resistant to 0.1 mM 4, 4'-diisothiocyano-2, 2'-stilbene-disulfonic acid (DIDS), furosemide and probenecid. The results of the present study indicate that clinically effective non-steroidal anti-inflammatory drugs (niflumic acid and tenidap) directly inhibit pendrin activity. © 2016 The Author(s) Published by S. Karger AG, Basel.

  20. Simultaneous human papilloma virus type 16 E7 and cdk inhibitor p21 expression induces apoptosis and cathepsin B activation

    International Nuclear Information System (INIS)

    Kaznelson, Dorte Wissing; Bruun, Silas; Monrad, Astrid; Gjerloev, Simon; Birk, Jesper; Roepke, Carsten; Norrild, Bodil

    2004-01-01

    Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation and induction of cell death. We have used the osteosarcoma cell line U2OS cells provided with E7 and the cdk2 inhibitor p21 (cip1/waf1) under inducible control, as a model system for the analysis of E7-mediated apoptosis. Our data shows that simultaneous expression of E7 and p21 proteins induces cell death, possibly because of conflicting growth control. Interestingly, E7/p21-induced cell death is associated with the activation of a newly identified mediator of apoptosis, namely cathepsin B. Activation of the cellular caspases is undetectable in cells undergoing E7/p21-induced apoptosis. To our knowledge, this is the first time a role for cathepsin B is reported in HPV-induced apoptotic signalling

  1. Enhancement of auranofin-induced apoptosis in MCF-7 human breast cells by selenocystine, a synergistic inhibitor of thioredoxin reductase.

    Directory of Open Access Journals (Sweden)

    Chaoran Liu

    Full Text Available Thioredoxin system plays an important role in regulation of intracellular redox balance and various signaling pathways. Thioredoxin reductase (TrxR is overexpressed in many cancer cells and has been identified as a potential target of anticancer drugs. Auranofin (AF is potent TrxR inhibitor with novel in vitro and in vivo anticancer activities. Selenocystine (SeC is a nutritionally available selenoamino acid with selective anticancer effects through induction of apoptosis. In the present study, we demonstrated the synergistic effects and the underlying molecular mechanisms of SeC in combination with AF on MCF-7 human breast cancer cells. The results showed that SeC and AF synergistically inhibited the cancer cell growth through induction of ROS-dependent apoptosis with the involvement of mitochondrial dysfunction. DNA damage-mediated p53 phosphorylation and down-regulation of phosphorylated AKT and ERK also contributed to cell apoptosis. Moreover, we demonstrated the important role of TrxR activity in the synergistic action of SeC and AF. Taken together, our results suggest the strategy to use SeC and AF in combination could be a highly efficient way to achieve anticancer synergism by targeting TrxR.

  2. Autophagy Regulates Proteasome Inhibitor-Induced Pigmentation in Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells.

    Science.gov (United States)

    Juuti-Uusitalo, Kati; Koskela, Ali; Kivinen, Niko; Viiri, Johanna; Hyttinen, Juha M T; Reinisalo, Mika; Koistinen, Arto; Uusitalo, Hannu; Sinha, Debasish; Skottman, Heli; Kaarniranta, Kai

    2017-05-19

    The impairment of autophagic and proteasomal cleansing together with changes in pigmentation has been documented in retinal pigment epithelial (RPE) cell degeneration. However, the function and co-operation of these mechanisms in melanosome-containing RPE cells is still unclear. We show that inhibition of proteasomal degradation with MG-132 or autophagy with bafilomycin A1 increased the accumulation of premelanosomes and autophagic structures in human embryonic stem cell (hESC)-derived RPE cells. Consequently, upregulation of the autophagy marker p62 (also known as sequestosome-1, SQSTM1) was confirmed in Western blot and perinuclear staining. Interestingly, cells treated with the adenosine monophosphatedependent protein kinase activator, AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), decreased the proteasome inhibitor-induced accumulation of premelanosomes, increased the amount of autophagosomes and eradicated the protein expression of p62 and LC3 (microtubule-associated protein 1A/1B-light chain 3). These results revealed that autophagic machinery is functional in hESC-RPE cells and may regulate cellular pigmentation with proteasomes.

  3. Effect of Known Inhibitors of Ion Transport on Pendrin (SLC26A4 Activity in a Human Kidney Cell Line

    Directory of Open Access Journals (Sweden)

    Emanuele Bernardinelli

    2016-05-01

    Full Text Available Background/Aims: Pendrin is a Cl-/I-/HCO3- exchanger playing a fundamental role in controlling blood pressure and airway function, therefore representing an attractive target for the treatment of hypertensive states and respiratory distresses. A review of the literature regarding the ability of some compounds (namely several known inhibitors of ion transport to block pendrin activity revealed discordant findings. These incongruous findings may be due, in part, to the concentration of compound and/or the nature of the model system used in the study. Methods: Pendrin activity was evaluated by measuring pendrin-dependent iodide influx following overexpression of the transporter in a human kidney cell line, in the presence of selected test compounds or the respective vehicles. Results: Pendrin activity was significantly hampered by 0.1 mM 5-nitro-2-[(3-phenylpropylamino]benzoic acid (NPPB, niflumic acid and tenidap, but was resistant to 0.1 mM 4, 4′-diisothiocyano-2, 2′-stilbene-disulfonic acid (DIDS, furosemide and probenecid. Conclusions: The results of the present study indicate that clinically effective non-steroidal anti-inflammatory drugs (niflumic acid and tenidap directly inhibit pendrin activity.

  4. [Effects of HSP90 inhibitor 17-AAG on cell cycle and apoptosis of human gastric cancer cell lines SGC-7901].

    Science.gov (United States)

    Chen, Meini; Xu, Jinghong; Zhao, Jumei

    2013-02-01

    To study the effect of the HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on cell proliferation and apoptosis of human cancer SGC-7901 cells and explore the mechanisms. The inhibitory effect of 17-AAG on the proliferation and morphology of SGC-7901 cells was assessed with MTT assay and DNA-PI staining, respectively. Flow cytometry was employed to analyze the changes in cell cycle and apoptosis of the cells following 17-AAG exposure. The cellular expression of Fas protein was detected by immunohistochemistry. 17-AAG significantly suppressed the proliferation of SGC-7901 cells in a time- and dose-dependent manner. After treatment with 17-AAG for 48 h, SGC-7901 cells showed cell cycle arrested at G(2)/M stage, and the cell apoptosis rate increased with the 17-AAG concentration. The expression of Fas protein in the cytoplasm of SGC-7901 cells increased gradually with the increase of 17-AAG concentration. 17-AAG can induce apoptosis, alters the cell cycle distribution and up-regulates the expression of Fas protein in SGC-7901 cells to suppress the cell proliferation.

  5. Thiol protease-specific inhibitor E-64 arrests human epidermoid carcinoma A431 cells at mitotic metaphase

    International Nuclear Information System (INIS)

    Shoji-Kasai, Y.; Senshu, M.; Iwashita, S.; Imahori, K.

    1988-01-01

    E-64-d /ethyl (2S, 3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butylcarbamoyl]oxirane-2-carboxylate/, a membrane-permeant derivative of the thiol protease-specific inhibitor E-64, was found to arrest human epidermoid carcinoma A431 cells at mitotic metaphase. This effect was dose-dependent with a threshold of 20μg/ml in chemically defined culture medium. Cell cycle analysis by flow cytometry showed that the relative proportion of the G 2 /M population increased 2.5-fold after treatment of the cells with E-64 (100 μg/ml) for 5 hr. In addition, time-lapse video analysis showed that E-64-treated cells remained at metaphase for an extended period after rounding-up, whereas untreated cells were able to complete mitosis within 42.0 +/- 5.7 min. Some treated cells were able to complete mitosis, while others did not do so within limits of the authors observation. An approach to the molecular basis of this phenomenon, they have shown that several cellular proteins can be labeled by incubation of cells with radioactive E-64-d

  6. ARQ 197, a novel and selective inhibitor of the human c-Met receptor tyrosine kinase with antitumor activity.

    Science.gov (United States)

    Munshi, Neru; Jeay, Sébastien; Li, Youzhi; Chen, Chang-Rung; France, Dennis S; Ashwell, Mark A; Hill, Jason; Moussa, Magdi M; Leggett, David S; Li, Chiang J

    2010-06-01

    The met proto-oncogene is functionally linked with tumorigenesis and metastatic progression. Validation of the receptor tyrosine kinase c-Met as a selective anticancer target has awaited the emergence of selective c-Met inhibitors. Herein, we report ARQ 197 as the first non-ATP-competitive small molecule that selectively targets the c-Met receptor tyrosine kinase. Exposure to ARQ 197 resulted in the inhibition of proliferation of c-Met-expressing cancer cell lines as well as the induction of caspase-dependent apoptosis in cell lines with constitutive c-Met activity. These cellular responses to ARQ 197 were phenocopied by RNAi-mediated c-Met depletion and further demonstrated by the growth inhibition of human tumors following oral administration of ARQ 197 in multiple mouse xenograft efficacy studies. Cumulatively, these data suggest that ARQ 197, currently in phase II clinical trials, is a promising agent for targeting cancers in which c-Met-driven signaling is important for their survival and proliferation.

  7. Insights into the Molecular Mechanism of Polymerization and Nucleoside Reverse Transcriptase Inhibitor Incorporation by Human PrimPol.

    Science.gov (United States)

    Mislak, Andrea C; Anderson, Karen S

    2016-01-01

    Human PrimPol is a newly identified DNA and RNA primase-polymerase of the archaeo-eukaryotic primase (AEP) superfamily and only the second known polymerase in the mitochondria. Mechanistic studies have shown that interactions of the primary mitochondrial DNA polymerase γ (mtDNA Pol γ) with nucleoside reverse transcriptase inhibitors (NRTIs), key components in treating HIV infection, are a major source of NRTI-associated toxicity. Understanding the interactions of host polymerases with antiviral and anticancer nucleoside analog therapies is critical for preventing life-threatening adverse events, particularly in AIDS patients who undergo lifelong treatment. Since PrimPol has only recently been discovered, the molecular mechanism of polymerization and incorporation of natural nucleotide and NRTI substrates, crucial for assessing the potential for PrimPol-mediated NRTI-associated toxicity, has not been explored. We report for the first time a transient-kinetic analysis of polymerization for each nucleotide and NRTI substrate as catalyzed by PrimPol. These studies reveal that nucleotide selectivity limits chemical catalysis while the release of the elongated DNA product is the overall rate-limiting step. Remarkably, PrimPol incorporates four of the eight FDA-approved antiviral NRTIs with a kinetic profile distinct from that of mtDNA Pol γ that may manifest in toxicity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  8. A Small Molecule Inhibitor Selectively Induces Apoptosis in Cells Transformed by High Risk Human Papilloma Viruses.

    Directory of Open Access Journals (Sweden)

    Amy K Sheaffer

    Full Text Available A phenotypic high-throughput cell culture screen was performed to identify compounds that prevented proliferation of the human Papilloma virus type 16 (HPV-16 transformed cell line Ca Ski. A series of quinoxaline compounds exemplified by Compound 1 was identified. Testing against a panel of cell lines demonstrated that Compound 1 selectively inhibited replication of all HPV-16, HPV-18, and HPV-31 transformed cell lines tested with 50% Inhibitory Concentration (IC50 values of 2 to 8 μM relative to IC50 values of 28 to 73 μM in HPV-negative cell lines. Treatment with Compound 1 resulted in a cascade of multiple apoptotic events, including selective activation of effector caspases 3 and 7, fragmentation of cellular DNA, and PARP (poly(ADP-ribose polymerase cleavage in HPV-positive cells relative to HPV-negative cells. Unregulated proliferation of HPV transformed cells is dependent on the viral oncogenes, E6 and E7. Treatment with Compound 1 resulted in a decrease in HPV E7 protein in Ca Ski cells. However, the timing of this reduction relative to other effects of compound treatment suggests that this was a consequence, rather than a cause, of the apoptotic cascade. Likewise, compound treatment resulted in no obvious effects on the E6- and E7- mediated down regulation of p53 and Rb, or their downstream effectors, p21 or PCNA. Further investigation of apoptotic signals induced by Compound 1 revealed cleavage of Caspase-8 in HPV-positive cells as early as 2 hours post-treatment, suggesting the compound initiates apoptosis through the extrinsic, death receptor-mediated, pathway of cell death. These studies provide proof of concept that cells transformed by oncogenic Papillomaviruses can be selectively induced to undergo apoptosis by compound treatment.

  9. A Small Molecule Inhibitor Selectively Induces Apoptosis in Cells Transformed by High Risk Human Papilloma Viruses.

    Science.gov (United States)

    Sheaffer, Amy K; Lee, Min S; Qi, Huilin; Chaniewski, Susan; Zheng, Xiaofan; Farr, Glen A; Esposito, Kim; Harden, David; Lei, Ming; Schweizer, Liang; Friborg, Jacques; Agler, Michele; McPhee, Fiona; Gentles, Robert; Beno, Brett R; Chupak, Lou; Mason, Stephen

    2016-01-01

    A phenotypic high-throughput cell culture screen was performed to identify compounds that prevented proliferation of the human Papilloma virus type 16 (HPV-16) transformed cell line Ca Ski. A series of quinoxaline compounds exemplified by Compound 1 was identified. Testing against a panel of cell lines demonstrated that Compound 1 selectively inhibited replication of all HPV-16, HPV-18, and HPV-31 transformed cell lines tested with 50% Inhibitory Concentration (IC50) values of 2 to 8 μM relative to IC50 values of 28 to 73 μM in HPV-negative cell lines. Treatment with Compound 1 resulted in a cascade of multiple apoptotic events, including selective activation of effector caspases 3 and 7, fragmentation of cellular DNA, and PARP (poly(ADP-ribose) polymerase) cleavage in HPV-positive cells relative to HPV-negative cells. Unregulated proliferation of HPV transformed cells is dependent on the viral oncogenes, E6 and E7. Treatment with Compound 1 resulted in a decrease in HPV E7 protein in Ca Ski cells. However, the timing of this reduction relative to other effects of compound treatment suggests that this was a consequence, rather than a cause, of the apoptotic cascade. Likewise, compound treatment resulted in no obvious effects on the E6- and E7- mediated down regulation of p53 and Rb, or their downstream effectors, p21 or PCNA. Further investigation of apoptotic signals induced by Compound 1 revealed cleavage of Caspase-8 in HPV-positive cells as early as 2 hours post-treatment, suggesting the compound initiates apoptosis through the extrinsic, death receptor-mediated, pathway of cell death. These studies provide proof of concept that cells transformed by oncogenic Papillomaviruses can be selectively induced to undergo apoptosis by compound treatment.

  10. Inhibitors of cysteine cathepsin and calpain do not prevent ultraviolet-B-induced apoptosis in human keratinocytes and HeLa cells

    DEFF Research Database (Denmark)

    Bang, Bo; Baadsgaard, Ole; Skov, Lone

    2004-01-01

    been demonstrated to play a role in the execution of programmed cell death induced by other stimuli, e.g. TNF-alpha. The purpose of the present study was therefore to investigate whether inhibitors of cysteine cathepsins and calpains could prevent UVB-induced apoptosis in HeLa cells and keratinocytes....... This was done by investigating the effect of the irreversible cysteine protease inhibitor zFA-fmk, the cathepsin B inhibitor CA-074-Me and the calpain inhibitor ALLN on the viability of UVB-irradiated human keratinocytes and HeLa cells. At concentrations of 10 microM and above zVAD-fmk conferred partial dose......-dependent protection against UVB-induced apoptosis in HeLa cells and keratinocytes. Moreover, caspase-3 activity was completely blocked at zVAD-fmk concentrations of 1 microM in HeLa cells. This indicates that caspase-independent mechanisms could be involved in UVB-induced apoptosis. However, the protease inhibitors z...

  11. Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

    Directory of Open Access Journals (Sweden)

    Sanne H. Olesen

    2015-01-01

    Full Text Available The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein–protein interaction (PPI inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2 did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM, while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.

  12. Novel Histone Deacetylase Inhibitors

    National Research Council Canada - National Science Library

    Strobl, Jeannie

    2001-01-01

    The research goal is to demonstrate HDACl is a new chemotherapeutic target for human breast tumor cells and to identify new HDACl inhibitors on the basis of the structure of quinoline antimalarials...

  13. The cathepsin B inhibitor, z-FA-CMK is toxic and readily induced cell death in human T lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Liow, K.Y.; Chow, S.C., E-mail: chow.sek.chuen@monash.edu

    2013-11-01

    The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-chloromethylketone (z-FA-CMK) was found to be toxic and readily induced cell death in the human T cell line, Jurkat, whereas two other analogs benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone (z-FA-FMK) and benzyloxycarbonyl-phenylalanine-alanine-diazomethylketone (z-FA-DMK) were not toxic. The toxicity of z-FA-CMK requires not only the CMK group, but also the presence of alanine in the P1 position and the benzyloxycarbonyl group at the N-terminal. Dose–response studies showed that lower concentrations of z-FA-CMK induced apoptosis in Jurkat T cells whereas higher concentrations induced necrosis. In z-FA-CMK-induced apoptosis, both initiator caspases (-8 and -9) and effector caspases (-3, -6 and -7) were processed to their respective subunits in Jurkat T cells. However, only the pro-form of the initiator caspases were reduced in z-FA-CMK-induced necrosis and no respective subunits were apparent. The caspase inihibitor benzyloxycarbonyl-valine-alanine-aspartic acid-(O-methyl)-fluoromehylketone (z-VAD-FMK) inhibits apoptosis and caspase processing in Jurkat T cells treated with low concentration of z-FA-CMK but has no effect on z-FA-CMK-induced necrosis and the loss of initiator caspases. This suggests that the loss of initiator caspases in Jurkat T cells during z-FA-CMK-induced necrosis is not a caspase-dependent process. Taken together, we have demonstrated that z-FA-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. - Highlights: • z-FA-CMK is toxic and induce cell death in the human T cells. • z-FA-CMK toxicity requires the CMK group, alanine and the benzyloxycarbonyl group. • z-FA-CMK induced apoptosis at low concentration and necrosis at high concentration.

  14. Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation.

    Science.gov (United States)

    Molica, Filippo; Nolli, Séverine; Fontana, Pierre; Kwak, Brenda Renata

    2017-04-06

    Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor

  15. The cathepsin B inhibitor, z-FA-CMK is toxic and readily induced cell death in human T lymphocytes

    International Nuclear Information System (INIS)

    Liow, K.Y.; Chow, S.C.

    2013-01-01

    The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-chloromethylketone (z-FA-CMK) was found to be toxic and readily induced cell death in the human T cell line, Jurkat, whereas two other analogs benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone (z-FA-FMK) and benzyloxycarbonyl-phenylalanine-alanine-diazomethylketone (z-FA-DMK) were not toxic. The toxicity of z-FA-CMK requires not only the CMK group, but also the presence of alanine in the P1 position and the benzyloxycarbonyl group at the N-terminal. Dose–response studies showed that lower concentrations of z-FA-CMK induced apoptosis in Jurkat T cells whereas higher concentrations induced necrosis. In z-FA-CMK-induced apoptosis, both initiator caspases (-8 and -9) and effector caspases (-3, -6 and -7) were processed to their respective subunits in Jurkat T cells. However, only the pro-form of the initiator caspases were reduced in z-FA-CMK-induced necrosis and no respective subunits were apparent. The caspase inihibitor benzyloxycarbonyl-valine-alanine-aspartic acid-(O-methyl)-fluoromehylketone (z-VAD-FMK) inhibits apoptosis and caspase processing in Jurkat T cells treated with low concentration of z-FA-CMK but has no effect on z-FA-CMK-induced necrosis and the loss of initiator caspases. This suggests that the loss of initiator caspases in Jurkat T cells during z-FA-CMK-induced necrosis is not a caspase-dependent process. Taken together, we have demonstrated that z-FA-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. - Highlights: • z-FA-CMK is toxic and induce cell death in the human T cells. • z-FA-CMK toxicity requires the CMK group, alanine and the benzyloxycarbonyl group. • z-FA-CMK induced apoptosis at low concentration and necrosis at high concentration

  16. Inhibition of P-glycoprotein by HIV protease inhibitors increases intracellular accumulation of berberine in murine and human macrophages.

    Directory of Open Access Journals (Sweden)

    Weibin Zha

    Full Text Available HIV protease inhibitor (PI-induced inflammatory response in macrophages is a major risk factor for cardiovascular diseases. We have previously reported that berberine (BBR, a traditional herbal medicine, prevents HIV PI-induced inflammatory response through inhibiting endoplasmic reticulum (ER stress in macrophages. We also found that HIV PIs significantly increased the intracellular concentrations of BBR in macrophages. However, the underlying mechanisms of HIV PI-induced BBR accumulation are unknown. This study examined the role of P-glycoprotein (P-gp in HIV PI-mediated accumulation of BBR in macrophages.Cultured mouse RAW264.7 macrophages, human THP-1-derived macrophages, Wild type MDCK (MDCK/WT and human P-gp transfected (MDCK/P-gp cells were used in this study. The intracellular concentration of BBR was determined by HPLC. The activity of P-gp was assessed by measuring digoxin and rhodamine 123 (Rh123 efflux. The interaction between P-gp and BBR or HIV PIs was predicated by Glide docking using Schrodinger program. The results indicate that P-gp contributed to the efflux of BBR in macrophages. HIV PIs significantly increased BBR concentrations in macrophages; however, BBR did not alter cellular HIV PI concentrations. Although HIV PIs did not affect P-gp expression, P-gp transport activities were significantly inhibited in HIV PI-treated macrophages. Furthermore, the molecular docking study suggests that both HIV PIs and BBR fit the binding pocket of P-gp, and HIV PIs may compete with BBR to bind P-gp.HIV PIs increase the concentration of BBR by modulating the transport activity of P-gp in macrophages. Understanding the cellular mechanisms of potential drug-drug interactions is critical prior to applying successful combinational therapy in the clinic.

  17. Metformin increases antitumor activity of MEK inhibitors through GLI1 downregulation in LKB1 positive human NSCLC cancer cells.

    Science.gov (United States)

    Della Corte, Carminia Maria; Ciaramella, Vincenza; Di Mauro, Concetta; Castellone, Maria Domenica; Papaccio, Federica; Fasano, Morena; Sasso, Ferdinando Carlo; Martinelli, Erika; Troiani, Teresa; De Vita, Ferdinando; Orditura, Michele; Bianco, Roberto; Ciardiello, Fortunato; Morgillo, Floriana

    2016-01-26

    Metformin, widely used as antidiabetic drug, showed antitumoral effects expecially in combination with chemotherapy. Our group recently has demonstrated that metformin and gefitinib are synergistic in LKB1-wild-type NSCLC cells. In these models, metformin as single agent induced an activation and phosphorylation of mitogen-activated-protein-kinase (MAPK) through an increased C-RAF/B-RAF heterodimerization. Since single agent metformin enhances proliferating signals through the RAS/RAF/MAPK pathway, and several MEK inhibitors (MEK-I) demonstrated clinical efficacy in combination with other agents in NSCLC, we tested the effects of metformin plus MEK-I (selumetinib or pimasertib) on proliferation, invasiveness, migration abilities in vitro and in vivo in LKB1 positive NSCLC models harboring KRAS wild type and mutated gene. The combination of metformin with MEK-I showed a strong anti-proliferative and proapoptotic effect in Calu-3, H1299, H358 and H1975 human NSCLC cell lines, independently from the KRAS mutational status. The combination reduced the metastatic behaviour of NSCLC cells, via a downregulation of GLI1 trascritional activity, thus affecting the transition from an epithelial to a mesenchymal phenotype. Metformin and MEK-Is combinations also decreased the production and activity of MMP-2 and MMP-9 by reducing the NF-jB (p65) binding to MMP-2 and MMP-9 promoters. Metformin potentiates the antitumor activity of MEK-Is in human LKB1-wild-type NSCLC cell lines, independently from the KRAS mutational status, through GLI1 downregulation and by reducing the NF-jB (p65)-mediated transcription of MMP-2 and MMP-9.

  18. Protein C inhibitor acts as a procoagulant by inhibiting the thrombomodulin-induced activation of protein C in human plasma

    NARCIS (Netherlands)

    Elisen, M. G.; von dem Borne, P. A.; Bouma, B. N.; Meijers, J. C.

    1998-01-01

    Protein C inhibitor (PCI), which was originally identified as an inhibitor of activated protein C, also efficiently inhibits coagulation factors such as factor Xa and thrombin. Recently it was found, using purified proteins, that the anticoagulant thrombin-thrombomodulin complex was also inhibited

  19. Overexpression of SERBP1 (Plasminogen activator inhibitor 1 RNA binding protein in human breast cancer is correlated with favourable prognosis

    Directory of Open Access Journals (Sweden)

    Serce Nuran Bektas

    2012-12-01

    Full Text Available Abstract Background Plasminogen activator inhibitor 1 (PAI-1 overexpression is an important prognostic and predictive biomarker in human breast cancer. SERBP1, a protein that is supposed to regulate the stability of PAI-1 mRNA, may play a role in gynaecological cancers as well, since upregulation of SERBP1 was described in ovarian cancer recently. This is the first study to present a systematic characterisation of SERBP1 expression in human breast cancer and normal breast tissue at both the mRNA and the protein level. Methods Using semiquantitative realtime PCR we analysed SERBP1 expression in different normal human tissues (n = 25, and in matched pairs of normal (n = 7 and cancerous breast tissues (n = 7. SERBP1 protein expression was analysed in two independent cohorts on tissue microarrays (TMAs, an initial evaluation set, consisting of 193 breast carcinomas and 48 normal breast tissues, and a second large validation set, consisting of 605 breast carcinomas. In addition, a collection of benign (n = 2 and malignant (n = 6 mammary cell lines as well as breast carcinoma lysates (n = 16 were investigated for SERBP1 expression by Western blot analysis. Furthermore, applying non-radioisotopic in situ hybridisation a subset of normal (n = 10 and cancerous (n = 10 breast tissue specimens from the initial TMA were analysed for SERBP1 mRNA expression. Results SERBP1 is not differentially expressed in breast carcinoma compared to normal breast tissue, both at the RNA and protein level. However, recurrence-free survival analysis showed a significant correlation (P = 0.008 between abundant SERBP1 expression in breast carcinoma and favourable prognosis. Interestingly, overall survival analysis also displayed a tendency (P = 0.09 towards favourable prognosis when SERBP1 was overexpressed in breast cancer. Conclusions The RNA-binding protein SERBP1 is abundantly expressed in human breast cancer and may represent a

  20. Validation of Simultaneous Quantitative Method of HIV Protease Inhibitors Atazanavir, Darunavir and Ritonavir in Human Plasma by UPLC-MS/MS

    Directory of Open Access Journals (Sweden)

    Tulsidas Mishra

    2014-01-01

    Full Text Available Objectives. HIV protease inhibitors are used in the treatment of patients suffering from AIDS and they act at the final stage of viral replication by interfering with the HIV protease enzyme. The paper describes a selective, sensitive, and robust method for simultaneous determination of three protease inhibitors atazanavir, darunavir and ritonavir in human plasma by ultra performance liquid chromatography-tandem mass spectrometry. Materials and Methods. The sample pretreatment consisted of solid phase extraction of analytes and their deuterated analogs as internal standards from 50 μL human plasma. Chromatographic separation of analytes was performed on Waters Acquity UPLC C18 (50 × 2.1 mm, 1.7 μm column under gradient conditions using 10 mM ammonium formate, pH 4.0, and acetonitrile as the mobile phase. Results. The method was established over a concentration range of 5.0–6000 ng/mL for atazanavir, 5.0–5000 ng/mL for darunavir and 1.0–500 ng/mL for ritonavir. Accuracy, precision, matrix effect, recovery, and stability of the analytes were evaluated as per US FDA guidelines. Conclusions. The efficiency of sample preparation, short analysis time, and high selectivity permit simultaneous estimation of these inhibitors. The validated method can be useful in determining plasma concentration of these protease inhibitors for therapeutic drug monitoring and in high throughput clinical studies.

  1. Noncanonical Wnt signaling promotes osteoclast differentiation and is facilitated by the human immunodeficiency virus protease inhibitor ritonavir

    International Nuclear Information System (INIS)

    Santiago, Francisco; Oguma, Junya; Brown, Anthony M.C.; Laurence, Jeffrey

    2012-01-01

    Highlights: ► First demonstration of direct role for noncanonical Wnt in osteoclast differentiation. ► Demonstration of Ryk as a Wnt5a/b receptor in inhibition of canonical Wnt signaling. ► Modulation of noncanonical Wnt signaling by a clinically important drug, ritonavir. ► Establishes a mechanism for an important clinical problem: HIV-associated bone loss. -- Abstract: Wnt proteins that signal via the canonical Wnt/β-catenin pathway directly regulate osteoblast differentiation. In contrast, most studies of Wnt-related effects on osteoclasts involve indirect changes. While investigating bone mineral density loss in the setting of human immunodeficiency virus (HIV) infection and its treatment with the protease inhibitor ritonavir (RTV), we observed that RTV decreased nuclear localization of β-catenin, critical to canonical Wnt signaling, in primary human and murine osteoclast precursors. This occurred in parallel with upregulation of Wnt5a and Wnt5b transcripts. These Wnts typically stimulate noncanonical Wnt signaling, and this can antagonize the canonical Wnt pathway in many cell types, dependent upon Wnt receptor usage. We now document RTV-mediated upregulation of Wnt5a/b protein in osteoclast precursors. Recombinant Wnt5b and retrovirus-mediated expression of Wnt5a enhanced osteoclast differentiation from human and murine monocytic precursors, processes facilitated by RTV. In contrast, canonical Wnt signaling mediated by Wnt3a suppressed osteoclastogenesis. Both RTV and Wnt5b inhibited canonical, β-catenin/T cell factor-based Wnt reporter activation in osteoclast precursors. RTV- and Wnt5-induced osteoclast differentiation were dependent upon the receptor-like tyrosine kinase Ryk, suggesting that Ryk may act as a Wnt5a/b receptor in this context. This is the first demonstration of a direct role for Wnt signaling pathways and Ryk in regulation of osteoclast differentiation, and its modulation by a clinically important drug, ritonavir. These studies

  2. Molecular Docking and Molecular Dynamics to Identify a Novel Human Immunodeficiency Virus Inhibitor from Alkaloids of Toddalia asiatica.

    Science.gov (United States)

    Priya, R; Sumitha, Rajendrarao; Doss, C George Priya; Rajasekaran, C; Babu, S; Seenivasan, R; Siva, R

    2015-10-01

    Acquired immunodeficiency syndrome caused by human immunodeficiency virus (HIV) is an immunosuppressive disease. Over the past decades, it has plagued human health due to the grave consequences in its harness. For this reason, anti-HIV agents are imperative, and the search for the same from natural resources would assure the safety. In this investigation we have performed molecular docking, molecular property prediction, drug-likeness score, and molecular dynamics (MD) simulation to develop a novel anti-HIV drug. We have screened 12 alkaloids from a medicinal plant Toddalia asiatica for its probabilistic binding with the active site of the HIV-1-reverse transcriptase (HIV-1-RT) domain (the major contributor to the onset of the disease). The docking results were evaluated based on free energies of binding (ΔG), and the results suggested toddanol, toddanone, and toddalenone to be potent inhibitors of HIV-1-RT. In addition, the alkaloids were subjected to molecular property prediction analysis. Toddanol and toddanone with more rotatable bonds were found to have a drug-likeness score of 0.23 and 0.11, respectively. These scores were comparable with the standard anti-HIV drug zidovudine with a model score 0.28. Finally, two characteristic protein-ligand complexes were exposed to MD simulation to determine the stability of the predicted conformations. The toddanol-RT complex showed higher stability and stronger H-bonds than toddanone-RT complex. Based on these observations, we firmly believe that the alkaloid toddanol could aid in efficient HIV-1 drug discovery. In the present study, the molecular docking and MD simulations are performed to explore the possible binding mode of HIV 1 RT with 12 alkaloids of T. asiatica. Molecular docking by AutoDock4 revealed three alkaloids toddanol, toddanone, and toddalenone with highest binding affinity towards HIV 1 RT. The drug likeness model score revealed a positive score for toddanol and toddanone which is comparable to the drug

  3. Antiproliferative and pro-apoptotic effects afforded by novel Src-kinase inhibitors in human neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    Angelucci Adriano

    2010-11-01

    Full Text Available Abstract Background Neuroblastoma (NB is the second most common solid malignancy of childhood that usually undergoes rapid progression with a poor prognosis upon metastasis. The Src-family tyrosine kinases (SFKs are a group of proteins involved in cancer development and invasiveness that seem to play an important role in the NB carcinogenesis. Methods To determine cell proliferation, the growth rate was evaluated by both MTT test and cells counted. Analysis of DNA content was performed for the evaluation of the cell cycle and apoptosis. To characterize the mechanisms underlying the antiproliferative effects induced by SI 34, a novel pyrazolo-pyrimidine derivative provided with Src inhibitory activity, the involvement of some cellular pathways that are important for cell proliferation and survival was investigated by western blot assays. In particular, the contribution of cyclins, Src and ERK were examined. Finally, experiments of cell adhesion and invasiveness were performed. Results Treatment of SH-SY5Y human NB cells and CHP100 human neuroepithelioma (NE cultures with three novel pyrazolo[3,4-d]pyrimidine derivatives, namely SI 34, SI 35 and SI 83, inhibits the cell proliferation in a time and concentration-dependent manner. The maximal effect was obtained after 72 hours incubation with SI 34 10 μM. Fluorescence microscopy experiments, flow cytometry analysis and determination of caspase-3 activity by fluorimetric assays showed that SI 34 induced SH-SY5Y apoptosis. Moreover, SI 34 determined cell cycle arrest at the G0/G1 phase, paralleled by a decreased expression of cyclin D1. Furthermore, our data indicate that SI 34 reduces the SH-SY5Y cells adhesion and invasiveness. Evidence that SI 34 inhibits the Src and the ERK-phosphorylation, suggests the mechanism through which it exerts its effects in SH-SY5Y cells. Conclusions Our study shows the ability of this pyrazolo-pyrimidine Src inhibitor in reducing the growth and the invasiveness of

  4. Suppression of human T cell proliferation by the caspase inhibitors, z-VAD-FMK and z-IETD-FMK is independent of their caspase inhibition properties

    Energy Technology Data Exchange (ETDEWEB)

    Lawrence, C.P. [Medical Research Council Toxicology Unit, Hodgkin Building, Lancaster Road, University of Leicester, Leicester LE1 9HN (United Kingdom); Chow, S.C., E-mail: chow.sek.chuen@monash.edu [School of Science, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, 46150 Selangor Darul Ehsan (Malaysia)

    2012-11-15

    The caspase inhibitors, benzyloxycarbony (Cbz)-l-Val-Ala-Asp (OMe)-fluoromethylketone (z-VAD-FMK) and benzyloxycarbonyl (Cbz)-Ile-Glu (OMe)-Thr-Asp (OMe)-FMK (z-IETD-FMK) at non-toxic doses were found to be immunosuppressive and inhibit human T cell proliferation induced by mitogens and IL-2 in vitro. Both caspase inhibitors were shown to block NF-κB in activated primary T cells, but have little inhibitory effect on the secretion of IL-2 and IFN-γ during T cell activation. However, the expression of IL-2 receptor α-chain (CD25) in activated T cells was inhibited by both z-VAD-FMK and z-IETD-FMK, whereas the expression of the early activated T cell marker, CD69 was unaffected. During primary T cell activation via the antigen receptor, both caspase-8 and caspase-3 were activated and processed to their respective subunits, but neither caspase inhibitors had any effect on the processing of these two caspases. In sharp contrast both caspase inhibitors readily blocked apoptosis and the activation of caspases during FasL-induced apoptosis in activated primary T cells and Jurkat T cells. Collectively, the results demonstrate that both z-VAD-FMK and z-IETD-FMK are immunosuppressive in vitro and inhibit T cell proliferation without blocking the processing of caspase-8 and caspase-3. -- Highlights: ► Caspase-8 and caspase-3 were activated during T cell activation and proliferation. ► T cell proliferation was blocked by caspase inhibitors. ► Caspase activation during T cell proliferation was not block by caspase inhibitors.

  5. Maternal-Fetal Transfer and Amniotic Fluid Accumulation of Nucleoside Analogue Reverse Transcriptase Inhibitors in Human Immunodeficiency Virus-Infected Pregnant Women

    OpenAIRE

    Chappuy, Hélène; Tréluyer, Jean-Marc; Jullien, Vincent; Dimet, Jérôme; Rey, Elisabeth; Fouché, Maria; Firtion, Ghislaine; Pons, Gérard; Mandelbrot, Laurent

    2004-01-01

    This study was performed to investigate placental transfer of nucleoside analogue reverse transcriptase inhibitors (NRTIs) and their concentrations in amniotic fluid when given to human immunodeficiency virus (HIV)-infected pregnant women. A total of 100 HIV type 1-infected mothers receiving antiretroviral therapy, including one or more NRTIs, for clinical indications at the time of delivery were enrolled. Maternal blood samples and amniotic fluid were obtained during delivery or cesarean sec...

  6. Reversible Inhibitors of Monoamine Oxidase-A (RIMAs): Robust, Reversible Inhibition of Human Brain MAO-A by CX157

    Science.gov (United States)

    Fowler, Joanna S; Logan, Jean; Azzaro, Albert J; Fielding, Robert M; Zhu, Wei; Poshusta, Amy K; Burch, Daniel; Brand, Barry; Free, James; Asgharnejad, Mahnaz; Wang, Gene-Jack; Telang, Frank; Hubbard, Barbara; Jayne, Millard; King, Payton; Carter, Pauline; Carter, Scott; Xu, Youwen; Shea, Colleen; Muench, Lisa; Alexoff, David; Shumay, Elena; Schueller, Michael; Warner, Donald; Apelskog-Torres, Karen

    2010-01-01

    Reversible inhibitors of monoamine oxidase-A (RIMA) inhibit the breakdown of three major neurotransmitters, serotonin, norepinephrine and dopamine, offering a multi-neurotransmitter strategy for the treatment of depression. CX157 (3-fluoro-7-(2,2,2-trifluoroethoxy)phenoxathiin-10,10-dioxide) is a RIMA, which is currently in development for the treatment of major depressive disorder. We examined the degree and reversibility of the inhibition of brain monoamine oxidase-A (MAO-A) and plasma CX157 levels at different times after oral dosing to establish a dosing paradigm for future clinical efficacy studies, and to determine whether plasma CX157 levels reflect the degree of brain MAO-A inhibition. Brain MAO-A levels were measured with positron emission tomography (PET) imaging and [11C]clorgyline in 15 normal men after oral dosing of CX157 (20–80 mg). PET imaging was conducted after single and repeated doses of CX157 over a 24-h time course. We found that 60 and 80 mg doses of CX157 produced a robust dose-related inhibition (47–72%) of [11C]clorgyline binding to brain MAO-A at 2 h after administration and that brain MAO-A recovered completely by 24 h post drug. Plasma CX157 concentration was highly correlated with the inhibition of brain MAO-A (EC50: 19.3 ng/ml). Thus, CX157 is the first agent in the RIMA class with documented reversible inhibition of human brain MAO-A, supporting its classification as a RIMA, and the first RIMA with observed plasma levels that can serve as a biomarker for the degree of brain MAO-A inhibition. These data were used to establish the dosing regimen for a current clinical efficacy trial with CX157. PMID:19890267

  7. Enhanced Chondrogenic Differentiation of Human Umbilical Cord Wharton's Jelly Derived Mesenchymal Stem Cells by GSK-3 Inhibitors.

    Directory of Open Access Journals (Sweden)

    Prapot Tanthaisong

    Full Text Available Articular cartilage is an avascular, alymphatic, and aneural system with very low regeneration potential because of its limited capacity for self-repair. Mesenchymal stem cells (MSCs are the preferred choice for cell-based therapies. Glycogen synthase kinase 3 (GSK-3 inhibitors are compounds that can induce the Wnt signaling pathway, which is involved in chondrogenesis and cartilage development. Here, we investigated the influence of lithium chloride (LiCl and SB216763 synergistically with TGF-β3 on chondrogenic differentiation in human mesenchymal stem cells derived from Wharton's jelly tissue (hWJ-MSCs. hWJ-MSCs were cultured and chondrogenic differentiation was induced in monolayer and pellet experiments using chondrogenic medium, chondrogenic medium supplemented with LiCl, or SB216763 for 4 weeks. After in vitro differentiation, cultured cells were examined for the expression of Sox9, ACAN, Col2a1, and β-catenin markers. Glycosaminoglycan (GAG accumulation was also examined by Alcian blue staining. The results indicated that SB216763 was more effective than LiCl as evidenced by a higher up-regulation of the expression of cartilage-specific markers, including Sox9, ACAN, Col2a1 as well as GAG accumulation. Moreover, collagen type II expression was strongly observed in cells cultured in the chondrogenic medium + SB216763 as evidenced by western blot analysis. Both treatments appeared to mediate the Wnt signaling pathway by up-regulating β-catenin gene expression. Further analyses showed that all treatments suppressed the progression of chondrocyte hypertrophy, determined by decreased expression of Col10a1 and Runx2. These results indicate that LiCl and SB216763 are potential candidates for further in vivo therapeutic trials and would be of great importance for cartilage regeneration.

  8. AT-406, an orally active antagonist of multiple inhibitor of apoptosis proteins, inhibits progression of human ovarian cancer.

    Science.gov (United States)

    Brunckhorst, Melissa K; Lerner, Dimitry; Wang, Shaomeng; Yu, Qin

    2012-07-01

    Ovarian carcinoma is the most deadly gynecological malignancy. Current chemotherapeutic drugs are only transiently effective and patients with advance disease often develop resistance despite significant initial responses. Mounting evidence suggests that anti-apoptotic proteins, including those of the inhibitor of apoptosis protein (IAP) family, play important roles in the chemoresistance. There has been a recent emergence of compounds that block the IAP functions. Here, we evaluated AT-406, a novel and orally active antagonist of multiple IAP proteins, in ovarian cancer cells as a single agent and in the combination with carboplatin for therapeutic efficacy and mechanism of action. We demonstrate that AT-406 has significant single agent activity in 60% of human ovarian cancer cell lines examined in vitro and inhibits ovarian cancer progression in vivo and that 3 out of 5 carboplatin-resistant cell lines are sensitive to AT-406, highlighting the therapeutic potential of AT-406 for patients with inherent or acquired platinum resistance. Additionally, our in vivo studies show that AT-406 enhances the carboplatin-induced ovarian cancer cell death and increases survival of the experimental mice, suggesting that AT-406 sensitizes the response of these cells to carboplatin. Mechanistically, we demonstrate that AT-406 induced apoptosis is correlated with its ability to down-regulate XIAP whereas AT-406 induces cIAP1 degradation in both AT-406 sensitive and resistance cell lines. Together, these results demonstrate, for the first time, the anti-ovarian cancer efficacy of AT-406 as a single agent and in the combination with carboplatin, suggesting that AT-406 has potential as a novel therapy for ovarian cancer patients, especially for patients exhibiting resistance to the platinum-based therapies.

  9. Proteasome inhibitor MG132 enhances the antigrowth and antimetastasis effects of radiation in human nonsmall cell lung cancer cells.

    Science.gov (United States)

    Liu, Jing; Shen, Wenhao; Tang, Yiting; Zhou, Jundong; Li, Ming; Zhu, Wei; Yang, Hongying; Wu, Jinchang; Zhang, Shuyu; Cao, Jianping

    2014-08-01

    The current treatment for advanced nonsmall cell lung cancer (NSCLC) remains unsatisfactory due to resistance to chemotherapy and ionizing radiation. The ubiquitin-proteasome system (UPS) regulates multiple cellular processes that are crucial for the proliferation and survival of all kinds of cells. Carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132), a specific and selective reversible inhibitor of the 26S proteasome, represents a novel approach for cancer therapy. However, whether MG132 can potentiate the effect of radiation against the growth and metastasis of NSCLC is not clear. We found that MG132 inhibited the proliferation of human NSCLC cell lines (A549 and H1299) in a dose- and time-dependent manner by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Then MG132 at a nontoxic dose (100 nM) was selected for following studies. Pretreatment of A549 and H1299 cells with 100 nM MG132 before ionizing radiation (IR) potentiated the anticancer effect of IR. Moreover, pretreatment with 100 nM MG132 before IR-enhanced radiation induced cell cycle arrest by decreased CyclinD1 but increased Wee1 expression in A549 and H1299 cells. In addition, pretreatment of MG132 combined with IR significantly suppressed cell migration and invasion abilities in NSCLC cell lines, which was accompanied by decreased expression of matrix metalloproteinase (MMP)-2 and MMP-9 in NSCLC cell lines. Taken together, our results demonstrate that MG132 enhances the antigrowth and antimetastatic effects of irradiation in NSCLC cells by modulating expression of cell cycle and invasion- related genes.

  10. New approaches of PARP-1 inhibitors in human lung cancer cells and cancer stem-like cells by some selected anthraquinone-derived small molecules.

    Directory of Open Access Journals (Sweden)

    Yu-Ru Lee

    Full Text Available Poly (ADP-ribose polymerase-1 (PARP-1 and telomerase, as well as DNA damage response pathways are targets for anticancer drug development, and specific inhibitors are currently under clinical investigation. The purpose of this work is to evaluate anticancer activities of anthraquinone-derived tricyclic and tetracyclic small molecules and their structure-activity relationships with PARP-1 inhibition in non-small cell lung cancer (NSCLC and NSCLC-overexpressing Oct4 and Nanog clone, which show high-expression of PARP-1 and more resistance to anticancer drug. We applied our library selected compounds to NCI's 60 human cancer cell-lines (NCI-60 in order to generate systematic profiling data. Based on our analysis, it is hypothesized that these drugs might be, directly and indirectly, target components to induce mitochondrial permeability transition and the release of pro-apoptotic factors as potential anti-NSCLC or PARP inhibitor candidates. Altogether, the most active NSC747854 showed its cytotoxicity and dose-dependent PARP inhibitory manner, thus it emerges as a promising structure for anti-cancer therapy with no significant negative influence on normal cells. Our studies present evidence that telomere maintenance should be taken into consideration in efforts not only to overcome drug resistance, but also to optimize the use of telomere-based therapeutics. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and telomerase inhibitors, in particular. Together, the data presented here expand our insight into the PARP inhibitors and support the resource-demanding lead optimization of structurally related small molecules for human cancer therapy.

  11. New Approaches of PARP-1 Inhibitors in Human Lung Cancer Cells and Cancer Stem-Like Cells by Some Selected Anthraquinone-Derived Small Molecules

    Science.gov (United States)

    Yu, Dah-Shyong; Huang, Kuo-Feng; Chou, Shih-Jie; Chen, Tsung-Chih; Lee, Chia-Chung; Chen, Chun-Liang; Chiou, Shih-Hwa; Huang, Hsu-Shan

    2013-01-01

    Poly (ADP-ribose) polymerase-1 (PARP-1) and telomerase, as well as DNA damage response pathways are targets for anticancer drug development, and specific inhibitors are currently under clinical investigation. The purpose of this work is to evaluate anticancer activities of anthraquinone-derived tricyclic and tetracyclic small molecules and their structure-activity relationships with PARP-1 inhibition in non-small cell lung cancer (NSCLC) and NSCLC-overexpressing Oct4 and Nanog clone, which show high-expression of PARP-1 and more resistance to anticancer drug. We applied our library selected compounds to NCI's 60 human cancer cell-lines (NCI-60) in order to generate systematic profiling data. Based on our analysis, it is hypothesized that these drugs might be, directly and indirectly, target components to induce mitochondrial permeability transition and the release of pro-apoptotic factors as potential anti-NSCLC or PARP inhibitor candidates. Altogether, the most active NSC747854 showed its cytotoxicity and dose-dependent PARP inhibitory manner, thus it emerges as a promising structure for anti-cancer therapy with no significant negative influence on normal cells. Our studies present evidence that telomere maintenance should be taken into consideration in efforts not only to overcome drug resistance, but also to optimize the use of telomere-based therapeutics. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and telomerase inhibitors, in particular. Together, the data presented here expand our insight into the PARP inhibitors and support the resource-demanding lead optimization of structurally related small molecules for human cancer therapy. PMID:23451039

  12. First-in-human trial of multikinase VEGF inhibitor regorafenib and anti-EGFR antibody cetuximab in advanced cancer patients

    OpenAIRE

    Subbiah, Vivek; Khawaja, Muhammad Rizwan; Hong, David S.; Amini, Behrang; Yungfang, Jiang; Liu, Hui; Johnson, Adrienne; Schrock, Alexa B.; Ali, Siraj M.; Sun, James X.; Fabrizio, David; Piha-Paul, Sarina; Fu, Siqing; Tsimberidou, Apostolia M.; Naing, Aung

    2017-01-01

    BACKGROUND. The combination of multikinase VEGF inhibitor regorafenib and anti-EGFR antibody cetuximab overcomes intrinsic and acquired resistance in both EGFR-sensitive and EGFR-resistant preclinical models of colorectal cancer (CRC).

  13. Trisubstituted purine inhibitors of PDGFR alpha and their antileukemic activity in the human eosinophilic cell line EOL-1

    Czech Academy of Sciences Publication Activity Database

    Malínková, Veronika; Řezníčková, Eva; Jorda, Radek; Gucký, T.; Kryštof, Vladimír

    2017-01-01

    Roč. 25, č. 24 (2017), s. 6523-6535 ISSN 0968-0896 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : dependent kinase inhibitors * src tyrosine kinase * 2,6,9-trisubstituted purines * therapeutic target * potent inhibitor * imatinib * leukemia * mutations * mutant * domain Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Hematology Impact factor: 2.930, year: 2016

  14. Human HDAC isoform selectivity achieved via exploitation of the acetate release channel with structurally unique small molecule inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Whitehead, Lewis; Dobler, Markus R.; Radetich, Branko; Zhu, Yanyi; Atadja, Peter W.; Claiborne, Tavina; Grob, Jonathan E.; McRiner, Andrew; Pancost, Margaret R.; Patnaik, Anup; Shao, Wenlin; Shultz, Michael; Tichkule, Ritesh; Tommasi, Ruben A.; Vash, Brian; Wang, Ping; Stams, Travis (Novartis)

    2013-11-20

    Herein we report the discovery of a family of novel yet simple, amino-acid derived class I HDAC inhibitors that demonstrate isoform selectivity via access to the internal acetate release channel. Isoform selectivity criteria is discussed on the basis of X-ray crystallography and molecular modeling of these novel inhibitors bound to HDAC8, potentially revealing insights into the mechanism of enzymatic function through novel structural features revealed at the atomic level.

  15. Identification of novel human dipeptidyl peptidase-IV inhibitors of natural origin (Part II: in silico prediction in antidiabetic extracts.

    Directory of Open Access Journals (Sweden)

    Laura Guasch

    Full Text Available BACKGROUND: Natural extracts play an important role in traditional medicines for the treatment of diabetes mellitus and are also an essential resource for new drug discovery. Dipeptidyl peptidase IV (DPP-IV inhibitors are potential candidates for the treatment of type 2 diabetes mellitus, and the effectiveness of certain antidiabetic extracts of natural origin could be, at least partially, explained by the inhibition of DPP-IV. METHODOLOGY/PRINCIPAL FINDINGS: Using an initial set of 29,779 natural products that are annotated with their natural source and an experimentally validated virtual screening procedure previously developed in our lab (Guasch et al.; 2012 [1], we have predicted 12 potential DPP-IV inhibitors from 12 different plant extracts that are known to have antidiabetic activity. Seven of these molecules are identical or similar to molecules with described antidiabetic activity (although their role as DPP-IV inhibitors has not been suggested as an explanation for their bioactivity. Therefore, it is plausible that these 12 molecules could be responsible, at least in part, for the antidiabetic activity of these extracts through their inhibitory effect on DPP-IV. In addition, we also identified as potential DPP-IV inhibitors 6 molecules from 6 different plants with no described antidiabetic activity but that share the same genus as plants with known antidiabetic properties. Moreover, none of the 18 molecules that we predicted as DPP-IV inhibitors exhibits chemical similarity with a group of 2,342 known DPP-IV inhibitors. CONCLUSIONS/SIGNIFICANCE: Our study identified 18 potential DPP-IV inhibitors in 18 different plant extracts (12 of these plants have known antidiabetic properties, whereas, for the remaining 6, antidiabetic activity has been reported for other plant species from the same genus. Moreover, none of the 18 molecules exhibits chemical similarity with a large group of known DPP-IV inhibitors.

  16. Bone marrow and tumor cell colony-forming units and human tumor xenograft efficacy of noncamptothecin and camptothecin topoisomerase I inhibitors.

    Science.gov (United States)

    Kurtzberg, Leslie S; Battle, Traci; Rouleau, Cecile; Bagley, Rebecca G; Agata, Naoki; Yao, Min; Schmid, Steven; Roth, Stephanie; Crawford, Jennifer; Krumbholz, Roy; Ewesuedo, Reginald; Yu, Xian-Jie; Wang, Fei; Lavoie, Edmond J; Teicher, Beverly A

    2008-10-01

    Topoisomerase I (TopoI), an established anticancer target, is an enzyme producing a single-strand DNA break during transcription. Several noncamptothecin TopoI inhibitors have been identified. One of these, ARC-111, was compared with two clinically used camptothecins, topotecan and irinotecan/SN-38. In mouse and human bone marrow colony formation [colony-forming units granulocyte-macrophage (CFU-GM)] assays, the IC(90) values were 519 and 331 nmol/L for topotecan and SN-38 mouse CFU-GM and were 19 and 26 nmol/L for human CFU-GM, giving mouse to human differentials of 28- and 13-fold. ARC-111 produced IC(90) values of 28 nmol/L in mouse and 6.2 nmol/L in human CFU-GM, thus only a 4.5-fold differential between species. Human bone marrow CFU-GM was more sensitive to topotecan than were several human cancer cell lines, but ARC-111 cytotoxicity was similar for human bone marrow CFU-GM and the seven human tumor cell lines tested. In HCT-116 xenografts, tumor growth delays (TGD) were 17 days for irinotecan and 20 days for ARC-111. In HT-29 xenografts, the TGD was 9 days for both irinotecan and ARC-111. Both ARC-111 and docetaxel had a TGD of 21 days in NCI-H460 xenografts, and both ARC-111 and gemcitabine had a TGD of 7 days in MiaPaCa2 xenograft. Current TopoI inhibitors have broad antitumor activity in human tumor xenografts that is not achieved in the clinic. This may be due to greater sensitivity of human bone marrow than mouse to the cytotoxicity of these agents. It may be possible to achieve similar levels of ARC-111 in patients as in mice allowing improved antitumor activity.

  17. Effects of inhibitors of DNA repair on the frequencies of chromosomal aberrations induced by x-rays or alkylating agents in cultured human lymphocytes

    International Nuclear Information System (INIS)

    Kihlman, B.A.; Andersson, H.C.

    1986-01-01

    In the first part of this presentation the authors give examples of the synergistic enhancements that are obtained with various inhibitor combinations in G/sub 2/. The second part of the presentation deals with the effects of two agents, also well known for their capacity to potentiate the frequency of chromosomal aberrations induced by physical and chemical agents, but with a different mechanism of action. These agents are caffeine and 3-aminobenzamide (3AB). Caffeine has for decades been used as an inhibitor of DNA repair although its mechanism of action has not been fully understood. 3AB has more recently come into focus as an efficient inhibitor of the synthesis of poly-(ADP-ribose), a substance believed to be of importance in connection with the repair of certain types of DNA damage. The results presented do not quite fit in with the general idea about the mode of action of these agents. All experiments were carried out with whole-blood cultures of human lymphocytes. When inhibitors were used as post-treatments, chromosomal aberrations were induced by X-rays or by the alkylating agents thiotepa (TT) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). X-rays were generated by a Siemens Stabilipan 200 apparatus, at a dose rate of 0.5 Gy/min. The tube (TR 200f) was operated at 180 kV, 10 mA and the radiation filtered through 4 mm Al

  18. Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma.

    Science.gov (United States)

    Calero, R; Morchon, E; Martinez-Argudo, I; Serrano, R

    2017-10-10

    Drug resistance by MAPK signaling recovery or activation of alternative signaling pathways, such as PI3K/AKT/mTOR, is an important factor that limits the long-term efficacy of targeted therapies in melanoma patients. In the present study, we investigated the phospho-proteomic profile of RTKs and its correlation with downstream signaling pathways in human melanoma. We found that tyrosine kinase receptors expression correlated with the expression of pivotal downstream components of the RAS/RAF/MAPK and PI3K/AKT/mTOR pathways in melanoma cell lines and tumors. We also found high expression of HSP90 and the PI3K/AKT/mTOR pathway proteins, 4EBP1 and AKT compared with healthy tissue and this correlated with poor overall survival of melanoma patients. The combination of the HSP90 inhibitor 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 showed a synergistic activity decreasing melanoma cell growth, inducing apoptosis and targeting simultaneously the MAPK and PI3K/AKT/mTOR pathways. These results demonstrate that the combination of HSP90 and PI3K/mTOR inhibitors could be an effective therapeutic strategy that target the main survival pathways in melanoma and must be considered to overcome resistance to BRAF inhibitors in melanoma patients. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Solution structure of the complex between poxvirus-encoded CC chemokine inhibitor vCCI and human MIP-1β

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Li; DeRider, Michele; McCornack, Milissa A.; Jao, Chris; Isern, Nancy G.; Ness, Traci; Moyer, Richard; Liwang, Patricia J.

    2006-09-19

    Chemokines (chemotactic cytokines) comprise a large family of proteins that recruit and activate leukocytes, giving chemokines a major role in both the immune response and inflammation-related diseases. The poxvirus-encoded viral CC chemokine inhibitor (vCCI) binds to many CC chemokines with high affinity, acting as a potent inhibitor of chemokine action. We have used heteronuclear multidimensional NMR to determine the first structure of an orthopoxvirus vCCI in complex with a human CC chemokine MIP-1β. vCCI binds to the chemokine with 1:1 stoichiometry, using residues from its β-sheet II to interact with the a surface of MIP-1β that includes the N-terminus, the following residues in the so-called N-loop20’s region, and the 40’s loop. This structure reveals a general strategy of vCCI for selective chemokine binding, as vCCI appears to interact most stronglyinteracts most directly with residues that are conserved among a subset of CC chemokines, but are not conservednot among the other chemokine subfamilies. This structure reveals a general strategy of vCCI for selective chemokine binding. Chemokines play critical roles in the immune system, causing chemotaxis of a variety of cells to sites of infection and inflammation, as well as mediating cell homing and immune system development 1(Baggiolini 2001). To date, about 50 chemokines have been identified, and these small proteins (7-14 kDa) are believed to function by binding with endothelial or matrix glycosaminoglycans to form a concentration gradient that is then sensed by high affinity, 7-transmembrane domain G-protein coupled chemokine receptors on the surface of immune cells surface. The chemokine system is critical for host defense in healthy individuals, butand can also lead to diseases including asthma, arthritis, and atherosclerosis in the case of malfunction, often due to inappropriate inflammation and subsequent tissue damage 2(Gerard and Rollins 2001). There are four subfamilies of chemokines, CC

  20. Biological and Molecular Effects of Small Molecule Kinase Inhibitors on Low-Passage Human Colorectal Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Falko Lange

    2014-01-01

    Full Text Available Low-passage cancer cell lines are versatile tools to study tumor cell biology. Here, we have employed four such cell lines, established from primary tumors of colorectal cancer (CRC patients, to evaluate effects of the small molecule kinase inhibitors (SMI vemurafenib, trametinib, perifosine, and regorafenib in an in vitro setting. The mutant BRAF (V600E/V600K inhibitor vemurafenib, but also the MEK1/2 inhibitor trametinib efficiently inhibited DNA synthesis, signaling through ERK1/2 and expression of genes downstream of ERK1/2 in BRAF mutant cells only. In case of the AKT inhibitor perifosine, three cell lines showed a high or intermediate responsiveness to the drug while one cell line was resistant. The multikinase inhibitor regorafenib inhibited proliferation of all CRC lines with similar efficiency and independent of the presence or absence of KRAS, BRAF, PIK3CA, and TP53 mutations. Regorafenib action was associated with broad-range inhibitory effects at the level of gene expression but not with a general inhibition of AKT or MEK/ERK signaling. In vemurafenib-sensitive cells, the antiproliferative effect of vemurafenib was enhanced by the other SMI. Together, our results provide insights into the determinants of SMI efficiencies in CRC cells and encourage the further use of low-passage CRC cell lines as preclinical models.

  1. Repurposing the clinically approved calcium antagonist manidipine dihydrochloride as a new early inhibitor of human cytomegalovirus targeting the Immediate-Early 2 (IE2) protein.

    Science.gov (United States)

    Mercorelli, Beatrice; Luganini, Anna; Celegato, Marta; Palù, Giorgio; Gribaudo, Giorgio; Loregian, Arianna

    2018-02-01

    Currently, there are no therapeutic alternatives to DNA polymerase inhibitors to treat human cytomegalovirus (HCMV) infections, a major threat for immunocompromised patients and pregnant women. Here, we explored the potential to repurpose manidipine dihydrochloride (MND), a calcium antagonist clinically approved to treat hypertension, as a new anti-HCMV agent. MND emerged in a previous drug repurposing screen to find early inhibitors of HCMV replication, and now we confirm that it inhibits in the low micromolar range the replication of different HCMV strains, including clinical isolates and viruses resistant to approved DNA polymerase inhibitors. The antiviral activity of MND is specific for HCMV over different both DNA and RNA viruses. Further experiments in HCMV-infected cells testing the effects of MND on viral DNA synthesis and viral proteins expression revealed that it halts the progression of the virus cycle prior to viral DNA replication and E genes expression, but after IE proteins expression. According to these results, we observed that the overall antiviral activity of MND involves a specific interference with the transactivating functions of the viral Immediate-Early 2 (IE-2) protein, an essential viral transcription factor required for the progression of HCMV replication. Given that the inhibitory concentration against HCMV is in the range of clinically relevant concentrations of MND in humans, and the mechanism of action differs from that of the other available therapeutics, this already approved drug is an attractive candidate for repurposing in alternative anti-HCMV therapeutic protocols. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Strong synergism between small molecule inhibitors of HER2, PI3K, mTOR and Bcl-2 in human breast cancer cells.

    Science.gov (United States)

    Hamunyela, Roswita H; Serafin, Antonio M; Akudugu, John M

    2017-02-01

    Targeting pro-survival cell signaling components has been promising in cancer therapy, but the benefit of targeting with single agents is limited. For malignancies such as triple-negative breast cancer, there is a paucity of targets that are amenable to existing interventions as they are devoid of the human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR), and estrogen receptor (ER). Concurrent targeting of cell signaling entities other than HER2, PR and ER with multiple agents may be more effective. Evaluating modes of interaction between agents can inform efficient selection of agents when used in cocktails. Using clonogenic cell survival, interaction between inhibitors of HER2 (TAK-165), phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) (NVP-BEZ235), and the pro-survival gene (Bcl-2) (ABT-263) in three human breast cell lines (MDA-MB-231, MCF-7 and MCF-12A) ranged from strong to very strong synergism. The strongest synergy was demonstrated in PR and ER negative cells. Inhibition of PI3K, mTOR and Bcl-2 could potentially be effective in the treatment of triple-negative cancers. The very strong synergy observed even at lowest concentrations of inhibitors indicates that these cocktails might be able to be used at a minimised risk of systemic toxicity. Concurrent use of multiple inhibitors can potentiate conventional interventions like radiotherapy and chemotherapy. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Discovering Bisdemethoxycurcumin from Curcuma longa rhizome as a potent small molecule inhibitor of human pancreatic α-amylase, a target for type-2 diabetes.

    Science.gov (United States)

    Ponnusamy, Sudha; Zinjarde, Smita; Bhargava, Shobha; Rajamohanan, P R; Ravikumar, Ameeta

    2012-12-15

    Curcuma longa rhizome is used extensively in culinary preparations in Far East and South-East Asia. Health benefits of curcuminoids from C. longa as antioxidants, anti-cancer and anti-inflammatory molecules have been well documented. We report here for the first time that Bisdemethoxycurcumin (BDMC) from C. longa, acts as an inhibitor to inactivate human pancreatic α-amylase, a therapeutic target for oral hypoglycemic agents in type-2 diabetes. Bioactivity guided isolation of rhizome isopropanol extract led to the identification by HPLC and NMR of BDMC as a lead small molecule inhibitor of porcine and human pancreatic α-amylase with an IC(50) value of 0.026 and 0.025 mM, respectively. Kinetic analysis revealed that using starch as the substrate, HPA exhibited an uncompetitive mode of inhibition with an apparent K(i) of 3.0 μM. The study gains importance as BDMC could be a good drug candidate in development of new inhibitors of HPA and of functional foods for controlling starch digestion in order to reduce post-prandial hyperglycemia. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Sulfur mustard-stimulated proteases and their inhibitors in a cultured normal human epidermal keratinocytes model: A potential approach for anti-vesicant drug development.

    Science.gov (United States)

    Jin, Xiannu; Ray, Radharaman; Ray, Prabhati

    2016-01-01

    Protease stimulation in cultured normal human epidermal keratinocytes (NHEK) due to sulfur mustard (SM) exposure is well documented. However, the specific protease(s) stimulated by SM and the protease substrates remain to be determined. In this study, we observed that SM stimulates several proteases and the epidermal-dermal attachment protein laminin-5 is one of the substrates. We propose that following SM exposure of the skin, laminin-5 degradation causes the detachment of the epidermis from the dermis and, therefore, vesication. We utilized gelatin zymography, Western blotting, immuno-fluorescence staining, and real-time polymerase chain reaction (RT-PCR) analyses to study the SM-stimulated proteases and laminin-5 degradation in NHEK. Two major protease bands (64 kDa and 72 kDa) were observed by zymography in SM-exposed cells. Addition of serine protease inhibitor (aprotinin, 100 μM), or the metalloprotease inhibitor (amastatin, 100 μM) to NHEK cultures prior to SM exposure decreased the SM-stimulated protease bands seen by zymography. These inhibitors completely or partially prevented SM-induced laminin-5 γ2 degradation as seen by Western blotting as well as immuno-fluorescence staining. Our results from Western blotting and RT-PCR studies also indicated that the membrane-type matrix metalloproteinase-1 (MT-MM-1) may be involved in SM-induced skin blistering. To summarize, our results in the NHEK model indicate the following: (a) SM stimulates multiple proteases including serine protease(s), and metalloproteases; (b) SM decreases the level of laminin-5 γ2, which is prevented by either a serine protease inhibitor or a metalloprotease inhibitor and (c) MT-MMP-1 maybe one of the proteases that is involved in skin blistering due to SM exposure.

  5. Sulfur mustard-stimulated proteases and their inhibitors in a cultured normal human epidermal keratinocytes model: A potential approach for anti-vesicant drug development

    Directory of Open Access Journals (Sweden)

    Xiannu Jin

    Full Text Available Protease stimulation in cultured normal human epidermal keratinocytes (NHEK due to sulfur mustard (SM exposure is well documented. However, the specific protease(s stimulated by SM and the protease substrates remain to be determined. In this study, we observed that SM stimulates several proteases and the epidermal-dermal attachment protein laminin-5 is one of the substrates. We propose that following SM exposure of the skin, laminin-5 degradation causes the detachment of the epidermis from the dermis and, therefore, vesication. We utilized gelatin zymography, Western blotting, immuno-fluorescence staining, and real-time polymerase chain reaction (RT-PCR analyses to study the SM-stimulated proteases and laminin-5 degradation in NHEK. Two major protease bands (64 kDa and 72 kDa were observed by zymography in SM-exposed cells. Addition of serine protease inhibitor (aprotinin, 100 μM, or the metalloprotease inhibitor (amastatin, 100 μM to NHEK cultures prior to SM exposure decreased the SM-stimulated protease bands seen by zymography. These inhibitors completely or partially prevented SM-induced laminin-5 γ2 degradation as seen by Western blotting as well as immuno-fluorescence staining. Our results from Western blotting and RT-PCR studies also indicated that the membrane-type matrix metalloproteinase-1 (MT-MM-1 may be involved in SM-induced skin blistering.To summarize, our results in the NHEK model indicate the following: (a SM stimulates multiple proteases including serine protease(s, and metalloproteases; (b SM decreases the level of laminin-5 γ2, which is prevented by either a serine protease inhibitor or a metalloprotease inhibitor and (c MT-MMP-1 maybe one of the proteases that is involved in skin blistering due to SM exposure. Keywords: Sulfur mustard, Serine protease, Metalloprotease, Protease inhibiter, Zymography, Laminin-5 γ2

  6. Gene expression levels of matrix metalloproteinases in human atherosclerotic plaques and evaluation of radiolabeled inhibitors as imaging agents for plaque vulnerability

    International Nuclear Information System (INIS)

    Müller, Adrienne; Krämer, Stefanie D.; Meletta, Romana; Beck, Katharina; Selivanova, Svetlana V.; Rancic, Zoran; Kaufmann, Philipp A.; Vos, Bernhard; Meding, Jörg; Stellfeld, Timo; Heinrich, Tobias K.; Bauser, Marcus; Hütter, Joachim; Dinkelborg, Ludger M.; Schibli, Roger; Ametamey, Simon M.

    2014-01-01

    Introduction: Atherosclerotic plaque rupture is the primary cause for myocardial infarction and stroke. During plaque progression macrophages and mast cells secrete matrix-degrading proteolytic enzymes, such as matrix metalloproteinases (MMPs). We studied levels of MMPs and tissue inhibitor of metalloproteinases-3 (TIMP-3) in relation to the characteristics of carotid plaques. We evaluated in vitro two radiolabeled probes targeting active MMPs towards non-invasive imaging of rupture-prone plaques. Methods: Human carotid plaques obtained from endarterectomy were classified into stable and vulnerable by visual and histological analysis. MMP-1, MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MMP-14, TIMP-3, and CD68 levels were investigated by quantitative polymerase chain reaction. Immunohistochemistry was used to localize MMP-2 and MMP-9 with respect to CD68-expressing macrophages. Western blotting was applied to detect their active forms. A fluorine-18-labeled MMP-2/MMP-9 inhibitor and a tritiated selective MMP-9 inhibitor were evaluated by in vitro autoradiography as potential lead structures for non-invasive imaging. Results: Gene expression levels of all MMPs and CD68 were elevated in plaques. MMP-1, MMP-9, MMP-12 and MMP-14 were significantly higher in vulnerable than stable plaques. TIMP-3 expression was highest in stable and low in vulnerable plaques. Immunohistochemistry revealed intensive staining of MMP-9 in vulnerable plaques. Western blotting confirmed presence of the active form in plaque lysates. In vitro autoradiography showed binding of both inhibitors to stable and vulnerable plaques. Conclusions: MMPs differed in their expression patterns among plaque phenotypes, providing possible imaging targets. The two tested MMP-2/MMP-9 and MMP-9 inhibitors may be useful to detect atherosclerotic plaques, but not the vulnerable lesions selectively

  7. Tissue Inhibitor of Metalloproteinase–3 (TIMP-3) induces FAS dependent apoptosis in human vascular smooth muscle cells

    Science.gov (United States)

    Ireland-Zecchini, Heather; Baker, Andrew H.; Littlewood, Trevor D.; Bennett, Martin R.; Murphy, Gillian

    2018-01-01

    Over expression of Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) in vascular smooth muscle cells (VSMCs) induces apoptosis and reduces neointima formation occurring after saphenous vein interposition grafting or coronary stenting. In studies to address the mechanism of TIMP-3-driven apoptosis in human VSMCs we find that TIMP-3 increased activation of caspase-8 and apoptosis was inhibited by expression of Cytokine response modifier A (CrmA) and dominant negative FAS-Associated protein with Death Domain (FADD). TIMP-3 induced apoptosis did not cause mitochondrial depolarisation, increase activation of caspase-9 and was not inhibited by over-expression of B-cell Lymphoma 2 (Bcl2), indicating a mitochondrial independent/type-I death receptor pathway. TIMP-3 increased levels of the First Apoptosis Signal receptor (FAS) and depletion of FAS with shRNA showed TIMP-3-induced apoptosis was FAS dependent. TIMP-3 induced formation of the Death-Inducing Signalling Complex (DISC), as detected by immunoprecipitation and by immunofluorescence. Cellular-FADD-like IL-1 converting enzyme-Like Inhibitory Protein (c-FLIP) localised with FAS at the cell periphery in the absence of TIMP-3 and this localisation was lost on TIMP-3 expression with c-FLIP adopting a perinuclear localisation. Although TIMP-3 inhibited FAS shedding, this did not increase total surface levels of FAS but instead increased FAS levels within localised regions at the cell surface. A Disintegrin And Metalloproteinase 17 (ADAM17) is inhibited by TIMP-3 and depletion of ADAM17 with shRNA significantly decreased FAS shedding. However ADAM17 depletion did not induce apoptosis or replicate the effects of TIMP-3 by increasing localised clustering of cell surface FAS. ADAM17-depleted cells could activate caspase-3 when expressing levels of TIMP-3 that were otherwise sub-apoptotic, suggesting a partial role for ADAM17 mediated ectodomain shedding in TIMP-3 mediated apoptosis. We conclude that TIMP-3 induced apoptosis

  8. The role of human equilibrative nucleoside transporter 1 on the cellular transport of the DNA methyltransferase inhibitors 5-azacytidine and CP-4200 in human leukemia cells.

    Science.gov (United States)

    Hummel-Eisenbeiss, Johanna; Hascher, Antje; Hals, Petter-Arnt; Sandvold, Marit Liland; Müller-Tidow, Carsten; Lyko, Frank; Rius, Maria

    2013-09-01

    The nucleoside analog 5-azacytidine is an archetypical drug for epigenetic cancer therapy, and its clinical effectiveness has been demonstrated in the treatment of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). However, therapy resistance in patients with MDS/AML remains a challenging issue. Membrane proteins that are involved in drug uptake are potential mediators of drug resistance. The responsible proteins for the transport of 5-azacytidine into MDS/AML cells are unknown. We have now systematically analyzed the expression and activity of various nucleoside transporters. We identified the human equilibrative nucleoside transporter 1 (hENT1) as the most abundant nucleoside transporter in leukemia cell lines and in AML patient samples. Transport assays using [¹⁴C]5-azacytidine demonstrated Na⁺-independent uptake of the drug into the cells, which was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a hENT1 inhibitor. The cellular toxicity of 5-azacytidine and its DNA demethylating activity were strongly reduced after hENT1 inhibition. In contrast, the cellular activity of the 5-azacytidine derivative 5-azacytidine-5'-elaidate (CP-4200), a nucleoside transporter-independent drug, persisted after hENT1 inhibition. A strong dependence of 5-azacytidine-induced DNA demethylation on hENT1 activity was also confirmed by array-based DNA methylation profiling, which uncovered hundreds of loci that became demethylated only when hENT1-mediated transport was active. Our data establish hENT1 as a key transporter for the cellular uptake of 5-azacytidine in leukemia cells and raise the possibility that hENT1 expression might be a useful biomarker to predict the efficiency of 5-azacytidine treatments. Furthermore, our data suggest that CP-4200 may represent a valuable compound for the modulation of transporter-related 5-azacytidine resistances.

  9. Angiomotin stabilization by tankyrase inhibitors antagonizes constitutive TEAD-dependent transcription and proliferation of human tumor cells with Hippo pathway core component mutations.

    Science.gov (United States)

    Troilo, Albino; Benson, Erica K; Esposito, Davide; Garibsingh, Rachel-Ann A; Reddy, E Premkumar; Mungamuri, Sathish Kumar; Aaronson, Stuart A

    2016-05-17

    The evolutionarily conserved Hippo inhibitory pathway plays critical roles in tissue homeostasis and organ size control, while mutations affecting certain core components contribute to tumorigenesis. Here we demonstrate that proliferation of Hippo pathway mutant human tumor cells exhibiting high constitutive TEAD transcriptional activity was markedly inhibited by dominant negative TEAD4, which did not inhibit the growth of Hippo wild-type cells with low levels of regulatable TEAD-mediated transcription. The tankyrase inhibitor, XAV939, identified in a screen for inhibitors of TEAD transcriptional activity, phenocopied these effects independently of its other known functions by stabilizing angiomotin and sequestering YAP in the cytosol. We also identified one intrinsically XAV939 resistant Hippo mutant tumor line exhibiting lower and less durable angiomotin stabilization. Thus, angiomotin stabilization provides a new mechanism for targeting tumors with mutations in Hippo pathway core components as well as a biomarker for sensitivity to such therapy.

  10. Synergy of amlodipine and angiotensin-converting enzyme inhibitors in regulating myocardial oxygen consumption in normal canine and failing human hearts.

    Science.gov (United States)

    Mital, S; Loke, K E; Slater, J P; Addonizio, L; Gersony, W M; Hintze, T H

    1999-06-17

    The production of endogenous nitric oxide, which regulates myocardial oxygen consumption, is decreased in heart failure. As with angiotensin-converting enzyme (ACE) inhibitors, amlodipine, a calcium antagonist, increases kinin-mediated nitric oxide production in coronary microvessels. We investigated the possibility of synergy between ACE inhibitors and amlodipine in regulating myocardial oxygen consumption. Left ventricular myocardium was isolated from 6 healthy dog hearts and 5 human hearts with end-stage heart failure at the time of orthotopic heart transplantation. Myocardial oxygen consumption was measured before and after administration of bradykinin, S-nitroso N-acetyl penicillamine (SNAP, a nitric oxide donor), ramiprilat (an ACE inhibitor), amlodipine, and the combination of a sub-threshold dose of ramiprilat (10(-8) md/L) + amlodipine. These experiments were repeated with L-nitro-arginine methyl ester (L-NAME, an inhibitor of nitric oxide synthesis), dichloroisocoumarin (an inhibitor of kinin synthesis), and HOE 140 (a B2 kinin-receptor antagonist). Baseline myocardial oxygen consumption in canine hearts was 182 +/- 21 nmol/g/min. Bradykinin and SNAP caused dose-dependent reductions in myocardial oxygen consumption (p <0.05). Ramiprilat and amlodipine caused a 10 +/- 3.2% and 11 +/- 0.8% reduction in myocardial oxygen consumption, respectively, when used alone (p <0.05). In the presence of a subthreshold dose of ramiprilat, amlodipine caused a larger (15 +/- 1.7%) reduction in myocardial oxygen consumption compared with either drug used alone (p <0.05). In human hearts, baseline myocardial oxygen consumption was 248 +/- 57 nmol/g/min. Amlodipine caused a larger reduction in myocardial oxygen consumption when used with ramiprilat (22 +/- 3.2%) as compared with amlodipine alone (15 +/- 2.6%). The effect of both drugs was attenuated by L-NAME, dichloroisocoumarin, and HOE 140 (p <0.05). In conclusion, ACE inhibitors and amlodipine act synergistically to

  11. 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....... At all reporter positions, fluorescence changes observed upon substrate application were distinctly different from those observed upon inhibitor application, with respect to relative amplitude or direction. Furthermore, escitalopram, fluoxetine, and cocaine induced a very similar pattern of fluorescent...

  12. Murinoglobulin, a novel protease inhibitor from murine plasma. Isolation, characterization, and comparison with murine alpha-macroglobulin and human alpha-2-macroglobulin.

    Science.gov (United States)

    Saito, A; Sinohara, H

    1985-01-25

    Two glycoproteins having trypsin-protein esterase activity were purified to apparent homogeneity from murine plasma. One was alpha-macroglobulin, a homologue of human alpha-2-macroglobulin, while the other, tentatively named murinoglobulin, did not correspond to any of the known plasma protease inhibitors that have been well characterized in men or other mammals. Murinoglobulin contained about 7.6% carbohydrate and was composed of a single-polypeptide chain of Mr = 180,000 as judged by the equilibrium sedimentation analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Murinoglobulin did not cross-react immunologically with mouse alpha-macroglobulin nor with human alpha-2-macroglobulin. Protease-inhibiting properties of murinoglobulin were compared with those of mouse alpha-macroglobulin and human alpha-2-macroglobulin. All the three proteins inhibited trypsin, papain, and thermolysin, although they differed considerably in both the degree of inhibition and the binding stoichiometry of protease-inhibitor complexes. The two macroglobulins inhibited pepsin at pH 5.5, whereas murinoglobulin was inactivated at this pH. Murinoglobulin was more sensitive to methylamine than the two macroglobulins. No protein corresponding to murinoglobulin was detected in human plasma.

  13. Targeting nitric oxide signaling with nNOS inhibitors as a novel strategy for the therapy and prevention of human melanoma.

    Science.gov (United States)

    Yang, Zhen; Misner, Bobbye; Ji, Haitao; Poulos, Thomas L; Silverman, Richard B; Meyskens, Frank L; Yang, Sun

    2013-08-10

    Our previous studies have shown that nitric oxide (NO) plays an important role in increasing the invasion and proliferation of human melanoma cells, suggesting that targeting NO signaling may facilitate therapy and prevention. Neuronal nitric oxide synthase (nNOS) is present in melanocytes, a cell type that originates from the neural crest. The aims of this study were to determine the role of nNOS in melanoma progression and the potential antitumor effects of novel synthesized nNOS inhibitors. In vitro studies demonstrated abundant expression of nNOS in melanoma compared to melanocytes, which was inducible by ultraviolet radiation and was associated with increased NO generation. nNOS was also detected in melanoma biopsies that increased with disease stage. Knockdown of nNOS in melanoma cells diminished L-arginine-induced NO production; the metastatic capacity was also reduced as well as the levels of MMP-1, Bcl-2, JunD, and APE/Ref-1. Similar inhibition of NO and invasion potential was observed utilizing novel, highly selective nNOS inhibitors. In three-dimensional human skin reconstructs, the nNOS inhibitor cpd8 effectively reversed the melanoma overgrowth stimulated by NO stress. Our work lays the foundation for development of clinical "drug-like" nNOS inhibitors as a new and promising strategy for the chemoprevention of early melanoma progression and the inhibition of secondary melanoma in high-risk individuals. Based on our observations, we propose that nNOS in melanoma results in constitutive overproduction of NO, which stimulates proliferation and increases invasion potential, leading to subsequent development of metastases.

  14. The Human Tyrosyl-DNA Phosphodiesterase 1 (hTdp1) Inhibitor NSC120686 as an Exploratory Tool to Investigate Plant Tdp1 Genes.

    Science.gov (United States)

    Macovei, Anca; Pagano, Andrea; Sabatini, Maria Elisa; Grandi, Sofia; Balestrazzi, Alma

    2018-03-28

    The hTdp1 (human tyrosyl-DNA phosphodiesterase 1) inhibitor NSC120686 has been used, along with topoisomerase inhibitors, as a pharmacophoric model to restrain the Tdp1 activity as part of a synergistic treatment for cancer. While this compound has an end-point application in medical research, in plants, its application has not been considered so far. The originality of our study consists in the use of hTdp1 inhibitor in Medicago truncatula cells, which, unlike human cells, contain two Tdp1 genes. Hence, the purpose of this study was to test the hTdp1 inhibitor NSC120686 as an exploratory tool to investigate the plant Tdp1 genes, since their characterization is still in incipient phases. To do so, M. truncatula calli were exposed to increasing (75, 150, 300 μM) concentrations of NSC120686. The levels of cell mortality and DNA damage, measured via diffusion assay and comet assay, respectively, were significantly increased when the highest doses were used, indicative of a cytotoxic and genotoxic threshold. In addition, the NSC120686-treated calli and untreated MtTdp1α -depleted calli shared a similar response in terms of programmed cell death (PCD)/necrosis and DNA damage. Interestingly, the expression profiles of MtTdp1α and MtTdp1β genes were differently affected by the NSC120686 treatment, as MtTdp1α was upregulated while MtTdp1β was downregulated. The NSC120686 treatment affected not only the MtTdp1 genes but also other genes with roles in alternative DNA repair pathways. Since the expression patterns of these genes were different than what was observed in the MtTdp1α -depleted plants, it could be hypothesized that the NSC120686 treatment exerts a different influence compared to that resulting from the lack of the MtTdp1α gene function.

  15. The pan-ErbB tyrosine kinase inhibitor canertinib induces caspase-mediated cell death in human T-cell leukemia (Jurkat) cells

    Energy Technology Data Exchange (ETDEWEB)

    Trinks, Cecilia, E-mail: Cecilia.trinks@liu.se [Division of Oncology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Severinsson, Emelie A., E-mail: Emelie.severinsson@liu.se [Division of Oncology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Holmlund, Birgitta, E-mail: Birgitta.holmlund@lio.se [Department of Oncology, County Council of Ostergoetland, Linkoeping (Sweden); Green, Anna, E-mail: Anna.green@liu.se [Division of Cell Biology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Green, Henrik, E-mail: Henrik.green@liu.se [Clinical Pharmacology, Division of Drug Research, Department of Medical and Health Sciences, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Joensson, Jan-Ingvar, E-mail: Jan-ingvar.jonsson@liu.se [Experimental Hematology Unit, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Hallbeck, Anna-Lotta, E-mail: Anna-Lotta.Hallbeck@lio.se [Division of Oncology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Department of Oncology, County Council of Ostergoetland, Linkoeping (Sweden); Walz, Thomas M., E-mail: Thomas.Walz@lio.se [Division of Oncology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Department of Oncology, County Council of Ostergoetland, Linkoeping (Sweden)

    2011-07-08

    Highlights: {yields} Canertinib induces caspase-mediated apoptosis in T-cell leukemia cells in vitro. {yields} Canertinib mediates activation of the intrinsic apoptotic pathway. {yields} Canertinib induces apoptosis in an ErbB receptor independent manner. {yields} Lymphocyte specific proteins as well as survival kinases are inhibited. {yields} Canertinib may act as a multi-kinase inhibiting drug in human T-cell malignancies. -- Abstract: Canertinib is a novel ErbB-receptor inhibitor currently in clinical development for the treatment of solid tumors overexpressing ErbB-receptors. We have recently demonstrated that canertinib displays anti-proliferative and pro-apoptotic effects in human myeloid leukemia cells devoid of ErbB-receptors. The mechanism mediating these effects are however unknown. In this study, we show that canertinib is able to act as a multi-kinase inhibitor by inhibition of several intracellular kinases involved in T-cell signaling such as Akt, Erk1/2 and Zap-70, and reduced Lck protein expression in the human T-cell leukemia cell line Jurkat. Treatment with canertinib at a concentration of 2 {mu}M caused accumulation of Jurkat cells in the G{sub 1} cell cycle phase and increased doses induced apoptosis in a time-dependent manner. Apoptotic signs of treated cells were detected by Annexin V staining and cleavage of PARP, caspase-3, -8, -9, -10 and Bid. A subset of the pro-apoptotic signals mediated by canertinib could be significantly reduced by specific caspase inhibitors. Taken together, these results demonstrate the dual ability of canertinib to downregulate important signaling pathways and to activate caspase-mediated intrinsic apoptosis pathway in human T-cell leukemia cells.

  16. Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells.

    Directory of Open Access Journals (Sweden)

    Luyuan Li

    Full Text Available Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2 were recently described in several cancers including chondrosarcomas. The IDH1 inhibitor AGI-5198 abrogates the ability of mutant IDH1 to produce the oncometabolite D-2 hydroxyglutarate (D-2HG in gliomas. We sought to determine if treatment with AGI-5198 would similarly inhibit tumorigenic activity and D-2HG production in IDH1-mutant human chondrosarcoma cells. Two human chondrosarcoma cell lines, JJ012 and HT1080 with endogenous IDH1 mutations and a human chondrocyte cell line C28 with wild type IDH1 were employed in our study. Mutation analysis of IDH was performed by PCR-based DNA sequencing, and D-2HG was detected using tandem mass spectrometry. We confirmed that JJ012 and HT1080 harbor IDH1 R132G and R132C mutation, respectively, while C28 has no mutation. D-2HG was detectable in cell pellets and media of JJ012 and HT1080 cells, as well as plasma and urine from an IDH-mutant chondrosarcoma patient, which decreased after tumor resection. AGI-5198 treatment decreased D-2HG levels in JJ012 and HT1080 cells in a dose-dependent manner, and dramatically inhibited colony formation and migration, interrupted cell cycling, and induced apoptosis. In conclusion, our study demonstrates anti-tumor activity of a mutant IDH1 inhibitor in human chondrosarcoma cell lines, and suggests that D-2HG is a potential biomarker for IDH mutations in chondrosarcoma cells. Thus, clinical trials of mutant IDH inhibitors are warranted for patients with IDH-mutant chondrosarcomas.

  17. The S-enantiomer of R,S-citalopram, increases inhibitor binding to the human serotonin transporter by an allosteric mechanism. Comparison with other serotonin transporter inhibitors

    DEFF Research Database (Denmark)

    Chen, Fenghua; Larsen, Mads Breum; Sánchez, Connie

    2005-01-01

    The interaction of the S- and R-enantiomers (escitalopram and R-citalopram) of citalopram, with high- and low-affinity binding sites in COS-1 cell membranes expressing human SERT (hSERT) were investigated. Escitalopram affinity for hSERT and its 5-HT uptake inhibitory potency was in the nanomolar...... range and approximately 40-fold more potent than R-citalopram. Escitalopram considerably stabilised the [3H]-escitalopram/SERT complex via an allosteric effect at a low-affinity binding site. The stereoselectivity between escitalopram and R-citalopram was approximately 3:1 for the [3H]-escitalopram....../hSERT complex. The combined effect of escitalopram and R-citalopram was additive. Paroxetine and sertraline mainly stabilised the [3H]-paroxetine/hSERT complex. Fluoxetine, duloxetine and venlafaxine have only minor effects. 5-HT stabilised the [125I]-RTI-55, [3H]-MADAM, [3H]-paroxetine, [3H]-fluoxetine and [3H...

  18. Joint bleeds increase the inhibitor response to human factor VIII in a rat model of severe haemophilia A

    DEFF Research Database (Denmark)

    Löfgren, Karin Maria; Søndergaard, H.; Skov, Søren

    2016-01-01

    Introduction The most serious complication in haemophilia A (HA) replacement therapy with coagulation factor VIII (FVIII) is neutralizing antibodies, i.e. inhibitors. It has been hypothesized that danger signals generated during a bleed might have an adjuvant effect on the immune response to FVIII...

  19. S1 pocket fingerprints of human and bacterial methionine aminopeptidases determined using fluorogenic libraries of substrates and phosphorus based inhibitors

    Czech Academy of Sciences Publication Activity Database

    Poreba, M.; Gajda, A.; Pícha, Jan; Jiráček, Jiří; Marschner, A.; Klein, Ch. D.; Salvesen, G. S.; Drag, M.

    2012-01-01

    Roč. 94, č. 3 (2012), s. 704-710 ISSN 0300-9084 R&D Projects: GA MŠk(CZ) LC06077 Institutional research plan: CEZ:AV0Z40550506 Keywords : methionine aminopeptidase * substrate library * protease * enzyme * inhibitor * substrate specificity Subject RIV: CC - Organic Chemistry Impact factor: 3.142, year: 2012

  20. Combining combinatorial chemistry and affinity chromatography: highly selective inhibitors of human betaine:homocysteine S-methyltransferase

    Czech Academy of Sciences Publication Activity Database

    Collinsová, Michaela; Castro, C.; Garrow, T. A.; Yiotakis, A.; Dive, V.; Jiráček, Jiří

    2003-01-01

    Roč. 10, - (2003), s. 113-122 ISSN 1074-5521 R&D Projects: GA AV ČR IAB4055003 Institutional research plan: CEZ:AV0Z4055905 Keywords : BHMT * inhibitor * phosphinic Subject RIV: CE - Biochemistry Impact factor: 6.129, year: 2003

  1. Structure based classification for bile salt export pump (BSEP) inhibitors using comparative structural modeling of human BSEP

    Science.gov (United States)

    Jain, Sankalp; Grandits, Melanie; Richter, Lars; Ecker, Gerhard F.

    2017-06-01

    The bile salt export pump (BSEP) actively transports conjugated monovalent bile acids from the hepatocytes into the bile. This facilitates the formation of micelles and promotes digestion and absorption of dietary fat. Inhibition of BSEP leads to decreased bile flow and accumulation of cytotoxic bile salts in the liver. A number of compounds have been identified to interact with BSEP, which results in drug-induced cholestasis or liver injury. Therefore, in silico approaches for flagging compounds as potential BSEP inhibitors would be of high value in the early stage of the drug discovery pipeline. Up to now, due to the lack of a high-resolution X-ray structure of BSEP, in silico based identification of BSEP inhibitors focused on ligand-based approaches. In this study, we provide a homology model for BSEP, developed using the corrected mouse P-glycoprotein structure (PDB ID: 4M1M). Subsequently, the model was used for docking-based classification of a set of 1212 compounds (405 BSEP inhibitors, 807 non-inhibitors). Using the scoring function ChemScore, a prediction accuracy of 81% on the training set and 73% on two external test sets could be obtained. In addition, the applicability domain of the models was assessed based on Euclidean distance. Further, analysis of the protein-ligand interaction fingerprints revealed certain functional group-amino acid residue interactions that could play a key role for ligand binding. Though ligand-based models, due to their high speed and accuracy, remain the method of choice for classification of BSEP inhibitors, structure-assisted docking models demonstrate reasonably good prediction accuracies while additionally providing information about putative protein-ligand interactions.

  2. A preclinical orthotopic model for glioblastoma recapitulates key features of human tumors and demonstrates sensitivity to a combination of MEK and PI3K pathway inhibitors

    Directory of Open Access Journals (Sweden)

    Rajaa El Meskini

    2015-01-01

    Full Text Available Current therapies for glioblastoma multiforme (GBM, the highest grade malignant brain tumor, are mostly ineffective, and better preclinical model systems are needed to increase the successful translation of drug discovery efforts into the clinic. Previous work describes a genetically engineered mouse (GEM model that contains perturbations in the most frequently dysregulated networks in GBM (driven by RB, KRAS and/or PI3K signaling and PTEN that induce development of Grade IV astrocytoma with properties of the human disease. Here, we developed and characterized an orthotopic mouse model derived from the GEM that retains the features of the GEM model in an immunocompetent background; however, this model is also tractable and efficient for preclinical evaluation of candidate therapeutic regimens. Orthotopic brain tumors are highly proliferative, invasive and vascular, and express histology markers characteristic of human GBM. Primary tumor cells were examined for sensitivity to chemotherapeutics and targeted drugs. PI3K and MAPK pathway inhibitors, when used as single agents, inhibited cell proliferation but did not result in significant apoptosis. However, in combination, these inhibitors resulted in a substantial increase in cell death. Moreover, these findings translated into the in vivo orthotopic model: PI3K or MAPK inhibitor treatment regimens resulted in incomplete pathway suppression and feedback loops, whereas dual treatment delayed tumor growth through increased apoptosis and decreased tumor cell proliferation. Analysis of downstream pathway components revealed a cooperative effect on target downregulation. These concordant results, together with the morphologic similarities to the human GBM disease characteristics of the model, validate it as a new platform for the evaluation of GBM treatment.

  3. Reversible dual inhibitor against G9a and DNMT1 improves human iPSC derivation enhancing MET and facilitating transcription factor engagement to the genome.

    Directory of Open Access Journals (Sweden)

    Juan Roberto Rodriguez-Madoz

    Full Text Available The combination of defined factors with small molecules targeting epigenetic factors is a strategy that has been shown to enhance optimal derivation of iPSCs and could be used for disease modelling, high throughput screenings and/or regenerative medicine applications. In this study, we showed that a new first-in-class reversible dual G9a/DNMT1 inhibitor compound (CM272 improves the efficiency of human cell reprogramming and iPSC generation from primary cells of healthy donors and patient samples, using both integrative and non-integrative methods. Moreover, CM272 facilitates the generation of human iPSC with only two factors allowing the removal of the most potent oncogenic factor cMYC. Furthermore, we demonstrated that mechanistically, treatment with CM272 induces heterochromatin relaxation, facilitates the engagement of OCT4 and SOX2 transcription factors to OSKM refractory binding regions that are required for iPSC establishment, and enhances mesenchymal to epithelial transition during the early phase of cell reprogramming. Thus, the use of this new G9a/DNMT reversible dual inhibitor compound may represent an interesting alternative for improving cell reprogramming and human iPSC derivation for many different applications while providing interesting insights into reprogramming mechanisms.

  4. Reversible dual inhibitor against G9a and DNMT1 improves human iPSC derivation enhancing MET and facilitating transcription factor engagement to the genome.

    Science.gov (United States)

    Rodriguez-Madoz, Juan Roberto; San Jose-Eneriz, Edurne; Rabal, Obdulia; Zapata-Linares, Natalia; Miranda, Estibaliz; Rodriguez, Saray; Porciuncula, Angelo; Vilas-Zornoza, Amaia; Garate, Leire; Segura, Victor; Guruceaga, Elizabeth; Agirre, Xabier; Oyarzabal, Julen; Prosper, Felipe

    2017-01-01

    The combination of defined factors with small molecules targeting epigenetic factors is a strategy that has been shown to enhance optimal derivation of iPSCs and could be used for disease modelling, high throughput screenings and/or regenerative medicine applications. In this study, we showed that a new first-in-class reversible dual G9a/DNMT1 inhibitor compound (CM272) improves the efficiency of human cell reprogramming and iPSC generation from primary cells of healthy donors and patient samples, using both integrative and non-integrative methods. Moreover, CM272 facilitates the generation of human iPSC with only two factors allowing the removal of the most potent oncogenic factor cMYC. Furthermore, we demonstrated that mechanistically, treatment with CM272 induces heterochromatin relaxation, facilitates the engagement of OCT4 and SOX2 transcription factors to OSKM refractory binding regions that are required for iPSC establishment, and enhances mesenchymal to epithelial transition during the early phase of cell reprogramming. Thus, the use of this new G9a/DNMT reversible dual inhibitor compound may represent an interesting alternative for improving cell reprogramming and human iPSC derivation for many different applications while providing interesting insights into reprogramming mechanisms.

  5. Vorinostat, an HDAC inhibitor attenuates epidermoid squamous cell carcinoma growth by dampening mTOR signaling pathway in a human xenograft murine model.

    Science.gov (United States)

    Kurundkar, Deepali; Srivastava, Ritesh K; Chaudhary, Sandeep C; Ballestas, Mary E; Kopelovich, Levy; Elmets, Craig A; Athar, Mohammad

    2013-01-15

    Histone deacetylase (HDAC) inhibitors are potent anticancer agents and show efficacy against various human neoplasms. Vorinostat is a potent HDAC inhibitor and has shown potential to inhibit growth of human xenograft tumors. However, its effect on the growth of skin neoplasm remains undefined. In this study, we show that vorinostat (2 μM) reduced expression of HDAC1, 2, 3, and 7 in epidermoid carcinoma A431 cells. Consistently, it increased acetylation of histone H3 and p53. Vorinostat (100mg/kg body weight, IP) treatment reduced human xenograft tumor growth in highly immunosuppressed nu/nu mice. Histologically, the vorinostat-treated tumor showed features of well-differentiation with large necrotic areas. Based on proliferating cell nuclear antigen (PCNA) staining and expression of cyclins D1, D2, E, and A, vorinostat seems to impair proliferation by down-regulating the expression of these proteins. However, it also induced apoptosis. The mechanism by which vorinostat blocks proliferation and makes tumor cells prone to apoptosis, involved inhibition of mTOR signaling which was accompanied by reduction in cell survival AKT and extracellular-signal regulated kinase (ERK) signaling pathways. Our data provide a novel mechanism-based therapeutic intervention for cutaneous squamous cell carcinoma (SCC). Vorinostat may be utilized to cure skin neoplasms in organ transplant recipient (OTR). These patients have high morbidity and surgical removal of these lesions which frequently develop in these patients, is difficult. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Backbone and side-chain 1H, 13C and 15N assignments of the ubiquitin-associated domain of human X-linked inhibitor of apoptosis protein

    Science.gov (United States)

    Hui, Sin-Kam; Tse, Man-Kit; Yang, Yinhua; Wong, Benjamin Chun-Yu

    2009-01-01

    X-linked inhibitor of apoptosis protein (XIAP), a leading member of the family of inhibitor of apoptosis (IAP) proteins, is considered as the most potent and versatile inhibitor of caspases and apoptosis. It has been reported that XIAP is frequently overexpressed in cancer and its expression level is implicated in contributing to tumorigenesis, disease progression, chemoresistance and poor patient-survival. Therefore, XIAP is one of the leading targets in drug development for cancer therapy. Recently, based on bioinformatics study, a previously unrecognized but evolutionarily conserved ubiquitin-associated (UBA) domain in IAPs was identified. The UBA domain is found to be essential for the oncogenic potential of IAP, to maintain endothelial cell survival and to protect cells from TNF-α-induced apoptosis. Moreover, the UBA domain is required for XIAP to activate NF-κB. In the present study, we report the near complete resonance assignments of the UBA domain-containing region of human XIAP protein. Secondary structure prediction based on chemical shift index (CSI) analysis reveals that the protein is predominately α-helical, which is consistent with the structures of known UBA proteins. PMID:19916060

  7. Apoptosis-related molecular differences for response to tyrosin kinase inhibitors in drug-sensitive and drug-resistant human bladder cancer cells

    Directory of Open Access Journals (Sweden)

    Jixia Li

    2013-01-01

    Full Text Available Context: The epidermal growth factor receptor (EGFR family is reportedly overexpressed in bladder cancer, and tyrosine kinaseinhibitors (TKIs have been suggested as treatment. Gefitinib is a selective inhibitor of the EGFR and lapatinib is a dual inhibitor of both the EGFR and HER2 (human EGFR type 2 receptor. Both compounds compete with the binding of adenosine triphosphate (ATP to the tyrosine kinase domain of the respective receptors to inhibit receptor autophosphorylation causing suppression of signal transduction. Unfortunately, resistance to these inhibitors is a major clinical problem. Aims: To compare the apoptosis signaling pathway(s induced by gefitinib and lapatinib, in UM-UC-5 (drug-sensitive and UM-UC-14 (drug-resistant bladder cancer cells and to identify molecular differences that might be useful predictors of their efficacy. Materials and Methods: Cell proliferation, cell cycle and apoptosis assay were used to detect the effect of TKIs on UM-UC-5 and UM-UC-14 cells. Molecular differences for response to TKIs were examined by protein array. Results: TKIs strongly inhibited cell proliferation and induced cell cycle G1 arrest and apoptosis in UM-UC-5 cells. Most notable apoptosis molecular differences included decreased claspin, trail, and survivin by TKIs in the sensitive cells. In contrast, TKIs had no effect on resistant cells. Conclusions: Claspin, trail, and survivin might be used to determine the sensitivity of bladder cancers to TKIs.

  8. Substrate Specificity, Inhibitor Selectivity and Structure-Function Relationships of Aldo-Keto Reductase 1B15: A Novel Human Retinaldehyde Reductase.

    Directory of Open Access Journals (Sweden)

    Joan Giménez-Dejoz

    Full Text Available Human aldo-keto reductase 1B15 (AKR1B15 is a newly discovered enzyme which shares 92% amino acid sequence identity with AKR1B10. While AKR1B10 is a well characterized enzyme with high retinaldehyde reductase activity, involved in the development of several cancer types, the enzymatic activity and physiological role of AKR1B15 are still poorly known. Here, the purified recombinant enzyme has been subjected to substrate specificity characterization, kinetic analysis and inhibitor screening, combined with structural modeling. AKR1B15 is active towards a variety of carbonyl substrates, including retinoids, with lower kcat and Km values than AKR1B10. In contrast to AKR1B10, which strongly prefers all-trans-retinaldehyde, AKR1B15 exhibits superior catalytic efficiency with 9-cis-retinaldehyde, the best substrate found for this enzyme. With ketone and dicarbonyl substrates, AKR1B15 also shows higher catalytic activity than AKR1B10. Several typical AKR inhibitors do not significantly affect AKR1B15 activity. Amino acid substitutions clustered in loops A and C result in a smaller, more hydrophobic and more rigid active site in AKR1B15 compared with the AKR1B10 pocket, consistent with distinct substrate specificity and narrower inhibitor selectivity for AKR1B15.

  9. Curcumin binds in silico to anti-cancer drug target enzyme MMP-3 (human stromelysin-1) with affinity comparable to two known inhibitors of the enzyme.

    Science.gov (United States)

    Jerah, Ahmed; Hobani, Yahya; Kumar, B Vinod; Bidwai, Anil

    2015-01-01

    In silico interaction of curcumin with the enzyme MMP-3 (human stromelysin-1) was studied by molecular docking using AutoDock 4.2 as the docking software application. AutoDock 4.2 software serves as a valid and acceptable docking application to study the interactions of small compounds with proteins. Interactions of curcumin with MMP-3 were compared to those of two known inhibitors of the enzyme, PBSA and MPPT. The calculated free energy of binding (ΔG binding) shows that curcumin binds with affinity comparable to or better than the two known inhibitors. Binding interactions of curcumin with active site residues of the enzyme are also predicted. Curcumin appears to bind in an extendended conformation making extensive VDW contacts in the active site of the enzyme. Hydrogen bonding and pi-pi interactions with key active site residues is also observed. Thus, curcumin can be considered as a good lead compound in the development of new inhibitors of MMP-3 which is a potential target of anticancer drugs. The results of these studies can serve as a starting point for further computational and experimental studies.

  10. A miR-21 inhibitor enhances apoptosis and reduces G2-M accumulation induced by ionizing radiation in human glioblastoma U251 cells

    International Nuclear Information System (INIS)

    Li, Yi; Li, Qiang; Asai, Akio; Kawamoto, Keiji; Zhao Shiguang; Zhen Yunbo; Teng Lei

    2011-01-01

    MicroRNAs (miRNAs) are small noncoding RNAs that take part in diverse biological processes by suppressing target gene expression. Elevated expression of miR-21 has been reported in many types of human cancers. Radiotherapy is a standard adjuvant treatment for patients with glioblastoma. However, the resistance of glioblastoma cells to radiation limits the success of this treatment. In this study, we found that miR-21 expression was upregulated in response to ionizing radiation (IR) in U251 cells, which suggested that miR-21 could be involved in the response of U251 cells to radiation. We showed that a miR-21 inhibitor enhanced IR-induced glioblastoma cell growth arrest and increased the level of apoptosis, which was probably caused by abrogation of the G 2 -M arrest induced by IR. Further research demonstrated that the miR-21 inhibitor induced the upregulation of Cdc25A. Taken together, these findings suggest that miR-21 inhibitor can increase IR-induced growth arrest and apoptosis in U251 glioblastoma cells, at least in part by abrogating G 2 -M arrest, and that Cdc25A is a potential target of miR-21. (author)

  11. Improved, selective, human intestinal carboxylesterase inhibitors designed to modulate 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (irinotecan; CPT-11) toxicity

    OpenAIRE

    Hicks, Latorya D.; Hyatt, Janice L; Stoddard, Shana; Tsurkan, Lyudmila; Edwards, Carol C.; Wadkins, Randy M.; Potter, Philip M.

    2009-01-01

    CPT-11 is an antitumor prodrug that is hydrolyzed by carboxylesterases (CE) to yield SN-38, a potent topoisomerase I poison. However, the dose limiting toxicity is delayed diarrhea that is thought to arise, in part, from activation of the prodrug by a human intestinal CE (hiCE). Therefore, we have sought to identify selective inhibitors of hiCE that may have utility in modulating drug toxicity. We have evaluated one such class of molecules (benzene sulfonamides), and developed QSAR models for...

  12. Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

    International Nuclear Information System (INIS)

    Gilmour, Peter S.; O'Shea, Patrick J.; Fagura, Malbinder; Pilling, James E.; Sanganee, Hitesh; Wada, Hiroki; Courtney, Paul F.; Kavanagh, Stefan; Hall, Peter A.; Escott, K. Jane

    2013-01-01

    Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging. GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH 1–34 or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption. - Highlights: • Wnt modulation with 3 novel GSK-3 inhibitors alters bone growth. • Human stem cell osteoblastogenesis and

  13. Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

    Energy Technology Data Exchange (ETDEWEB)

    Gilmour, Peter S., E-mail: Peter.Gilmour@astrazeneca.com [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); O' Shea, Patrick J.; Fagura, Malbinder [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Pilling, James E. [Discovery Sciences, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Sanganee, Hitesh [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Wada, Hiroki [R and I IMed, AstraZeneca R and D, Molndal (Sweden); Courtney, Paul F. [DMPK, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Kavanagh, Stefan; Hall, Peter A. [Safety Assessment, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom); Escott, K. Jane [New Opportunities Innovative Medicines group, AstraZeneca R and D, Alderley Park, Cheshire SK10 4TF (United Kingdom)

    2013-10-15

    Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging. GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH{sub 1–34} or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption. - Highlights: • Wnt modulation with 3 novel GSK-3 inhibitors alters bone growth. • Human stem cell osteoblastogenesis

  14. Protecting Gram-negative bacterial cell envelopes from human lysozyme: Interactions with Ivy inhibitor proteins from Escherichia coli and Pseudomonas aeruginosa.

    Science.gov (United States)

    Liu, Zhihong; García-Díaz, Beatriz; Catacchio, Bruno; Chiancone, Emilia; Vogel, Hans J

    2015-11-01

    Lysozymes play an important role in host defense by degrading peptidoglycan in the cell envelopes of pathogenic bacteria. Several Gram-negative bacteria can evade this mechanism by producing periplasmic proteins that inhibit the enzymatic activity of lysozyme. The Escherichia coli inhibitor of vertebrate lysozyme, Ivyc and its Pseudomonas aeruginosa homolog, Ivyp1 have been shown to be potent inhibitors of hen egg white lysozyme (HEWL). Since human lysozyme (HL) plays an important role in the innate immune response, we have examined the binding of HL to Ivyc and Ivyp1. Our results show that Ivyp1 is a weaker inhibitor of HL than Ivyc even though they inhibit HEWL with similar potency. Calorimetry experiments confirm that Ivyp1 interacts more weakly with HL than HEWL. Analytical ultracentrifugation studies revealed that Ivyp1 in solution is a monomer and forms a 30kDa heterodimer with both HL and HEWL, while Ivyc is a homodimer that forms a tetramer with both enzymes. The interaction of Ivyp1 with HL was further characterized by NMR chemical shift perturbation experiments. In addition to the characteristic His-containing Ivy inhibitory loop that binds into the active site of lysozyme, an extended loop (P2) between the final two beta-strands also participates in forming protein-protein interactions. The P2 loop is not conserved in Ivyc and it constitutes a flexible region in Ivyp1 that becomes more rigid in the complex with HL. We conclude that differences in the electrostatic interactions at the binding interface between Ivy inhibitors and distinct lysozymes determine the strength of this interaction. This article is part of a Special Issue entitled: Bacterial Resistance to Antimicrobial Peptides. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. The phosphodiesterase 3 inhibitor cilostazol dilates large cerebral arteries in humans without affecting regional cerebral blood flow

    DEFF Research Database (Denmark)

    Birk, Steffen; Kruuse, Christina Rostrup; Petersen, Kenneth A

    2004-01-01

    Cilostazol, an inhibitor of phosphodiesterase (PDE) type 3, is used clinically in peripheral artery disease. PDE3 inhibitors may be clinically useful in the treatment of delayed cerebral vasospasm after subarachnoid hemorrhage. The authors present the first results on the effect of cilostazol...... on cerebral hemodynamics in normal participants. In this double-blind, randomized, crossover study, 200 mg cilostazol or placebo was administered orally to 12 healthy participants. Cerebral blood flow was measured using 133Xe inhalation and single photon emission computerized tomography. Mean flow velocity...... in the middle cerebral arteries (VMCA) was measured with transcranial Doppler, and the superficial temporal and radial arteries diameters were measured with ultrasonography. During the 4-hour observation period, there was no effect on systolic blood pressure (P = 0.28), but diastolic blood pressure decreased...

  16. Switch in Site of Inhibition: A Strategy for Structure-Based Discovery of Human Topoisomerase IIα Catalytic Inhibitors.

    Science.gov (United States)

    Baviskar, Ashish T; Amrutkar, Suyog M; Trivedi, Neha; Chaudhary, Vikas; Nayak, Anmada; Guchhait, Sankar K; Banerjee, Uttam C; Bharatam, Prasad V; Kundu, Chanakya N

    2015-04-09

    A study of structure-based modulation of known ligands of hTopoIIα, an important enzyme involved in DNA processes, coupled with synthesis and in vitro assays led to the establishment of a strategy of rational switch in mode of inhibition of the enzyme's catalytic cycle. 6-Arylated derivatives of known imidazopyridine ligands were found to be selective inhibitors of hTopoIIα, while not showing TopoI inhibition and DNA binding. Interestingly, while the parent imidazopyridines acted as ATP-competitive inhibitors, arylated derivatives inhibited DNA cleavage similar to merbarone, indicating a switch in mode of inhibition from ATP-hydrolysis to the DNA-cleavage stage of catalytic cycle of the enzyme. The 6-aryl-imidazopyridines were relatively more cytotoxic than etoposide in cancer cells and less toxic to normal cells. Such unprecedented strategy will encourage research on "choice-based change" in target-specific mode of action for rapid drug discovery.

  17. Effect of Known Inhibitors of Ion Transport on Pendrin (SLC26A4) Activity in a Human Kidney Cell Line

    OpenAIRE

    Emanuele Bernardinelli; Roberta Costa; Charity Nofziger; Markus Paulmichl; Silvia Dossena

    2016-01-01

    Background/Aims: Pendrin is a Cl-/I-/HCO3- exchanger playing a fundamental role in controlling blood pressure and airway function, therefore representing an attractive target for the treatment of hypertensive states and respiratory distresses. A review of the literature regarding the ability of some compounds (namely several known inhibitors of ion transport) to block pendrin activity revealed discordant findings. These incongruous findings may be due, in part, to the concentration of compoun...

  18. In vitro differentiation of human monocytes to macrophages: change of PDE profile and its relationship to suppression of tumour necrosis factor-α release by PDE inhibitors

    Science.gov (United States)

    Gantner, Florian; Kupferschmidt, Rochus; Schudt, Christian; Wendel, Albrecht; Hatzelmann, Armin

    1997-01-01

    During in vitro culture in 10% human AB serum, human peripheral blood monocytes acquire a macrophage-like phenotype. The underlying differentiation was characterized by increased activities of the macrophage marker enzymes unspecific esterase (NaF-insensitive form) and acid phosphatase, as well as by a down-regulation in surface CD14 expression. In parallel, a dramatic change in the phosphodiesterase (PDE) profile became evident within a few days that strongly resembled that previously described for human alveolar macrophages. Whereas PDE1 and PDE3 activities were augmented, PDE4 activity, which represented the major cyclic AMP-hydrolysing activity of peripheral blood monocytes, rapidly declined. Monocytes and monocyte-derived macrophages responded to lipopolysaccharide (LPS) with the release of tumour necrosis factor-α (TNF). In line with the change in CD14 expression, the EC50 value of LPS for induction of TNF release increased from approximately 0.1 ng ml−1 in peripheral blood monocytes to about 2 ng ml−1 in macrophages. Both populations of cells were equally susceptible towards inhibition of TNF release by cyclic AMP elevating agents such as dibutyryl cyclic AMP, prostaglandin E2 (PGE2) or forskolin, which all led to a complete abrogation of TNF production in a concentration-dependent manner and which were more efficient than the glucocorticoid dexamethasone. In monocytes, PDE4 selective inhibitors (rolipram, RP73401) suppressed TNF formation by 80%, whereas motapizone, a PDE3 selective compound, exerted a comparatively weak effect (10–15% inhibition). Combined use of PDE3 plus PDE4 inhibitors resulted in an additive effect and fully abrogated LPS-induced TNF release as did the mixed PDE3/4 inhibitor tolafentrine. In monocyte-derived macrophages, neither PDE3- nor PDE4-selective drugs markedly affected TNF generation when used alone (<15% inhibition), whereas in combination, they led to a maximal inhibition of TNF formation by about 40–50

  19. Can Inhibitors of Snake Venom Phospholipases A₂ Lead to New Insights into Anti-Inflammatory Therapy in Humans? A Theoretical Study.

    Science.gov (United States)

    Sales, Thaís A; Marcussi, Silvana; da Cunha, Elaine F F; Kuca, Kamil; Ramalho, Teodorico C

    2017-10-25

    Human phospholipase A₂ ( h PLA₂) of the IIA group (HGIIA) catalyzes the hydrolysis of membrane phospholipids, producing arachidonic acid and originating potent inflammatory mediators. Therefore, molecules that can inhibit this enzyme are a source of potential anti-inflammatory drugs, with different action mechanisms of known anti-inflammatory agents. For the study and development of new anti-inflammatory drugs with this action mechanism, snake venom PLA₂ ( sv PLA₂) can be employed, since the sv PLA₂ has high similarity with the human PLA₂ HGIIA. Despite the high similarity between these secretory PLA₂s , it is still not clear if these toxins can really be employed as an experimental model to predict the interactions that occur with the human PLA₂ HGIIA and its inhibitors. Thus, the present study aims to compare and evaluate, by means of theoretical calculations, docking and molecular dynamics simulations, as well as experimental studies, the interactions of human PLA₂ HGIIA and two sv PLA₂s , Bothrops toxin II and Crotoxin B (BthTX-II and CB, respectively). Our theoretical findings corroborate experimental data and point out that the human PLA₂ HGIIA and sv PLA₂ BthTX-II lead to similar interactions with the studied compounds. From our results, the sv PLA₂ BthTX-II can be used as an experimental model for the development of anti-inflammatory drugs for therapy in humans.

  20. Comparative effect of two pan-class I PI3K inhibitors used as anticancer drugs on human T cell function.

    Science.gov (United States)

    Blanco, Belén; Herrero-Sánchez, Carmen; Rodríguez-Serrano, Concepción; Sánchez-Barba, Mercedes; Del Cañizo, María Consuelo

    2015-09-01

    The phosphatidylinositol 3-kinase (PI3K) pathway is commonly deregulated in cancer and, thus, PI3K has been recognized as an attractive molecular target for novel anti-cancer therapies. However, the effect of PI3K inhibitors on T-cell function, a key component of antitumor immunity, has been scantly explored. The objective of this study was to investigate the effect on human T-cell activation of two PI3K inhibitors currently being tested in clinical trials: PX-866 and BKM120. Their activity against a leukemic T cell line was also assessed. For that purpose, Jurkat cells or anti-CD3/anti-CD28 stimulated human peripheral blood mononuclear cells were cultured in the presence of different concentrations of PX-866 or BKM120 and their effect on T-cell proliferation, apoptosis, expression of activation markers and cytokine secretion was analyzed by flow cytometry. In addition, Akt and Erk phosphorylation was analyzed by Western blotting. Both PX-866 and BKM120 decreased viability of Jurkat cells and blocked cell cycle progression. Regarding primary T cells, both compounds similarly inhibited expression of activation markers and cytokine secretion, although they did not induce apoptosis of stimulated T cells. Interestingly, we found differences in their ability to block T-cell proliferation and IL-2 secretion, exerting BKM120 a more potent inhibition. These disparate effects could be related to differences observed in PI3K/Akt and RAS/MEK/ERK signaling between PX-866 and BKM120 treated cells. Our results suggest that, when selecting a PI3K inhibitor for cancer therapy, immunosuppressive characteristics should be taken into account in order to minimize detrimental effects on immune function. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Targeting both viral and host determinants of human immunodeficiency virus entry, using a new lentiviral vector coexpressing the T20 fusion inhibitor and a selective CCL5 intrakine.

    Science.gov (United States)

    Petit, Nicolas; Dorgham, Karim; Levacher, Béatrice; Burlion, Aude; Gorochov, Guy; Marodon, Gilles

    2014-08-01

    Numerous strategies targeting early and late steps of the HIV life cycle have been proposed for gene therapy. However, targeting viral and host determinants of HIV entry is the only strategy that would prevent viral DNA-mediated CD4(+) cell death while diminishing the possibility for the virus to escape. To this end, we devised a bicistronic lentiviral vector expressing the membrane-bound form of the T20 fusion inhibitor, referred to as the C46 peptide, and a CCR5 superagonist, modified to sequester CCR5 away from the cell surface, referred to as the P2-CCL5 intrakine. We tested the effects of the vector on HIV infection and replication, using the human CEMR5 cell line expressing CD4 and CCR5, and primary human T cells. Transduced cells expressed the C46 peptide, detected with the 2F5 monoclonal antibody by flow cytometry. Expression of the P2-CCL5 intrakine correlates with lower levels of cell surface CCR5. Complete protection against HIV infection could be observed in cells expressing the protective transgenes. Importantly, we show that the combination of the transgenes was more potent than either transgene alone, showing the interest of expressing two entry inhibitors to inhibit HIV infection. Last, genetically modified cells possessed a selective advantage over nonmodified cells on HIV challenge in vitro, showing that modified cells were protected from HIV-induced cell death. Our results demonstrate that lentiviral vectors coexpressing the T20 fusion inhibitor and the P2-CCL5 intrakine represent promising tools for HIV gene therapy.

  2. X-ray structures of human neutrophil collagenase complexed with peptide hydroxamate and peptide thiol inhibitors. Implications for substrate binding and rational drug design.

    Science.gov (United States)

    Grams, F; Reinemer, P; Powers, J C; Kleine, T; Pieper, M; Tschesche, H; Huber, R; Bode, W

    1995-03-15

    Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases involved in tissue remodeling. They have also been implicated in various disease processes including tumour invasion and joint destruction and are therefore attractive targets for inhibitor design. For rational drug design, information of inhibitor binding at the atomic level is essential. Recently, we have published the refined high-resolution crystal structure of the catalytic domain of human neutrophil collagenase (HNC) complexed with the inhibitor Pro-Leu-Gly-NHOH, which is a mimic for the unprimed (P3-P1) residues of a bound peptide substrate. We have now determined two additional HNC complexes formed with the thiol inhibitor HSCH2CH(CH2Ph)CO-L-Ala-Gly-NH2 and another hydroxamate inhibitor, HONHCOCH(iBu)CO-L-Ala-Gly-NH2, which were both refined to R-values of 0.183/0.198 at 0.240/0.225-nm resolution. The inhibitor thiol and hydroxamate groups ligand the catalytic zinc, giving rise to a slightly distorted tetrahedral and trigonal-bipyramidal coordination sphere, respectively. The thiol inhibitor diastereomer with S-configuration at the P1' residue (corresponding to an L-amino acid analog) binds to HNC. Its peptidyl moiety mimics binding of primed (P1'-P3') residues of the substrate. In combination with our first structure a continuous hexapeptide corresponding to a peptide substrate productively bound to HNC was constructed and energy-minimized. Proteolytic cleavage of this Michaelis complex is probably general base-catalyzed as proposed for thermolysin, i.e. a glutamate assists nucleophilic attack of a water molecule. Although there are many structural and mechanistic similarities to thermolysin, substrate binding to MMPs differs due to the interactions beyond S1'-P1'. While thermolysin binds substrates with a kink at P1', substrates are bound in an extended conformation in the collagenases. This property explains the tolerance of thermolysin for D-amino acid residues at the P1' position, in

  3. Discovery of highly potent and selective small molecule ADAMTS-5 inhibitors that inhibit human cartilage degradation via encoded library technology (ELT).

    Science.gov (United States)

    Deng, Hongfeng; O'Keefe, Heather; Davie, Christopher P; Lind, Kenneth E; Acharya, Raksha A; Franklin, G Joseph; Larkin, Jonathan; Matico, Rosalie; Neeb, Michael; Thompson, Monique M; Lohr, Thomas; Gross, Jeffrey W; Centrella, Paolo A; O'Donovan, Gary K; Bedard, Katie L Sargent; van Vloten, Kurt; Mataruse, Sibongile; Skinner, Steven R; Belyanskaya, Svetlana L; Carpenter, Tiffany Y; Shearer, Todd W; Clark, Matthew A; Cuozzo, John W; Arico-Muendel, Christopher C; Morgan, Barry A

    2012-08-23

    The metalloprotease ADAMTS-5 is considered a potential target for the treatment of osteoarthritis. To identify selective inhibitors of ADAMTS-5, we employed encoded library technology (ELT), which enables affinity selection of small molecule binders from complex mixtures by DNA tagging. Selection of ADAMTS-5 against a four-billion member ELT library led to a novel inhibitor scaffold not containing a classical zinc-binding functionality. One exemplar, (R)-N-((1-(4-(but-3-en-1-ylamino)-6-(((2-(thiophen-2-yl)thiazol-4-yl)methyl)amino)-1,3,5-triazin-2-yl)pyrrolidin-2-yl)methyl)-4-propylbenzenesulfonamide (8), inhibited ADAMTS-5 with IC(50) = 30 nM, showing >50-fold selectivity against ADAMTS-4 and >1000-fold selectivity against ADAMTS-1, ADAMTS-13, MMP-13, and TACE. Extensive SAR studies showed that potency and physicochemical properties of the scaffold could be further improved. Furthermore, in a human osteoarthritis cartilage explant study, compounds 8 and 15f inhibited aggrecanase-mediated (374)ARGS neoepitope release from aggrecan and glycosaminoglycan in response to IL-1β/OSM stimulation. This study provides the first small molecule evidence for the critical role of ADAMTS-5 in human cartilage degradation.

  4. Frequent expression loss of Inter-alpha-trypsin inhibitor heavy chain (ITIH) genes in multiple human solid tumors: A systematic expression analysis

    International Nuclear Information System (INIS)

    Hamm, Alexander; Knuechel, Ruth; Dahl, Edgar; Veeck, Juergen; Bektas, Nuran; Wild, Peter J; Hartmann, Arndt; Heindrichs, Uwe; Kristiansen, Glen; Werbowetski-Ogilvie, Tamra; Del Maestro, Rolando

    2008-01-01

    The inter-alpha-trypsin inhibitors (ITI) are a family of plasma protease inhibitors, assembled from a light chain – bikunin, encoded by AMBP – and five homologous heavy chains (encoded by ITIH1, ITIH2, ITIH3, ITIH4, and ITIH5), contributing to extracellular matrix stability by covalent linkage to hyaluronan. So far, ITIH molecules have been shown to play a particularly important role in inflammation and carcinogenesis. We systematically investigated differential gene expression of the ITIH gene family, as well as AMBP and the interacting partner TNFAIP6 in 13 different human tumor entities (of breast, endometrium, ovary, cervix, stomach, small intestine, colon, rectum, lung, thyroid, prostate, kidney, and pancreas) using cDNA dot blot analysis (Cancer Profiling Array, CPA), semiquantitative RT-PCR and immunohistochemistry. We found that ITIH genes are clearly downregulated in multiple human solid tumors, including breast, colon and lung cancer. Thus, ITIH genes may represent a family of putative tumor suppressor genes that should be analyzed in greater detail in the future. For an initial detailed analysis we chose ITIH2 expression in human breast cancer. Loss of ITIH2 expression in 70% of cases (n = 50, CPA) could be confirmed by real-time PCR in an additional set of breast cancers (n = 36). Next we studied ITIH2 expression on the protein level by analyzing a comprehensive tissue micro array including 185 invasive breast cancer specimens. We found a strong correlation (p < 0.001) between ITIH2 expression and estrogen receptor (ER) expression indicating that ER may be involved in the regulation of this ECM molecule. Altogether, this is the first systematic analysis on the differential expression of ITIH genes in human cancer, showing frequent downregulation that may be associated with initiation and/or progression of these malignancies

  5. Similar reductions in the risk of human colon cancer by selective and nonselective cyclooxygenase-2 (COX-2 inhibitors

    Directory of Open Access Journals (Sweden)

    Alshafie Galal A

    2008-08-01

    Full Text Available Abstract Background Epidemiologic and laboratory investigations suggest that aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs have chemopreventive effects against colon cancer perhaps due at least in part to their activity against cyclooxygenase-2 (COX-2, the rate-limiting enzyme of the prostaglandin cascade. Methods We conducted a case control study of colon cancer designed to compare effects of selective and non-selective COX-2 inhibitors. A total of 326 incident colon cancer patients were ascertained from the James Cancer Hospital, Columbus, Ohio, during 2003–2004 and compared with 652 controls with no history of cancer and matched to the cases at a 2:1 ratio on age, race, and county of residence. Data on the past and current use of prescription and over the counter medications and colon cancer risk factors were ascertained using a standardized risk factor questionnaire. Effects of COX-2 inhibiting agents were quantified by calculating odds ratios (OR and 95% confidence intervals. Results Results showed significant risk reductions for selective COX-2 inhibitors (OR = 0.31, 95% CI = 0.16–0.57, regular aspirin (OR = 0.33, 95% CI = 0.20–0.56, and ibuprofen or naproxen (0.28, 95% CI = 0.15–0.54. Acetaminophen, a compound with negligible COX-2 activity and low dose aspirin (81 mg produced no significant change in the risk of colon cancer. Conclusion These results suggest that both non-selective and selective COX-2 inhibitors produce significant reductions in the risk of colon cancer, underscoring their strong potential for colon cancer chemoprevention.

  6. Identification of novel human dipeptidyl peptidase-IV inhibitors of natural origin (part I: virtual screening and activity assays.

    Directory of Open Access Journals (Sweden)

    Laura Guasch

    Full Text Available BACKGROUND: There has been great interest in determining whether natural products show biological activity toward protein targets of pharmacological relevance. One target of particular interest is DPP-IV whose most important substrates are incretins that, among other beneficial effects, stimulates insulin biosynthesis and secretion. Incretins have very short half-lives because of their rapid degradation by DPP-IV and, therefore, inhibiting this enzyme improves glucose homeostasis. As a result, DPP-IV inhibitors are of considerable interest to the pharmaceutical industry. The main goals of this study were (a to develop a virtual screening process to identify potential DPP-IV inhibitors of natural origin; (b to evaluate the reliability of our virtual-screening protocol by experimentally testing the in vitro activity of selected natural-product hits; and (c to use the most active hit for predicting derivatives with higher binding affinities for the DPP-IV binding site. METHODOLOGY/PRINCIPAL FINDINGS: We predicted that 446 out of the 89,165 molecules present in the natural products subset of the ZINC database would inhibit DPP-IV with good ADMET properties. Notably, when these 446 molecules were merged with 2,342 known DPP-IV inhibitors and the resulting set was classified into 50 clusters according to chemical similarity, there were 12 clusters that contained only natural products for which no DPP-IV inhibitory activity has been previously reported. Nine molecules from 7 of these 12 clusters were then selected for in vitro activity testing and 7 out of the 9 molecules were shown to inhibit DPP-IV (where the remaining two molecules could not be solubilized, preventing the evaluation of their DPP-IV inhibitory activity. Then, the hit with the highest activity was used as a lead compound in the prediction of more potent derivatives. CONCLUSIONS/SIGNIFICANCE: We have demonstrated that our virtual-screening protocol was successful in identifying novel

  7. Monitoring processed, mature Human Immunodeficiency Virus type 1 particles immediately following treatment with a protease inhibitor-containing treatment regimen

    Directory of Open Access Journals (Sweden)

    Kuritzkes Daniel R

    2005-04-01

    Full Text Available Abstract Protease inhibitors (PIs block HIV-1 maturation into an infectious virus particle by inhibiting the protease processing of gag and gag-pol precursor proteins. We have used a simple anti-HIV-1 p24 Western blot to monitor the processing of p55gag precursor into the mature p24 capsid immediately following the first dosage of a PI-containing treatment regimen. Evidence of PI activity was observed in plasma virus as early as 72 hours post treatment-initiation and was predictive of plasma viral RNA decrease at 4 weeks.

  8. A Single Amino Acid Difference between Mouse and Human 5-Lipoxygenase Activating Protein (FLAP) Explains the Speciation and Differential Pharmacology of Novel FLAP Inhibitors*

    Science.gov (United States)

    Blevitt, Jonathan M.; Hack, Michael D.; Herman, Krystal; Chang, Leon; Keith, John M.; Mirzadegan, Tara; Rao, Navin L.; Lebsack, Alec D.; Milla, Marcos E.

    2016-01-01

    5-Lipoxygenase activating protein (FLAP) plays a critical role in the metabolism of arachidonic acid to leukotriene A4, the precursor to the potent pro-inflammatory mediators leukotriene B4 and leukotriene C4. Studies with small molecule inhibitors of FLAP have led to the discovery of a drug binding pocket on the protein surface, and several pharmaceutical companies have developed compounds and performed clinical trials. Crystallographic studies and mutational analyses have contributed to a general understanding of compound binding modes. During our own efforts, we identified two unique chemical series. One series demonstrated strong inhibition of human FLAP but differential pharmacology across species and was completely inactive in assays with mouse or rat FLAP. The other series was active across rodent FLAP, as well as human and dog FLAP. Comparison of rodent and human FLAP amino acid sequences together with an analysis of a published crystal structure led to the identification of amino acid residue 24 in the floor of the putative binding pocket as a likely candidate for the observed speciation. On that basis, we tested compounds for binding to human G24A and mouse A24G FLAP mutant variants and compared the data to that generated for wild type human and mouse FLAP. These studies confirmed that a single amino acid mutation was sufficient to reverse the speciation observed in wild type FLAP. In addition, a PK/PD method was established in canines to enable preclinical profiling of mouse-inactive compounds. PMID:27129215

  9. P1-Substituted Symmetry-Based Human Immunodeficiency Virus Protease Inhibitors with Potent Antiviral Activity against Drug-Resistant Viruses

    Energy Technology Data Exchange (ETDEWEB)

    DeGoey, David A.; Grampovnik, David J.; Chen, Hui-Ju; Flosi, William J.; Klein, Larry L.; Dekhtyar, Tatyana; Stoll, Vincent; Mamo, Mulugeta; Molla, Akhteruzzaman; Kempf, Dale J. (Abbott)

    2013-03-07

    Because there is currently no cure for HIV infection, patients must remain on long-term drug therapy, leading to concerns over potential drug side effects and the emergence of drug resistance. For this reason, new and safe antiretroviral agents with improved potency against drug-resistant strains of HIV are needed. A series of HIV protease inhibitors (PIs) with potent activity against both wild-type (WT) virus and drug-resistant strains of HIV was designed and synthesized. The incorporation of substituents with hydrogen bond donor and acceptor groups at the P1 position of our symmetry-based inhibitor series resulted in significant potency improvements against the resistant mutants. By this approach, several compounds, such as 13, 24, and 29, were identified that demonstrated similar or improved potencies compared to 1 against highly mutated strains of HIV derived from patients who previously failed HIV PI therapy. Overall, compound 13 demonstrated the best balance of potency against drug resistant strains of HIV and oral bioavailability in pharmacokinetic studies. X-ray analysis of an HIV PI with an improved resistance profile bound to WT HIV protease is also reported.

  10. 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.

  11. Use of bile correction factors for allometric prediction of human pharmacokinetic parameters of torcetrapib, a facile cholesteryl ester transfer protein inhibitor.

    Science.gov (United States)

    Mullangi, Ramesh; Ahlawat, Preeti; Trivedi, Ravi K; Srinivas, Nuggehally R

    2009-01-01

    Torcetrapib was the lead candidate belonging to the class of cholesteryl ester transfer protein (CETP) inhibitor which was being developed for the management of cardiovascular risk factors by raising HDL. The availability of pharmacokinetic parameters (clearance: CL/F, volume of distribution: Vd/F, elimination rate constant: K(el) and elimination half-life: t(l/2)) in mice, rats and monkeys, enabled the prediction of human parameter values using the well accepted tool of allometry. Although allometry work has been largely restricted to intravenous drugs, the present case of torcetrapib showed that allometry may be equally applicable to oral route. Simple allometry appeared to markedly inflate the human parameters for CL/F, Vd/F, K(el), and t(1/2). However, the application of bile correction factors provided allometric equations of 0.2486W(0.877) (R2 = 0.9416), 1.4723W(1.8263) (R2 = 0.8873), 0.1685W(-095) (R2 = 0.828) and 4.1044W(0.493) (R2 = 0.9337) for CL/F, Vd/F, K(el) and t(1/2), rendering a closer prediction of human parameter values. Accordingly, the predicted (observed) values of torcetrapib were 10.3 L/h (15.8 L/h), 3449 L (4810 L), 0.00298 h(-1) (0.00328 h(-1)) and 211 h (231 h) for CL/F, Vd/F, K(el) and t(1/2), respectively. In summary, the data suggested that allometry tool with appropriate bile correction factors could be effectively used in a prospective manner for other orally administered CETP inhibitors.

  12. Vorinostat, an HDAC inhibitor attenuates epidermoid squamous cell carcinoma growth by dampening mTOR signaling pathway in a human xenograft murine model

    Energy Technology Data Exchange (ETDEWEB)

    Kurundkar, Deepali; Srivastava, Ritesh K.; Chaudhary, Sandeep C. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States); Ballestas, Mary E. [Department of Pediatrics Infectious Disease, Children' s of Alabama, School of Medicine, University of Alabama at Birmingham, AL (United States); Kopelovich, Levy [Division of Cancer Prevention, National Cancer Institute, 6130 Executive Blvd., Suite 2114, Bethesda, MD 20892 (United States); Elmets, Craig A. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, 1530 3rd Avenue South, VH 509, Birmingham, AL 35294-0019 (United States)

    2013-01-15

    Histone deacetylase (HDAC) inhibitors are potent anticancer agents and show efficacy against various human neoplasms. Vorinostat is a potent HDAC inhibitor and has shown potential to inhibit growth of human xenograft tumors. However, its effect on the growth of skin neoplasm remains undefined. In this study, we show that vorinostat (2 μM) reduced expression of HDAC1, 2, 3, and 7 in epidermoid carcinoma A431 cells. Consistently, it increased acetylation of histone H3 and p53. Vorinostat (100 mg/kg body weight, IP) treatment reduced human xenograft tumor growth in highly immunosuppressed nu/nu mice. Histologically, the vorinostat-treated tumor showed features of well-differentiation with large necrotic areas. Based on proliferating cell nuclear antigen (PCNA) staining and expression of cyclins D1, D2, E, and A, vorinostat seems to impair proliferation by down-regulating the expression of these proteins. However, it also induced apoptosis. The mechanism by which vorinostat blocks proliferation and makes tumor cells prone to apoptosis, involved inhibition of mTOR signaling which was accompanied by reduction in cell survival AKT and extracellular-signal regulated kinase (ERK) signaling pathways. Our data provide a novel mechanism-based therapeutic intervention for cutaneous squamous cell carcinoma (SCC). Vorinostat may be utilized to cure skin neoplasms in organ transplant recipient (OTR). These patients have high morbidity and surgical removal of these lesions which frequently develop in these patients, is difficult. -- Highlights: ► Vorinostat reduces SCC growth in a xenograft murine model. ► Vorinostat dampens proliferation and induces apoptosis in tumor cells. ► Diminution in mTOR, Akt and ERK signaling underlies inhibition in proliferation. ► Vorinostat by inhibiting HDACs inhibits epithelial–mesenchymal transition.

  13. Vorinostat, an HDAC inhibitor attenuates epidermoid squamous cell carcinoma growth by dampening mTOR signaling pathway in a human xenograft murine model

    International Nuclear Information System (INIS)

    Kurundkar, Deepali; Srivastava, Ritesh K.; Chaudhary, Sandeep C.; Ballestas, Mary E.; Kopelovich, Levy; Elmets, Craig A.; Athar, Mohammad

    2013-01-01

    Histone deacetylase (HDAC) inhibitors are potent anticancer agents and show efficacy against various human neoplasms. Vorinostat is a potent HDAC inhibitor and has shown potential to inhibit growth of human xenograft tumors. However, its effect on the growth of skin neoplasm remains undefined. In this study, we show that vorinostat (2 μM) reduced expression of HDAC1, 2, 3, and 7 in epidermoid carcinoma A431 cells. Consistently, it increased acetylation of histone H3 and p53. Vorinostat (100 mg/kg body weight, IP) treatment reduced human xenograft tumor growth in highly immunosuppressed nu/nu mice. Histologically, the vorinostat-treated tumor showed features of well-differentiation with large necrotic areas. Based on proliferating cell nuclear antigen (PCNA) staining and expression of cyclins D1, D2, E, and A, vorinostat seems to impair proliferation by down-regulating the expression of these proteins. However, it also induced apoptosis. The mechanism by which vorinostat blocks proliferation and makes tumor cells prone to apoptosis, involved inhibition of mTOR signaling which was accompanied by reduction in cell survival AKT and extracellular-signal regulated kinase (ERK) signaling pathways. Our data provide a novel mechanism-based therapeutic intervention for cutaneous squamous cell carcinoma (SCC). Vorinostat may be utilized to cure skin neoplasms in organ transplant recipient (OTR). These patients have high morbidity and surgical removal of these lesions which frequently develop in these patients, is difficult. -- Highlights: ► Vorinostat reduces SCC growth in a xenograft murine model. ► Vorinostat dampens proliferation and induces apoptosis in tumor cells. ► Diminution in mTOR, Akt and ERK signaling underlies inhibition in proliferation. ► Vorinostat by inhibiting HDACs inhibits epithelial–mesenchymal transition.

  14. Synthesis of an Endogenous Steroidal Na Pump Inhibitor Marinobufagenin, Implicated in Human Cardiovascular Diseases, Is Initiated by CYP27A1 via Bile Acid Pathway.

    Science.gov (United States)

    Fedorova, Olga V; Zernetkina, Valentina I; Shilova, Victoria Y; Grigorova, Yulia N; Juhasz, Ondrej; Wei, Wen; Marshall, Courtney A; Lakatta, Edward G; Bagrov, Alexei Y

    2015-10-01

    The bioactive steroid, marinobufagenin, is an endogenous Na/K-ATPase bufadienolide inhibitor that is synthesized by adrenocortical and placental cells. Marinobufagenin binding to Na/K-ATPase initiates profibrotic cell signaling, and heightened marinobufagenin levels are implicated in the pathogenesis of hypertension, preeclampsia, and chronic kidney disease. Steroids are derived from cholesterol through the traditional steroidogenesis pathway initiated by enzyme CYP11A1, and via the acidic bile acid pathway, which is controlled by enzyme CYP27A1. The mechanism of marinobufagenin biosynthesis in mammals, however, remains unknown. Here, we show that post-transcriptional silencing of the CYP27A1 gene in human trophoblast and rat adrenocortical cells reduced the expression of CYP27A1 mRNA by 70%, reduced total bile acids 2-fold, and marinobufagenin levels by 67% when compared with nontreated cells or cells transfected with nontargeting siRNA. In contrast, silencing of the CYP11A1 gene did not affect marinobufagenin production in either cell culture, but suppressed production of progesterone 2-fold in human trophoblast cells and of corticosterone by 90% in rat adrenocortical cells when compared with cells transfected with nontargeting siRNA. In vivo, in a high-salt administration experiment, male and female Dahl salt-sensitive rats became hypertensive after 4 weeks on a high-NaCl diet, their plasma marinobufagenin levels doubled, and adrenocortical CYP27A1 mRNA and protein increased 1.6-fold and 2.0-fold. Therefore, the endogenous steroidal Na/K-ATPase inhibitor, marinobufagenin, is synthesized in mammalian placenta and adrenal cortex from cholesterol through the novel acidic bile acid pathway. These findings will help to understand the role of marinobufagenin in highly prevalent human cardiovascular diseases. © 2015 American Heart Association, Inc.

  15. Insight into the interaction mechanism of human SGLT2 with its inhibitors: 3D-QSAR studies, homology modeling, and molecular docking and molecular dynamics simulations.

    Science.gov (United States)

    Dong, Lili; Feng, Ruirui; Bi, Jiawei; Shen, Shengqiang; Lu, Huizhe; Zhang, Jianjun

    2018-03-06

    Human sodium-dependent glucose co-transporter 2 (hSGLT2) is a crucial therapeutic target in the treatment of type 2 diabetes. In this study, both comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were applied to generate three-dimensional quantitative structure-activity relationship (3D-QSAR) models. In the most accurate CoMFA-based and CoMSIA-based QSAR models, the cross-validated coefficients (r 2 cv ) were 0.646 and 0.577, respectively, while the non-cross-validated coefficients (r 2 ) were 0.997 and 0.991, respectively, indicating that both models were reliable. In addition, we constructed a homology model of hSGLT2 in the absence of a crystal structure. Molecular docking was performed to explore the bonding mode of inhibitors to the active site of hSGLT2. Molecular dynamics (MD) simulations and binding free energy calculations using MM-PBSA and MM-GBSA were carried out to further elucidate the interaction mechanism. With regards to binding affinity, we found that hydrogen-bond interactions of Asn51 and Glu75, located in the active site of hSGLT2, with compound 40 were critical. Hydrophobic and electrostatic interactions were shown to enhance activity, in agreement with the results obtained from docking and 3D-QSAR analysis. Our study results shed light on the interaction mode between inhibitors and hSGLT2 and may aid in the development of C-aryl glucoside SGLT2 inhibitors.

  16. The Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Cellular Senescence of Cultivated Human Corneal Endothelial Cells.

    Science.gov (United States)

    Hongo, Akane; Okumura, Naoki; Nakahara, Makiko; Kay, EunDuck P; Koizumi, Noriko

    2017-07-01

    We have begun a clinical trial of a cell-based therapy for corneal endothelial dysfunction in Japan. The purpose of this study was to investigate the usefulness of a p38 MAPK inhibitor for prevention cellular senescence in cultivated human corneal endothelial cells (HCECs). HCECs of 10 donor corneas were divided and cultured with or without SB203580 (a p38 MAPK inhibitor). Cell density and morphology were evaluated by phase-contrast microscopy. Expression of function-related proteins was examined by immunofluorescent staining. Cellular senescence was evaluated by SA-β-gal staining and Western blotting for p16 and p21. Senescence-associated factors were evaluated by membrane blotting array, quantitative PCR, and ELISA. Phase-contrast microscopy showed a significantly higher cell density for HCECs cultured with SB203580 than without SB203580 (2623 ± 657 cells/mm2 and 1752 ± 628 cells/mm2, respectively). The HCECs cultured with SB203580 maintained a hexagonal morphology and expressed ZO-1, N-cadherin, and Na+/K+-ATPase in the plasma membrane, whereas the control HCECs showed an altered staining pattern for these marker proteins. HCECs cultured without SB203580 showed high positive SA-β-gal staining, a low nuclear/cytoplasm ratio, and expression of p16 and p21. IL-6, IL-8, CCL2, and CXCL1 were observed at high levels in low cell density HCECs cultured without SB203580. Activation of p38 MAPK signaling due to culture stress might be a causative factor that induces cellular senescence; therefore, the use of p38 MAPK inhibitor to counteract senescence may achieve sufficient numbers of HCECs for tissue engineering therapy for corneal endothelial dysfunction.

  17. Influence of the tyrosine kinase inhibitors STI571 (Glivec), lavendustin A and genistein on human mast cell line (HMC-1(560)) activation.

    Science.gov (United States)

    Löber, Kristin; Alfonso, Amparo; Escribano, Luis; Botana, Luis M

    2008-03-01

    The human mast cell line (HMC-1(560)) was used to study the effects of tyrosine kinase (TyrK) inhibition on histamine release in consequence of intracellular Ca2+ or pH changes. This is important since the TyrK inhibitor STI571 (Glivec) inhibits proliferation and induces apoptosis in HMC-1(560). HMC-1(560) cells have a mutation in c-kit, which leads to a permanent phosphorylation of the KIT protein and their ligand-independent proliferation. The TyrK inhibitors STI571, lavendustin A and genistein decrease spontaneous histamine release in 24-h pre-incubated cells. Results are compared with those of the mast cell stabiliser cromoglycic acid, which also drops spontaneous histamine release. When exocytosis is stimulated by alkalinisation, STI571 pre-incubated cells release more histamine than non-pre-incubated cells. Alkalinisation-induced histamine release reaches still higher levels in STI571 cells with activated protein kinase C (PKC) by PMA. We do not observe modifications on histamine release in cells, treated with PKC inhibitors (rottlerin, Gf109203 or Gö6976). Lavendustin A- and genistein 24-h incubated cells behave similar to STI571 cells, whereas cromoglycic acid does not show effects after stimulation with alkalinisation. Stimulation of exocytosis with the Ca2+ ionophore ionomycin does not modify histamine response in TyrK inhibited cells. Ca2+ and pH changes are observed after long-time incubation with STI571. Results show that pH is still higher in STI571 pre-incubated cells after alkalinisation with NH4Cl, whereas intracellular Ca2+ concentration remains stable. This work further strength the importance of pHi as a cell signal and suggest that STI571 has transduction pathways in common with other TyrKs.

  18. Prevotella intermedia stimulates tissue-type plasminogen activator and plasminogen activator inhibitor-2 expression via multiple signaling pathways in human periodontal ligament cells.

    Science.gov (United States)

    Guan, Su-Min; He, Jian-Jun; Zhang, Ming; Shu, Lei

    2011-06-01

    Prevotella intermedia is an important periodontal pathogen that induces various inflammatory and immune responses. In this study, we investigated the effects of P. intermedia on the plasminogen system in human periodontal ligament (hPDL) cells and explored the signaling pathways involved. Using semi-quantitative reverse transcription (RT)-PCR and quantitative real-time RT-qPCR, we demonstrated that P. intermedia challenge increased tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI)-2 expression in a concentration- and time-dependent manner, but exerted no influence on urokinase-type plasminogen activator and PAI-1mRNA expression in hPDL cells. Prevotella intermedia stimulation also enhanced tPA protein secretion as confirmed by enzyme-linked immunosorbent assay. Western blot results revealed that P. intermedia treatment increased phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 kinase (p38). ERK, JNK and protein kinase C inhibitors significantly attenuated the P. intermedia-induced tPA and PAI-2 expression. Furthermore, p38 and phosphatidylinositol 3-kinase inhibitors markedly decreased PAI-2 expression, whereas they showed no or little inhibition on tPA expression. In contrast, inhibition of protein kinase A greatly enhanced the upregulatory effect of P. intermedia on tPA and PAI-2 expression. Our results suggest that P. intermedia may contribute to periodontal tissue destruction by upregulating tPA and PAI-2 expression in hPDL cells via multiple signaling pathways. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  19. Insight into the molecular basis of pathogen abundance: group A Streptococcus inhibitor of complement inhibits bacterial adherence and internalization into human cells.

    Science.gov (United States)

    Hoe, Nancy P; Ireland, Robin M; DeLeo, Frank R; Gowen, Brian B; Dorward, David W; Voyich, Jovanka M; Liu, Mengyao; Burns, Eugene H; Culnan, Derek M; Bretscher, Anthony; Musser, James M

    2002-05-28

    Streptococcal inhibitor of complement (Sic) is a secreted protein made predominantly by serotype M1 Group A Streptococcus (GAS), which contributes to persistence in the mammalian upper respiratory tract and epidemics of human disease. Unexpectedly, an isogenic sic-negative mutant adhered to human epithelial cells significantly better than the wild-type parental strain. Purified Sic inhibited the adherence of a sic negative serotype M1 mutant and of non-Sic-producing GAS strains to human epithelial cells. Sic was rapidly internalized by human epithelial cells, inducing cell flattening and loss of microvilli. Ezrin and moesin, human proteins that functionally link the cytoskeleton to the plasma membrane, were identified as Sic-binding proteins by affinity chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. Sic colocalized with ezrin inside epithelial cells and bound to the F-actin-binding site region located in the carboxyl terminus of ezrin and moesin. Synthetic peptides corresponding to two regions of Sic had GAS adherence-inhibitory activity equivalent to mature Sic and inhibited binding of Sic to ezrin. In addition, the sic mutant was phagocytosed and killed by human polymorphonuclear leukocytes significantly better than the wild-type strain, and Sic colocalized with ezrin in discrete regions of polymorphonuclear leukocytes. The data suggest that binding of Sic to ezrin alters cellular processes critical for efficient GAS contact, internalization, and killing. Sic enhances bacterial survival by enabling the pathogen to avoid the intracellular environment. This process contributes to the abundance of M1 GAS in human infections and their ability to cause epidemics.

  20. In silico-based identification of human α-enolase inhibitors to block cancer cell growth metabolically

    Directory of Open Access Journals (Sweden)

    Lung J

    2017-11-01

    s rule of five from the ZINC database were docked to α-enolase by virtual screening. Twenty-four chemical structures with docking scores better than that of the enolase substrate, 2-phosphoglycerate, were further screened by the absorption, distribution, metabolism, excretion, and toxicity (ADMET properties prediction. Four of them were classified as non-mutagenic, non-carcinogenic, and capable of oral administration where they showed steady interactions to α-enolase that were comparable, even superior, to the currently available inhibitors in molecular dynamics (MD simulation. These compounds may be considered promising leads for further development of the α-enolase inhibitors and could help fight cancer metabolically. Keywords: α-enolase inhibitor, virtual screening, molecular dynamics simulation, glycolysis, metabolism

  1. 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.

  2. A computational docking study for prediction of binding mode of diospyrin and derivatives: Inhibitors of human and leishmanial DNA topoisomerase-I.

    Science.gov (United States)

    Chhabra, Sandeep; Sharma, Pooja; Ghoshal, Nanda

    2007-08-15

    A computational approach was utilized to study the relative binding modes of diospyrin (bisnaphthoquinonoid) with the crystal structure of human DNA-TopoI and the recently reported Leishmania donavani DNA-TopoI. Additionally, the binding site interactions of amino derivatives of diospyrin with human TopoI were studied extensively. Based on the docking results, binding modes of diospyrin with the human and leishmanial TopoI catalytic core were predicted. The parallel use of two efficient and predictive docking programs, GOLD and Ligandfit, allowed mutual validation of the predicted binding poses. A reasonably good correlation coefficient between the calculated docking scores and the experimentally determined cytotoxicity helped in validating the docking method. Furthermore, a structure-based pharmacophore model was developed for L. donavani DNA-TopoI inhibition which helped in elucidating the topological and spatial requirements of the ligand-receptor interactions. This study provides an understanding of the structural basis of ligand binding to the topoisomerase receptor, which may be used for the structure-based design of potent and novel ligands for anticancer and antileishmanial therapy. To our knowledge, this is the first report of a binding mode exploration study for diospyrin and its derivatives as inhibitors of the leishmanial and human TopoI enzymes.

  3. Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models

    Science.gov (United States)

    Wen, Li; Voronina, Svetlana; Javed, Muhammad A.; Awais, Muhammad; Szatmary, Peter; Latawiec, Diane; Chvanov, Michael; Collier, David; Huang, Wei; Barrett, John; Begg, Malcolm; Stauderman, Ken; Roos, Jack; Grigoryev, Sergey; Ramos, Stephanie; Rogers, Evan; Whitten, Jeff; Velicelebi, Gonul; Dunn, Michael; Tepikin, Alexei V.; Criddle, David N.; Sutton, Robert

    2015-01-01

    Background & Aims Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release–activated calcium modulator ORAI1 is the most abundant Ca2+ entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice. Methods Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects. Results GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca2+ currents after Ca2+ release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis. Conclusions Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed

  4. Anti-VEGF strategies - from antibodies to tyrosine kinase inhibitors: background and clinical development in human cancer.

    LENUS (Irish Health Repository)

    Korpanty, Grzegorz

    2012-01-01

    Tumour angiogenesis (formation of new blood vessels supporting tumour growth and metastasis) is a result of complex interactions between the tumour and the surrounding microenvironment. Targeting tumours with anti-angiogenic therapy remains an exciting area of preclinical and clinical studies. Although many significant advances have been achieved and the clinical use of anti-angiogenic drugs is now well recognized in many solid malignancies, these therapies fall short of their anticipated clinical benefits and leave many unanswered questions like exact mechanism of action, patients\\' selection and monitoring response to anti-angiogenic drugs. Tumour angiogenesis is controlled by complex signaling cascades and ongoing research into molecular mechanisms of tumour angiogenesis not only helps to understand its basic mechanisms but hopefully will identify new therapeutic targets. In 2012, both monoclonal antibodies and small molecule tyrosine kinase inhibitors remain the two major clinically useful therapeutic options that interfere with tumour angiogenesis in many solid malignancies.

  5. New Human Monoamine Oxidase A Inhibitors with Potential Anti- Depressant Activity: Design, Synthesis, Biological Screening and Evaluation of Pharmacological Activity.

    Science.gov (United States)

    Evranos-Aksoz, Begum; Ucar, Gulberk; Tas, Sadik Taskin; Aksoz, Erkan; Yelekci, Kemal; Erikci, Acelya; Sara, Yildirim; Iskit, Alper Bektas

    2017-01-01

    Depression is a momentous disease that can greatly reduce the quality of life and cause death. In depression, neurotransmitter levels such as serotonine, dopamine and noradrenaline are impaired. Monoamine oxidases (MAO) are responsible for oxidative catalysis of these monoamine neurotransmitters. Because of this relation, MAO-A inhibitors show antidepressant activity by regulating neurotransmitter levels. This study was carried out to investigate the design, synthesis and activity of new antidepressant compounds in pyrazoline and hydrazone structure. Chalcones and hydrazides were heated under reflux to give new pyrazoline and hydrazone derivatives. Docking simulations were performed using AutoDock4.2. hMAO activities were determined by a fluorimetric method. To determine cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used. Behavioral activities of the three compounds were determined by using Forced Swim Test, Step-Through Passive Avoidance Test, Elevated Plus Maze and Open Field Arena Tests. According to in vitro tests, all of the synthesized compounds were found more potent than moclobemide and six of the synthesized compounds were found more selective than moclobemide. Three of the synthesized compounds were investigated for their behavioral activities comparing with moclobemide after 7 days of i.p. treatment at 30 mg/kg. One of the three compounds elicited significant antidepressant properties. All of the synthesized compounds were found potent hMAO-A inhibitors in in vitro screening tests. Only one of the in vivo tested three compounds, (3-(2-hydroxy-5-methylphenyl)-5- p-tolyl-4,5-dihydropyrazol-1-yl)(pyridin-4-yl) methanone indicated significant antidepressant activity. This article opens a window for further development of new pyrazoline and hydrazone derivatives as antidepressant agents. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  6. Functional recruitment of human complement inhibitor C4B-binding protein to outer membrane protein Rck of Salmonella.

    Directory of Open Access Journals (Sweden)

    Derek K Ho

    Full Text Available Resistance to complement mediated killing, or serum resistance, is a common trait of pathogenic bacteria. Rck is a 17 kDa outer membrane protein encoded on the virulence plasmid of Salmonella enterica serovars Typhimurium and Enteritidis. When expressed in either E. coli or S. enterica Typhimurium, Rck confers LPS-independent serum resistance as well as the ability to bind to and invade mammalian cells. Having recently shown that Rck binds the inhibitor of the alternative pathway of complement, factor H (fH, we hypothesized that Rck can also bind the inhibitor of the classical and lectin pathways, C4b-binding protein (C4BP. Using flow cytometry and direct binding assays, we demonstrate that E. coli expressing Rck binds C4BP from heat-inactivated serum and by using the purified protein. No binding was detected in the absence of Rck expression. C4BP bound to Rck is functional, as we observed factor I-mediated cleavage of C4b in cofactor assays. In competition assays, binding of radiolabeled C4BP to Rck was reduced by increasing concentrations of unlabeled protein. No effect was observed by increasing heparin or salt concentrations, suggesting mainly non-ionic interactions. Reduced binding of C4BP mutants lacking complement control protein domains (CCPs 7 or 8 was observed compared to wt C4BP, suggesting that these CCPs are involved in Rck binding. While these findings are restricted to Rck expression in E. coli, these data suggest that C4BP binding may be an additional mechanism of Rck-mediated complement resistance.

  7. A novel class I HDAC inhibitor, MPT0G030, induces cell apoptosis and differentiation in human colorectal cancer cells via HDAC1/PKCδ and E-cadherin.

    Science.gov (United States)

    Wang, Li-Ting; Liou, Jing-Ping; Li, Yu-Hsuan; Liu, Yi-Min; Pan, Shiow-Lin; Teng, Che-Ming

    2014-07-30

    Accumulation of genetic and epigenetic changes contributes to cancer development and progression. Compared with gene mutations or deletions, epigenetic changes are reversible, which alter the chromatin structure remodeling instead of changes in DNA sequence, and therefore become a promising strategy for chemotherapy. Histone deacetylases (HDACs) are a class of enzymes that responsible for the epigenetic regulation of gene expression. MPT0G030 is a potent and selective class I HDAC inhibitor which showed broad-spectrum cytotoxicity against various human cancer cell lines. in vitro fluorometric HDAC activity assay showed that MPT0G030 effectively inhibited Class I HDACs (HDAC1~3), which were overexpressed in many malignant neoplasms. Interestingly, MPT0G030 not only induced histone acetylation and tumor suppressor p21 transcription, but also redistributed E-cadherin and activated Protein Kinase C δ (PKCδ), which was linked to cell apoptosis and differentiation. Further, activation of PKCδ was demonstrated to be modulated through HDAC1. The in vivo anticancer activity of MPT0G030 and the importance of PKCδ were confirmed in the HT-29 tumor xenograft models. Taken together, those results indicate that MPT0G030, a class I HDAC inhibitor, has great potential as a new drug candidate for cancer therapy.

  8. Diverse small molecule inhibitors of human apurinic/apyrimidinic endonuclease APE1 identified from a screen of a large public collection.

    Directory of Open Access Journals (Sweden)

    Dorjbal Dorjsuren

    Full Text Available The major human apurinic/apyrimidinic endonuclease APE1 plays a pivotal role in the repair of base damage via participation in the DNA base excision repair (BER pathway. Increased activity of APE1, often observed in tumor cells, is thought to contribute to resistance to various anticancer drugs, whereas down-regulation of APE1 sensitizes cells to DNA damaging agents. Thus, inhibiting APE1 repair endonuclease function in cancer cells is considered a promising strategy to overcome therapeutic agent resistance. Despite ongoing efforts, inhibitors of APE1 with adequate drug-like properties have yet to be discovered. Using a kinetic fluorescence assay, we conducted a fully-automated high-throughput screen (HTS of the NIH Molecular Libraries Small Molecule Repository (MLSMR, as well as additional public collections, with each compound tested as a 7-concentration series in a 4 µL reaction volume. Actives identified from the screen were subjected to a panel of confirmatory and counterscreen tests. Several active molecules were identified that inhibited APE1 in two independent assay formats and exhibited potentiation of the genotoxic effect of methyl methanesulfonate with a concomitant increase in AP sites, a hallmark of intracellular APE1 inhibition; a number of these chemotypes could be good starting points for further medicinal chemistry optimization. To our knowledge, this represents the largest-scale HTS to identify inhibitors of APE1, and provides a key first step in the development of novel agents targeting BER for cancer treatment.

  9. Probing the Inhibitor versus Chaperone Properties of sp2-Iminosugars towards Human β-Glucocerebrosidase: A Picomolar Chaperone for Gaucher Disease

    Directory of Open Access Journals (Sweden)

    Teresa Mena-Barragán

    2018-04-01

    Full Text Available A series of sp2-iminosugar glycomimetics differing in the reducing or nonreducing character, the configurational pattern (d-gluco or l-ido, the architecture of the glycone skeleton, and the nature of the nonglycone substituent has been synthesized and assayed for their inhibition properties towards commercial glycosidases. On the basis of their affinity and selectivity towards GH1 β-glucosidases, reducing and nonreducing bicyclic derivatives having a hydroxylation profile of structural complementarity with d-glucose and incorporating an N′-octyl-isourea or -isothiourea segment were selected for further evaluation of their inhibitory/chaperoning potential against human glucocerebrosidase (GCase. The 1-deoxynojirimycin (DNJ-related nonreducing conjugates behaved as stronger GCase inhibitors than the reducing counterparts and exhibited potent chaperoning capabilities in Gaucher fibroblasts hosting the neuronopathic G188S/G183W mutation, the isothiourea derivative being indeed one of the most efficient chaperone candidates reported up to date (70% activity enhancement at 20 pM. At their optimal concentration, the four selected compounds promoted mutant GCase activity enhancements over 3-fold; yet, the inhibitor/chaperoning balance became unfavorable at much lower concentration for nonreducing as compared to reducing derivatives.

  10. Mechanism of cell death by 5-aminolevulinic acid-based photodynamic action and its enhancement by ferrochelatase inhibitors in human histiocytic lymphoma cell line U937.

    Science.gov (United States)

    Amo, Takashi; Kawanishi, Noriaki; Uchida, Masataka; Fujita, Hirofumi; Oyanagi, Eri; Utsumi, Toshihiko; Ogino, Tetsuya; Inoue, Keiji; Shuin, Taro; Utsumi, Kozo; Sasaki, Junzo

    2009-12-01

    Photodynamic therapy (PDT) for tumors is based on the tumor-selective accumulation of a photosensitizer, protoporphyrin IX (PpIX), followed by irradiation with visible light. However, the molecular mechanism of cell death caused by PDT has not been fully elucidated. The 5-aminolevulinic acid (ALA)-based photodynamic action (PDA) was dependent on the accumulation of PpIX, the level of which decreased rapidly by eliminating ALA from the incubation medium in human histiocytic lymphoma U937 cells. PDA induced apoptosis characterized by lipid peroxidation, increase in Bak and Bax/Bcl-xL, decrease in Bid, membrane depolarization, cytochrome c release, caspase-3 activation, phosphatidylserine (PS) externalization. PDT-induced cell death seemed to occur predominantly via apoptosis through distribution of PpIX in mitochondria. These cell death events were enhanced by ferrochelatase inhibitors. These results indicated that ALA-based-PDA induced apoptotic cell death through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors even at low concentrations of ALA.

  11. L-Chicoric acid inhibits human immunodeficiency virus type 1 integration in vivo and is a noncompetitive but reversible inhibitor of HIV-1 integrase in vitro

    International Nuclear Information System (INIS)

    Reinke, Ryan A.; Lee, Deborah J.; McDougall, Brenda R.; King, Peter J.; Victoria, Joseph; Mao Yingqun; Lei Xiangyang; Reinecke, Manfred G.; Robinson, W. Edward

    2004-01-01

    The human immunodeficiency virus (HIV) integrase (IN) must covalently join the viral cDNA into a host chromosome for productive HIV infection. L-Chicoric acid (L-CA) enters cells poorly but is a potent inhibitor of IN in vitro. Using quantitative real-time polymerase chain reaction (PCR), L-CA inhibits integration at concentrations from 500 nM to 10 μM but also inhibits entry at concentrations above 1 μM. Using recombinant HIV IN, steady-state kinetic analyses with L-CA were consistent with a noncompetitive or irreversible mechanism of inhibition. IN, in the presence or absence of L-CA, was successively washed. Inhibition of IN diminished, demonstrating that L-CA was reversibly bound to the protein. These data demonstrate that L-CA is a noncompetitive but reversible inhibitor of IN in vitro and of HIV integration in vivo. Thus, L-CA likely interacts with amino acids other than those which bind substrate

  12. Novel stable HBV producing cell line systems for expression and screening antiviral inhibitor of hepatitis B virus in human hepatoma cell line.

    Science.gov (United States)

    Ogura, Naoki; Ogawa, Kazuya; Watashi, Koichi; Ito, Takayoshi; Wakita, Takaji

    2018-03-25

    Chronic hepatitis B virus (HBV) infection is currently a major public health burden. Therefore, there is an urgent need for the development of novel antiviral inhibitors. The stable HBV-producing cell lines of genotype D are widely used to investigate the HBV life cycle and to evaluate antiviral agents. However, stable HBV-producing cell lines of different genotypes do not exist. To construct more convenient and efficient novel cell systems, stable cell lines of genotypes A, B, and C were established using a full-length HBV genome sequence isolated from chronic HBV patients in human hepatoma HepG2 cells. Novel HBV clones were identified and stable HBV-producing cell lines derived from these clones were constructed. HBV replication activities demonstrated time-dependent expression, and the novel cell lines were susceptible to several antiviral inhibitors with no cytotoxicity. Furthermore, infectious viruses were produced from these cell lines. In conclusion, we have established novel stable HBV-producing cell line systems of genotypes A, B, and C. These systems can provide valuable tools for screening antiviral agents and analyzing viral phenotypes in vitro. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Antitumor action of CDK inhibitor LS-007 as a single agent and in combination with ABT-199 against human acute leukemia cells.

    Science.gov (United States)

    Xie, Shao; Jiang, Hui; Zhai, Xiao-Wen; Wei, Fan; Wang, Shu-Dong; Ding, Jian; Chen, Yi

    2016-11-01

    LS-007 is a CDK inhibitor, which exhibits potent antitumor activity against chronic lymphocytic leukemia and ovarian cancer cells. In this study, we further evaluated the antitumor activity of LS-007 alone and in combination with a Bcl-2 inhibitor ABT-199 in acute leukemia (AL) cells. Cell viability was detected using resazurin assay, and cell apoptosis was examined using Annexin V/PI double staining and flow cytometry. The inhibition of LS-007 on kinases was evaluated with the mobility shift assay or ELISA. The expression of relevant signaling molecules was assessed using Western blotting and RT-PCR. Primary lymphocytes from patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were separated using Ficoll-Paque PLUS. LS-007 inhibited the proliferation of 6 AL cell lines with IC 50 values of 100-200 nmol/L, and decreased the survival of ALL and AML patient-derived lymphocytes with mean LD 50 value of 67 and 102 nmol/L, respectively. In kinase assays in vitro, LS-007 was more selective for the CDK family, inhibiting CDK2, CDK9, CDK1 and CDK4 at low nanomolar concentrations. In HL-60 and CCRF-CEM cells, LS-007 (0.1-0.4 μmol/L) dose-dependently induced cell apoptosis predominantly through CDK9 inhibition-related dephosphorylation at the ser2 residue of RNA pol II and the corresponding depletion of anti-apoptotic proteins, especially Mcl-1 and XIAP. LS-007 (0.2 and 0.4 μmol/L) also induced cell apoptosis in the patient-derived lymphocytes. In HL-60, CCRF-CEM and Molt-4 cells, combined application of LS-007 with ABT-199 (1 or 2 μmol/L) markedly increased cell apoptosis with a maximal decrease in the XIAP levels as compared with either drug used alone. CDK inhibitor LS-007 potently inhibits the established human AL cell lines and primary AL blasts, and it also shows remarkable synergy with Bcl-2 inhibitor ABT-199.

  14. Effects of PARP-1 inhibitors AG-014699 and AZD2281 on proliferation and apoptosis of human hepatoma cell line HepG2

    Directory of Open Access Journals (Sweden)

    DU Senrong

    2015-06-01

    Full Text Available ObjectiveTo observe the inhibitory and pro-apoptotic effects of two poly(ADP-ribose polymerase (PARP-1 inhibitors, AG-014699 and AZD2281, on human hepatoma HepG2 cells and preliminarily explore the mechanism by which AG-014699 induces HepG2 cell apoptosis, and to provide a new therapeutic target for hepatoma. MethodsThe effects of different concentrations of AG-014699 and AZD2281 on HepG2 cell proliferation were determined by MTT assay. The cell apoptosis rate was measured by flow cytometry. The expression levels of caspase-3 and caspase-8 were measured by Western Blot. Inter-group comparison was made by t test. ResultsBoth AG-014699 and AZD2281 suppressed HepG2 cell proliferation in a time- and dose-dependent manner. However, the sensitivity of HepG2 cells to the two PARP-1 inhibitors was different. The half-maximal inhibitory concentrations of AG-014699 and AZD2281 at 48 h determined by MTT assay were about 20 μmol/L and 400 μmol/L, respectively. Flow cytometry and Western blot were not used to evaluate the apoptosis of HepG2 cells exposed to AZD2281 to which these cells were not sensitive. HepG2 cell apoptosis could be induced by 10, 30, and 50 μmol/L AG-014699, and the highest apoptosis rate at 48 h was significantly higher than that of the control group (3100%±2.13% vs 09%±0013%, P<0.01. Compared with those in the control group, the protein levels of caspase-3 and caspase-8 in HepG2 cells after 48-h exposure to 30, and 50 μmol/L AG-014699 increased. ConclusionThe two PARP-1 inhibitors AG-014699 and AZD2281 can inhibit the proliferation of HepG2 cells, which showed different sensitivities to the two inhibitors. AG-014699 can induce HepG2 cell apoptosis by up-regulating the protein expression of caspase-3 and caspase-8.

  15. Poly(ADP-ribose) polymerase inhibitors suppress UV-induced human immunodeficiency virus type 1 gene expression at the posttranscriptional level

    International Nuclear Information System (INIS)

    Yamagoe, S.; Kohda, T.; Oishi, M.

    1991-01-01

    Gene expression of human immunodeficiency virus type 1 (HIV-1) is induced not only by trans activation mediated through a gene product (tat) encoded by the virus but also by treatment of virus-carrying cells with DNA-damaging agents such as UV light. Employing an artificially constructed DNA in which the chloramphenicol acetyltransferase gene was placed under the control of the HIV-1 long terminal repeat, we analyzed the induction process in HeLa cells and found that inhibitors of poly(ADP-ribose) polymerase suppressed UV-induced HIV-1 gene expression but not tat-mediated expression. We also found that suppression occurs at the posttranscriptional level. These results indicate that HIV-1 gene expression is activated by at least two different mechanisms, one of which involves poly-ADP ribosylation. A possible new role of poly-ADP ribosylation in the regulation of specific gene expression is also discussed

  16. B-cell lymphoma 2 inhibitor ABT-737 induces Beclin1- and reactive oxygen species-dependent autophagy in Adriamycin-resistant human hepatocellular carcinoma cells.

    Science.gov (United States)

    Yao, Xiaoxiao; Li, Xiaoning; Zhang, Dan; Xie, Yingjun; Sun, Baozhen; Li, Hang; Sun, Liankun; Zhang, Xuewen

    2017-03-01

    ABT-737, a B-cell lymphoma 2 homology 3 mimetic, not only induces cell apoptosis by inhibiting the interaction of B-cell lymphoma 2 and Bax but also induces cell autophagy by interrupting the interaction of B-cell lymphoma 2 and Beclin1. Several recent studies have reported that ABT-737 has antitumor efficacy in diverse cancers. However, another study showed that hepatocellular carcinoma cells with high B-cell lymphoma 2 expression were resistant to ABT-737 compared to hepatocellular carcinoma cells with low B-cell lymphoma 2 expression. It was also found that ABT-737-induced autophagy is crucial for drug resistance. Here, we observed that of B-cell lymphoma 2 expression in Adriamycin-resistant human hepatocellular carcinoma HepG2/ADM cells is higher than that in human hepatocellular carcinoma HepG2 cells. Therefore, we further confirmed the mechanism and effect of autophagy induced by ABT-737 on apoptosis in HepG2/ADM cells with high B-cell lymphoma 2 expression. Our results showed that ABT-737 induced apoptosis and autophagy in time- and dose-dependent manner in HepG2/ADM cells, and this ABT-737-induced autophagy was Beclin1-dependent. In addition, we demonstrated that ABT-737 induced reactive oxygen species-mediated autophagy, and the reactive oxygen species-inhibitor N-acetyl-l-cysteine suppressed the reactive oxygen species-induced autophagy and ABT-737-induced increase in HepG2/ADM cell apoptosis. Furthermore, autophagy inhibitors increased HepG2/ADM cell apoptosis. In conclusion, our study further confirms that Beclin1- and reactive oxygen species-dependent autophagy induced by ABT-737 also plays a protective function in HepG2/ADM cells, which show B-cell lymphoma 2 expression higher than that in HepG2 cells.

  17. Quantitative structure-activity relationship analysis of human neutrophil elastase inhibitors using shuffling classification and regression trees and adaptive neuro-fuzzy inference systems.

    Science.gov (United States)

    Asadollahi-Baboli, M

    2012-07-01

    The purpose of this study was to develop quantitative structure-activity relationship models for N-benzoylindazole derivatives as inhibitors of human neutrophil elastase. These models were developed with the aid of classification and regression trees (CART) and an adaptive neuro-fuzzy inference system (ANFIS) combined with a shuffling cross-validation technique using interpretable descriptors. More than one hundred meaningful descriptors, representing various structural characteristics for all 51 N-benzoylindazole derivatives in the data set, were calculated and used as the original variables for shuffling CART modelling. Five descriptors of average Wiener index, Kier benzene-likeliness index, subpolarity parameter, average shape profile index of order 2 and folding degree index selected by the shuffling CART technique have been used as inputs of the ANFIS for prediction of inhibition behaviour of N-benzoylindazole derivatives. The results of the developed shuffling CART-ANFIS model compared to other techniques, such as genetic algorithm (GA)-partial least square (PLS)-ANFIS and stepwise multiple linear regression (MLR)-ANFIS, are promising and descriptive. The satisfactory results r2p = 0.845, Q2(LOO) = 0.861, r2(L25%O) = 0.829, RMSE(LOO)  = 0.305 and RMSE(L25%O)  = 0.336) demonstrate that shuffling CART-ANFIS models present the relationship between human neutrophil elastase inhibitor activity and molecular descriptors, and they yield predictions in excellent agreement with the experimental values.

  18. Structural and biophysical analysis of interactions between cod and human uracil-DNA N-glycosylase (UNG) and UNG inhibitor (Ugi)

    Energy Technology Data Exchange (ETDEWEB)

    Assefa, Netsanet Gizaw [UiT The Arctic University of Norway, 9037 Tromsø (Norway); Niiranen, Laila [UiT The Arctic University of Norway, 9037 Tromsø (Norway); University of Turku, FIN-20014 Turku (Finland); Johnson, Kenneth A.; Leiros, Hanna-Kirsti Schrøder; Smalås, Arne Oskar; Willassen, Nils Peder [UiT The Arctic University of Norway, 9037 Tromsø (Norway); Moe, Elin, E-mail: elin.moe@uit.no [UiT The Arctic University of Norway, 9037 Tromsø (Norway); Universidade Nova de Lisboa, Avenida da Republica (EAN), 2780-157 Oeiras (Portugal)

    2014-08-01

    A structural and biophysical study of the interactions between cod and human uracil-DNA N-glycosylase (UNG) and their inhibitor Ugi is presented. The stronger interaction between cod UNG and Ugi can be explained by a greater positive electrostatic surface potential. Uracil-DNA N-glycosylase from Atlantic cod (cUNG) shows cold-adapted features such as high catalytic efficiency, a low temperature optimum for activity and reduced thermal stability compared with its mesophilic homologue human UNG (hUNG). In order to understand the role of the enzyme–substrate interaction related to the cold-adapted properties, the structure of cUNG in complex with a bacteriophage encoded natural UNG inhibitor (Ugi) has been determined. The interaction has also been analyzed by isothermal titration calorimetry (ITC). The crystal structure of cUNG–Ugi was determined to a resolution of 1.9 Å with eight complexes in the asymmetric unit related through noncrystallographic symmetry. A comparison of the cUNG–Ugi complex with previously determined structures of UNG–Ugi shows that they are very similar, and confirmed the nucleotide-mimicking properties of Ugi. Biophysically, the interaction between cUNG and Ugi is very strong and shows a binding constant (K{sub b}) which is one order of magnitude larger than that for hUNG–Ugi. The binding of both cUNG and hUNG to Ugi was shown to be favoured by both enthalpic and entropic forces; however, the binding of cUNG to Ugi is mainly dominated by enthalpy, while the entropic term is dominant for hUNG. The observed differences in the binding properties may be explained by an overall greater positive electrostatic surface potential in the protein–Ugi interface of cUNG and the slightly more hydrophobic surface of hUNG.

  19. Human kallikrein 2 (hK2), but not prostate-specific antigen (PSA), rapidly complexes with protease inhibitor 6 (PI-6) released from prostate carcinoma cells.

    Science.gov (United States)

    Saedi, M S; Zhu, Z; Marker, K; Liu, R S; Carpenter, P M; Rittenhouse, H; Mikolajczyk, S D

    2001-11-01

    Human kallikrein 2 (hK2) is a secreted, trypsin-like protease that shares 80% amino acid sequence identity with prostate-specific antigen (PSA). hK2 has been shown to be a serum marker for prostate cancer and may also play a role in cancer progression and metastasis. We have previously identified a novel complex between human kallikrein 2 (hK2) and protease inhibitor 6 (PI-6) in prostate cancer tissue. PI-6 is an intracellular serine protease inhibitor with both antitrypsin and antichymotrypsin activity. In the current study we have shown that PI-6 forms a rapid in vitro complex with hK2 but does not complex with PSA. Recombinant mammalian cells expressing both hK2 and PI-6 showed hK2-PI-6 complex in the spent media only after cell death and lysis. Similarly, LNCaP cells expressing endogenous hK2 and PI-6 showed extracellular hK2-PI-6 complex formation concurrently with cell death. Immunostaining of prostate cancer tissues with PI-6 monoclonal antibodies showed a marked preferential staining pattern in cancerous epithelial cells compared with noncancerous tissue. These results indicate that the hK2-PI-6 complex may be a naturally occurring marker of tissue damage and necrosis associated with neoplasia. Both hK2 and PI-6 were shed into the lumen of prostate cancer glands as granular material that appeared to be cellular necrotic debris. The differential staining pattern of PI6 in tissues suggests a complex regulation of PI-6 expression that may play a role in other aspects of neoplastic progression. Copyright 2001 Wiley-Liss, Inc.

  20. The hepatitis B virus ribonuclease H is sensitive to inhibitors of the human immunodeficiency virus ribonuclease H and integrase enzymes.

    Directory of Open Access Journals (Sweden)

    John E Tavis

    2013-01-01

    Full Text Available Nucleos(tide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(tide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(tide analogs. The HBV ribonuclease H (RNAseH is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 µM, the best compounds had low micromolar IC(50 values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 µM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug

  1. Selective killing of human immunodeficiency virus infected cells by non-nucleoside reverse transcriptase inhibitor-induced activation of HIV protease

    Directory of Open Access Journals (Sweden)

    Smeulders Liesbeth

    2010-10-01

    Full Text Available Abstract Background Current antiretroviral therapy against human immunodeficiency virus (HIV-1 reduces viral load and thereby prevents viral spread, but it cannot eradicate proviral genomes from infected cells. Cells in immunological sanctuaries as well as cells producing low levels of virus apparently contribute to a reservoir that maintains HIV persistence in the presence of highly active antiretroviral therapy. Thus, accelerated elimination of virus producing cells may represent a complementary strategy to control HIV infection. Here we sought to exploit HIV protease (PR related cytotoxicity in order to develop a strategy for drug induced killing of HIV producing cells. PR processes the viral Gag and Gag-Pol polyproteins during virus maturation, but is also implicated in killing of virus producing cells through off-target cleavage of host proteins. It has been observed previously that micromolar concentrations of certain non-nucleoside reverse transcriptase inhibitors (NNRTIs can stimulate intracellular PR activity, presumably by enhancing Gag-Pol dimerization. Results Using a newly developed cell-based assay we compared the degree of PR activation displayed by various NNRTIs. We identified inhibitors showing higher potency with respect to PR activation than previously described for NNRTIs, with the most potent compounds resulting in ~2-fold increase of the Gag processing signal at 250 nM. The degree of enhancement of intracellular Gag processing correlated with the compound's ability to enhance RT dimerization in a mammalian two-hybrid assay. Compounds were analyzed for their potential to mediate specific killing of chronically infected MT-4 cells. Levels of cytotoxicity on HIV infected cells determined for the different NNRTIs corresponded to the relative degree of drug induced intracellular PR activation, with CC50 values ranging from ~0.3 μM to above the tested concentration range (10 μM. Specific cytotoxicity was reverted by addition

  2. Is transforming growth factor-β signaling activated in human hypertrophied prostate treated by 5-alpha reductase inhibitor?

    Science.gov (United States)

    Kim, Hye Kyung; Zhao, Chen; Choi, Bo Ram; Chae, Han Jung; Kim, Do Sung; Park, Jong Kwan

    2013-01-01

    It is well known that androgen deprivation relates to penile fibrosis, so we hypothesize that long-term treatment with 5-alphareductase inhibitors (5ARIs) may increase the risk of fibrosis of prostate. Thirty-two BPH patients who underwent transurethral resection of the prostate were enrolled. The patients were divided into two groups: group one, 16 patients underwent TURP who had been treated with tamsulosin for 2 years; group two, 16 patients underwent TURP who had been treated with combination of tamsulosin and dutasteride for at least 1 year. We evaluated the expressions of nNOS, iNOS, eNOS, TGF-β1, TGF-β2, phosphorylated-Smad2/3 (p-Smad2/3), E-cadherin, N-cadherin, and α-smooth muscle actin in the resected prostate tissues by western blotting, and the TGF-β concentration was determined by ELISA kit. The expressions of 3 isoforms of NOS were significantly increased in group 2 except of eNOS in lateral prostate, and the expressions of TGF-β1, TGF-β2, and p-Smad2/3 increased about 2-fold compared with group 1. In group 2, the E-cadherin expression decreased while N-cadherin expression increased significantly. The overexpression of nNOS may contribute to prostate smooth muscle relaxation; however, long-time treatment with 5 ARI increases the risk of fibrosis of prostate.

  3. ETS1 mediates MEK1/2-dependent overexpression of cancerous inhibitor of protein phosphatase 2A (CIP2A in human cancer cells.

    Directory of Open Access Journals (Sweden)

    Anchit Khanna

    2011-03-01

    Full Text Available EGFR-MEK-ERK signaling pathway has an established role in promoting malignant growth and disease progression in human cancers. Therefore identification of transcriptional targets mediating the oncogenic effects of the EGFR-MEK-ERK pathway would be highly relevant. Cancerous inhibitor of protein phosphatase 2A (CIP2A is a recently characterized human oncoprotein. CIP2A promotes malignant cell growth and is over expressed at high frequency (40-80% in most of the human cancer types. However, the mechanisms inducing its expression in cancer still remain largely unexplored. Here we present systematic analysis of contribution of potential gene regulatory mechanisms for high CIP2A expression in cancer. Our data shows that evolutionary conserved CpG islands at the proximal CIP2A promoter are not methylated both in normal and cancer cells. Furthermore, sequencing of the active CIP2A promoter region from altogether seven normal and malignant cell types did not reveal any sequence alterations that would increase CIP2A expression specifically in cancer cells. However, treatment of cancer cells with various signaling pathway inhibitors revealed that CIP2A mRNA expression was sensitive to inhibition of EGFR activity as well as inhibition or activation of MEK-ERK pathway. Moreover, MEK1/2-specific siRNAs decreased CIP2A protein expression. Series of CIP2A promoter-luciferase constructs were created to identify proximal -27 to -107 promoter region responsible for MEK-dependent stimulation of CIP2A expression. Additional mutagenesis and chromatin immunoprecipitation experiments revealed ETS1 as the transcription factor mediating stimulation of CIP2A expression through EGFR-MEK pathway. Thus, ETS1 is probably mediating high CIP2A expression in human cancers with increased EGFR-MEK1/2-ERK pathway activity. These results also suggest that in addition to its established role in invasion and angiogenesis, ETS1 may support malignant cellular growth via regulation of

  4. Inhibition of human platelet aggregation by dihydropyrano- and dihydrofuranocoumarins, a new class of cAMP-phosphodiesterase inhibitors

    DEFF Research Database (Denmark)

    Thastrup, Ole; Knudsen, J B; Lemmich, J

    1985-01-01

    Certain esters of dihydropyranocoumarin and dihydrofuranocoumarin alcohols have previously been shown to inhibit the cAMP-phosphodiesterase from bovine heart. We now report that these naturally occurring coumarins inhibit the high affinity (Km = 1.1 microM) cAMP-phosphodiesterase from human...... platelets with activities that closely correlate with those obtained using phosphodiesterase from bovine heart tissue. Additionally the coumarins inhibit the aggregation of human platelets induced with ADP, adrenaline and collagen with activities comparable to those of dipyridamole. A lack of significant...

  5. The crystal structure of human dipeptidyl peptidase I (cathepsin C) in complex with the inhibitor Gly-Phe-CHN2

    DEFF Research Database (Denmark)

    Mølgaard, Anne; Arnau, Jose; Lauritzen, C.

    2007-01-01

    hDDPI (human dipeptidyl peptidase I) is a lysosomal cysteine protease involved in zymogen activation of granule-associated proteases, including granzymes A and B from cytotoxic T-lymphocytes and natural killer cells, cathepsin G and neutrophil elastase, and mast cell tryptase and chymase. In the ......hDDPI (human dipeptidyl peptidase I) is a lysosomal cysteine protease involved in zymogen activation of granule-associated proteases, including granzymes A and B from cytotoxic T-lymphocytes and natural killer cells, cathepsin G and neutrophil elastase, and mast cell tryptase and chymase...

  6. Simultaneous human papilloma virus type 16 E7 and cdk inhibitor p21 expression induces apoptosis and cathepsin B activation

    DEFF Research Database (Denmark)

    Kaznelson, Dorte Wissing; Bruun, Silas; Monrad, Astrid

    2004-01-01

    Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation and induc......Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation...

  7. Carbonic anhydrase inhibitors: Design, synthesis and structural characterization of new heteroaryl-N-carbonylbenzenesulfonamides targeting druggable human carbonic anhydrase isoforms

    Czech Academy of Sciences Publication Activity Database

    Buemi, M. R.; De Luca, L.; Ferro, S.; Bruno, E.; Ceruso, M.; Supuran, C. T.; Pospíšilová, K.; Brynda, Jiří; Řezáčová, Pavlína; Gitto, R.

    2015-01-01

    Roč. 102, Sep 18 (2015), s. 223-232 ISSN 0223-5234 Institutional support: RVO:61388963 Keywords : human carbonic anhydrase * isoquinoline * quinoline * X-ray * molecular docking Subject RIV: CE - Biochemistry Impact factor: 3.902, year: 2015

  8. Proteasome inhibitor MG132 sensitizes HPV-positive human cervical cancer cells to rhTRAIL-induced apoptosis

    NARCIS (Netherlands)

    Hougardy, BMT; Maduro, JH; van der Zee, AGJ; de Groot, DJA; van den Heuvel, FAJ; de Vries, EGE; de Jong, S

    2006-01-01

    In cervical carcinogenesis, the p53 tumor suppressor pathway is disrupted by HPV (human papilloma virus) E6 oncogene expression. E6 targets p53 for rapid proteasome-mediated degradation. We therefore investigated whether proteasome inhibition by MG132 could restore wild-type p53 levels and sensitize

  9. Liquid chromatography-tandem mass spectrometric assay for the nucleoside reverse transcriptase inhibitor emtricitabine in human plasma

    NARCIS (Netherlands)

    Sparidans, Rolf W.; Prins, Jan M.; Schellens, Jan H. M.; Beijnen, Jos H.

    2007-01-01

    A liquid chromatography-tandem mass spectrometric assay for the determination of the antiretroviral nucleoside emtricitabine in human plasma was developed and validated using a simple sample pre-treatment procedure. After addition of 5'-deoxy-5-fluorocytidine as the internal standard and protein

  10. Novel Substituted Heteroaromatic Piperazine and Piperidine Derivatives as Inhibitors of Human Enterovirus 71 and Coxsackievirus A16

    Directory of Open Access Journals (Sweden)

    Xian Zhang

    2013-04-01

    Full Text Available A series of substituted heteroaromatic piperazine and piperidine derivatives were found through virtual screening based on the structure of human enterovirus 71 capsid protein VP1. The preliminary biological evaluation revealed that compounds 8e and 9e have potent activity against EV71 and Coxsackievirus A16 with low cytotoxicity.

  11. Disposition and metabolism of [14C]-levomilnacipran, a serotonin and norepinephrine reuptake inhibitor, in humans, monkeys, and rats

    Directory of Open Access Journals (Sweden)

    Brunner V

    2015-06-01

    Full Text Available Valérie Brunner,1 Bernadette Maynadier,2 Laishun Chen,3 Louise Roques,2 Isabelle Hude,2 Sébastien Séguier,2 Laurence Barthe,1 Philippe Hermann11Pierre Fabre Médicament, Centre de R&D, Toulouse, 2Centre Experimental PreClinque, Campans, France; 3Forest Research Institute Inc., an affiliate of Actavis Inc., Jersey City, NJ, USAAbstract: Levomilnacipran is approved in the US for the treatment of major depressive disorder in adults. We characterized the metabolic profile of levomilnacipran in humans, monkeys, and rats after oral administration of [14C]-levomilnacipran. In vitro binding of levomilnacipran to human plasma proteins was also studied. Unchanged levomilnacipran was the major circulating compound after dosing in all species. Within 12 hours of dosing in humans, levomilnacipran accounted for 52.9% of total plasma radioactivity; the circulating metabolites N-desethyl levomilnacipran N-carbamoyl glucuronide, N-desethyl levomilnacipran, and levomilnacipran N-carbamoyl glucuronide accounted for 11.3%, 7.5%, and 5.6%, respectively. Similar results were seen in monkeys. N-Desethyl levomilnacipran and p-hydroxy levomilnacipran were the main circulating metabolites in rats. Mass balance results indicated that renal excretion was the major route of elimination with 58.4%, 35.5%, and 40.2% of total radioactivity being excreted as unchanged levomilnacipran in humans, monkeys, and rats, respectively. N-Desethyl levomilnacipran was detected in human, monkey, and rat urine (18.2%, 12.4%, and 7.9% of administered dose, respectively. Human and monkey urine contained measurable quantities of levomilnacipran glucuronide (3.8% and 4.1% of administered dose, respectively and N-desethyl levomilnacipran glucuronide (3.2% and 2.3% of administered dose, respectively; these metabolites were not detected in rat urine. The metabolites p-hydroxy levomilnacipran and p-hydroxy levomilnacipran glucuronide were detected in human urine (≤1.2% of administered dose

  12. Orlistat (Ro 18-0647), a lipase inhibitor, in the treatment of human obesity: a multiple dose study.

    Science.gov (United States)

    Drent, M L; Larsson, I; William-Olsson, T; Quaade, F; Czubayko, F; von Bergmann, K; Strobel, W; Sjöström, L; van der Veen, E A

    1995-04-01

    To evaluate efficacy and tolerability of the lipase inhibitor Orlistat (Ro 18-0647) in doses of 10, 60 and 120 mg three times a day in addition to a mild hypocaloric diet containing 30% of calories as fat. 4 week single-blind placebo run-in period of diet alone followed by a 12 week double-blind, placebo-controlled, randomized treatment period. Five European outpatient clinics specializing in endocrinology and/or the treatment of obesity, one central laboratory. Of 237 healthy obese subjects meeting the inclusion criteria, 188 showed compliance to the diet during the run-in period and were randomized for the treatment period. Primary efficacy criterion was the difference in weight loss after 12 weeks of treatment between the Orlistat treated groups and the diet alone group. Secondary efficacy criteria were changes in serum total, HDL- and LDL-cholesterol. Compared to placebo a mean (+/- s.e.) additional weight loss of 0.63 +/- 0.54 kg with 30 mg a day (P = 0.246), 0.71 +/- 0.55 kg with 180 mg a day (P = 0.190) and 1.75 +/- 0.54 kg with 360 mg a day was seen (P = 0.001) or Orlistat was observed. Overall data indicated dose-dependency. Small decreases were seen in total and LDL-cholesterol (significant in the 180 and 360 mg a day groups) and LDL- to HDL-cholesterol ratio (significant in the 360 mg a day group only). Mild, mostly gastrointestinal side effects were observed more frequently in the Orlistat groups and caused premature withdrawal from the study in only four patients. No marked laboratory abnormalities were shown, including the lipid-soluble vitamins A, D and E. Orlistat, in an apparently dose-dependent manner, leads to additional weight loss compared to diet alone and overall, is well tolerated.

  13. The STAT3 inhibitor pimozide impedes cell proliferation and induces ROS generation in human osteosarcoma by suppressing catalase expression.

    Science.gov (United States)

    Cai, Nan; Zhou, Wei; Ye, Lan-Lan; Chen, Jun; Liang, Qiu-Ni; Chang, Gang; Chen, Jia-Jie

    2017-01-01

    Currently, there is a considerable need to develop new treatments for osteosarcoma (OS), a very aggressive bone cancer. The activation of STAT3 signaling is positively associated with poor prognosis and aggressive progression in OS patients. Our previous study reported that the FDA-approved antipsychotic drug pimozide had anti-tumor activity against hepatocellular carcinoma and prostate cancer cells by suppressing STAT3 activity. Therefore, the aim of this study was to investigate the specific effect of pimozide on OS cells and the underlying molecular mechanism. Pimozide inhibited cell proliferation, colony formation, and sphere formation capacities of the OS cells in a dose-dependent manner, inducing G0/G1 phase cell cycle arrest. Pimozide reduced the percentage of side population cells representing cancer stem-like cells and enhanced the sensitivity of OS cells to 5-FU induced proliferative inhibition. In addition, pimozide induced apoptosis of U2OS cells, which showed increased expression of cleaved-PARP, a marker of programmed cell death. Moreover, pimozide suppressed Erk signaling in OS cells. Importantly, pimozide induced ROS generation by downregulating the expression of the antioxidant enzyme catalase (CAT). NAC treatment partially reversed the ROS generation and cytotoxic effects induced by pimozide. CAT treatment attenuated the pimozide-induced proliferation inhibition. The decrease of CAT expression induced by pimozide was potentially mediated through the suppression of cellular STAT3 activity in OS cells. Thus, pimozide may be a novel STAT3 inhibitor that suppresses cellular STAT3 activity to inhibit OS cells or stem-like cells and is a novel potential anti-cancer agent in OS treatment.

  14. Is Transforming Growth Factor-β Signaling Activated in Human Hypertrophied Prostate Treated by 5-Alpha Reductase Inhibitor?

    Directory of Open Access Journals (Sweden)

    Hye Kyung Kim

    2013-01-01

    Full Text Available Background and Aim. It is well known that androgen deprivation relates to penile fibrosis, so we hypothesize that long-term treatment with 5-alphareductase inhibitors (5ARIs may increase the risk of fibrosis of prostate. Patients and Methods. Thirty-two BPH patients who underwent transurethral resection of the prostate were enrolled. The patients were divided into two groups: group one, 16 patients underwent TURP who had been treated with tamsulosin for 2 years; group two, 16 patients underwent TURP who had been treated with combination of tamsulosin and dutasteride for at least 1 year. We evaluated the expressions of nNOS, iNOS, eNOS, TGF-β1, TGF-β2, phosphorylated-Smad2/3 (p-Smad2/3, E-cadherin, N-cadherin, and α-smooth muscle actin in the resected prostate tissues by western blotting, and the TGF-β concentration was determined by ELISA kit. Results. The expressions of 3 isoforms of NOS were significantly increased in group 2 except of eNOS in lateral prostate, and the expressions of TGF-β1, TGF-β2, and p-Smad2/3 increased about 2-fold compared with group 1. In group 2, the E-cadherin expression decreased while N-cadherin expression increased significantly. Conclusions. The overexpression of nNOS may contribute to prostate smooth muscle relaxation; however, long-time treatment with 5 ARI increases the risk of fibrosis of prostate.

  15. The effect of antenatal depression and selective serotonin reuptake inhibitor treatment on nerve growth factor signaling in human placenta.

    Directory of Open Access Journals (Sweden)

    Helena Kaihola

    Full Text Available Depressive symptoms during pregnancy are common and may have impact on the developing child. Selective serotonin reuptake inhibitors (SSRIs are the most prescribed antidepressant treatment, but unfortunately, these treatments can also negatively affect the behavioral development and health of a child during pregnancy. In addition, serotonin (5-HT exerts neurotrophic actions with thus far not fully known effects in the offspring. The neurotrophic growth factor (NGF is involved in neuronal cell survival and differentiation, and altered placenta levels have been found to increase the risk for pregnancy complications, similar to those found in women treated with SSRIs. We therefore investigated whether the NGF signaling pathway was altered in the placenta from women treated with SSRIs (n = 12 and compared them with placenta from depressed (n = 12 and healthy mothers (n = 12. Results from immunohistochemical stainings revealed that placental NGF protein levels of SSRI-treated women were increased in both trophoblasts and endothelial cells compared with depressed and control women. In addition, downstream of the NGF receptor TrkA, increased levels of the signaling proteins ROCK2 and phosphorylated Raf-1 were found in stromal cells and a tendency towards increased levels of ROCK2 in trophoblasts and endothelial cells in SSRI-treated women when compared to healthy controls. SSRI-treated women also displayed increased levels of phosphorylated ROCK2 in all placental cell types studied in comparison with depressed and control women. Interestingly, in placental endothelial cells from depressed women, NGF levels were significantly lower compared to control women, but ROCK2 levels were increased compared with control and SSRI-treated women. Taken together, these results show that the NGF signaling and downstream pathways in the placenta are affected by SSRI treatment and/or antenatal depression. This might lead to an altered placental function, although the

  16. Functional recruitment of the human complement inhibitor C4BP to Yersinia pseudotuberculosis outer membrane protein Ail.

    Science.gov (United States)

    Ho, Derek K; Riva, Rauna; Kirjavainen, Vesa; Jarva, Hanna; Ginström, Erica; Blom, Anna M; Skurnik, Mikael; Meri, Seppo

    2012-05-01

    Ail is a 17-kDa chromosomally encoded outer membrane protein that mediates serum resistance (complement resistance) in the pathogenic Yersiniae (Yersinia pestis, Y. enterocolitica, and Y. pseudotuberculosis). In this article, we demonstrate that Y. pseudotuberculosis Ail from strains PB1, 2812/79, and YPIII/pIB1 (serotypes O:1a, O:1b, and O:3, respectively) can bind the inhibitor of the classical and lectin pathways of complement, C4b-binding protein (C4BP). Binding was observed irrespective of serotype tested and independently of YadA, which is the primary C4BP receptor of Y. enterocolitica. Disruption of the ail gene in Y. pseudotuberculosis resulted in loss of C4BP binding. Cofactor assays revealed that bound C4BP is functional, because bound C4BP in the presence of factor I cleaved C4b. In the absence of YadA, Ail conferred serum resistance to strains PB1 and YPIII, whereas serum resistance was observed in strain 2812/79 in the absence of both YadA and Ail, suggesting additional serum resistance factors. Ail from strain YPIII/pIB1 alone can mediate serum resistance and C4BP binding, because its expression in a serum-sensitive laboratory strain of Escherichia coli conferred both of these phenotypes. Using a panel of C4BP mutants, each deficient in a single complement control protein domain, we observed that complement control protein domains 6-8 are important for binding to Ail. Binding of C4BP was unaffected by increasing heparin or salt concentrations, suggesting primarily nonionic interactions. These results indicate that Y. pseudotuberculosis Ail recruits C4BP in a functional manner, facilitating resistance to attack from complement.

  17. Histone deacetylase inhibitor FR901228 enhances the antitumor effect of telomerase-specific replication-selective adenoviral agent OBP-301 in human lung cancer cells.

    Science.gov (United States)

    Watanabe, Takanori; Hioki, Masayoshi; Fujiwara, Toshiya; Nishizaki, Masahiko; Kagawa, Shunsuke; Taki, Masaki; Kishimoto, Hiroyuki; Endo, Yoshikatsu; Urata, Yasuo; Tanaka, Noriaki; Fujiwara, Toshiyoshi

    2006-02-01

    Replication-competent oncolytic viruses are being developed for human cancer therapy. We previously reported that an attenuated adenovirus OBP-301 (Telomelysin), in which the human telomerase reverse transcriptase promoter element drives expression of E1A and E1B genes linked with an internal ribosome entry site, could replicate in and causes selective lysis of human cancer cells. Infection efficiency in target cancer cells is the most important factor that predicts the antitumor effects of OBP-301. The objectives of this study are to examine the effects of the histone deacetylase inhibitor FR901228 on the level of coxsackie and adenovirus receptor (CAR) expression and OBP-301-mediated oncolysis in human non-small cell lung cancer cell lines. Flow cytometric analysis revealed up-regulated CAR expression in A549 and H460 cells following treatment with 1 ng/ml of FR901228, which was associated with increased infection efficiency as confirmed by replication-deficient beta-galactosidase-expressing adenovirus vector. In contrast, neither CAR expression nor infection efficiency was affected by FR901228 in H1299 cells. To visualize and quantify viral replication in the presence of FR901228, we used OBP-401 (Telomelysin-GFP) that expresses the green fluorescent protein (GFP) reporter gene under the control of the cytomegalovirus promoter in the E3 region. Fluorescence microscopy and flow cytometry showed that FR901228 increased GFP expression in A549 and H460 cells following OBP-401 infection in a dose-dependent manner, but this effect did not occur in H1299 cells. In addition, OBP-301 and FR901228 demonstrated a synergistic antitumor effect in A549 cells in vitro, as confirmed by isobologram analysis. Our data indicate that FR901228 preferentially increases adenovirus infectivity via up-regulation of CAR expression, leading to a profound oncolytic effect, which may have a significant impact on the outcome of adenovirus-based oncolytic virotherapy.

  18. Epidermal Growth Factor Receptor Kinase Inhibitors Synergize with TCDD to Induce CYP1A1/1A2 in Human Breast Epithelial MCF10A Cells.

    Science.gov (United States)

    Joiakim, Aby; Mathieu, Patricia A; Shelp, Catherine; Boerner, Julie; Reiners, John J

    2016-05-01

    CYP1A1 and CYP1A2 are transcriptionally activated in the human normal breast epithelial cell line MCF10A following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Shifting MCF10A cultures to medium deficient in serum and epidermal growth factor (EGF) caused rapid reductions in the activated (i.e., phosphorylated) forms of extracellular regulated kinases (ERKs) and the epidermal growth factor receptor (EGFR). Shifting to serum/EGF-deficient medium also enhanced TCDD-mediated induction of cytochrome P450 (CYP)1A1 Treatment of cells cultured in complete medium with the EGFR inhibitors gefitinib (Iressa), AG1478, and CI-1033 resulted in concentration-dependent reductions of active EGFR and ERKs, and increased CYP1A1 mRNA content ∼3- to 18-fold above basal level. EGFR inhibitors synergized with TCDD and resulted in transient CYP1A1 and CYP1A2 mRNA accumulations ∼8-fold greater (maximum at 5 hours) than that achieved with only TCDD. AG1478, gefitinib, and TCDD individually induced small increases (∼1.2- to 2.5-fold) in CYP1A1 protein content but did not cause additive or synergistic accumulations of CYP1A1 protein when used in combination. The mitogen-activated protein kinase kinase inhibitor PD184352 inhibited ERK and EGFR activation in a concentration-dependent fashion without causing CYP1A1 mRNA accumulation. However, cotreatment with PD184352 potentiated TCDD-mediated CYP1A1 induction. TCDD-mediated induction of CYP1A1 in MCF7-TET on-EGFR cells, a MCF7 variant in which EGFR expression can be controlled, was not affected by the activity status of EGFR or ERKs. Hence, EGFR signaling mutes both basal and ligand-induced expression of two aryl hydrocarbon receptor-responsive P450s in MCF10A cultures. However, these effects are cell context-dependent. Furthermore, CYP1A1 mRNA and protein abundance are not closely coupled in MCF10A cultures. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  19. Carbonic anhydrase inhibitors: Design, synthesis and structural characterization of new heteroaryl-N-carbonylbenzenesulfonamides targeting druggable human carbonic anhydrase isoforms

    Czech Academy of Sciences Publication Activity Database

    Buemi, M. R.; De Luca, L.; Ferro, S.; Bruno, E.; Ceruso, M.; Supuran, C. T.; Pospíšilová, K.; Brynda, Jiří; Řezáčová, Pavlína; Gitto, R.

    2015-01-01

    Roč. 102, SEP 18 (2015), s. 223-232 ISSN 0223-5234 R&D Projects: GA ČR GA15-05677S Grant - others:Fondo di Ateneo per la Ricerca (PRA)(IT) ORME09SPNC Institutional support: RVO:68378050 Keywords : Human carbonic anhydrase * Isoquinoline * Quinoline * X-ray * Molecular docking Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.902, year: 2015

  20. A novel peptide derived from human apolipoprotein E is an inhibitor of tumor growth and ocular angiogenesis.

    Directory of Open Access Journals (Sweden)

    Partha S Bhattacharjee

    2011-01-01

    Full Text Available Angiogenesis is a hallmark of tumor development and metastasis and now a validated target for cancer treatment. We previously reported that a novel dimer peptide (apoEdp derived from the receptor binding region of human apolipoprotein E (apoE inhibits virus-induced angiogenesis. However, its role in tumor anti-angiogenesis is unknown. This study demonstrates that apoEdp has anti-angiogenic property in vivo through reduction of tumor growth in a mouse model and ocular angiogenesis in a rabbit eye model. Our in vitro studies show that apoEdp inhibits human umbilical vein endothelial cell proliferation, migration, invasion and capillary tube formation. We document that apoEdp inhibits vascular endothelial growth factor-induced Flk-1 activation as well as downstream signaling pathways that involve c-Src, Akt, eNOS, FAK, and ERK1/2. These in vitro data suggest potential sites of the apoE dipeptide inhibition that could occur in vivo.This is the first evidence that a synthetic dimer peptide mimicking human apoE has anti-angiogenesis functions and could be an anti-tumor drug candidate.

  1. Insulin-like growth factor binding protein 7 and tissue inhibitor of metalloproteinases-2: differential expression and secretion in human kidney tubule cells

    Science.gov (United States)

    Emlet, David R.; Pastor-Soler, Nuria; Marciszyn, Allison; Wen, Xiaoyan; Gomez, Hernando; Humphries, William H.; Morrisroe, Seth; Volpe, Jacob K.

    2017-01-01

    We have characterized the expression and secretion of the acute kidney injury (AKI) biomarkers insulin-like growth factor binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases-2 (TIMP-2) in human kidney epithelial cells in primary cell culture and tissue. We established cell culture model systems of primary kidney cells of proximal and distal tubule origin and observed that both proteins are indeed expressed and secreted in both tubule cell types in vitro. However, TIMP-2 is both expressed and secreted preferentially by cells of distal tubule origin, while IGFBP7 is equally expressed across tubule cell types yet preferentially secreted by cells of proximal tubule origin. In human kidney tissue, strong staining of IGFBP7 was seen in the luminal brush-border region of a subset of proximal tubule cells, and TIMP-2 stained intracellularly in distal tubules. Additionally, while some tubular colocalization of both biomarkers was identified with the injury markers kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin, both biomarkers could also be seen alone, suggesting the possibility for differential mechanistic and/or temporal profiles of regulation of these early AKI biomarkers from known markers of injury. Last, an in vitro model of ischemia-reperfusion demonstrated enhancement of secretion of both markers early after reperfusion. This work provides a rationale for further investigation of these markers for their potential role in the pathogenesis of acute kidney injury. PMID:28003188

  2. Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Petri, Marcelo H. [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden); Tellier, Céline; Michiels, Carine [NARILIS, URBC, University of Namur, Namur (Belgium); Ellertsen, Ingvill [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden); Dogné, Jean-Michel [Department of Pharmacy, Namur Thrombosis and Hemostasis Center, University of Namur, Namur (Belgium); Bäck, Magnus, E-mail: Magnus.Back@ki.se [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden)

    2013-11-15

    Highlights: •EV-077 reduced TNF-α induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A{sub 2} is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.

  3. The Novel CXCR4 Antagonist KRH-3955 Is an Orally Bioavailable and Extremely Potent Inhibitor of Human Immunodeficiency Virus Type 1 Infection: Comparative Studies with AMD3100▿

    Science.gov (United States)

    Murakami, Tsutomu; Kumakura, Sei; Yamazaki, Toru; Tanaka, Reiko; Hamatake, Makiko; Okuma, Kazu; Huang, Wei; Toma, Jonathan; Komano, Jun; Yanaka, Mikiro; Tanaka, Yuetsu; Yamamoto, Naoki

    2009-01-01

    The previously reported CXCR4 antagonist KRH-1636 was a potent and selective inhibitor of CXCR4-using (X4) human immunodeficiency virus type 1 (HIV-1) but could not be further developed as an anti-HIV-1 agent because of its poor oral bioavailability. Newly developed KRH-3955 is a KRH-1636 derivative that is bioavailable when administered orally with much more potent anti-HIV-1 activity than AMD3100 and KRH-1636. The compound very potently inhibits the replication of X4 HIV-1, including clinical isolates in activated peripheral blood mononuclear cells from different donors. It is also active against recombinant X4 HIV-1 containing resistance mutations in reverse transcriptase and protease and envelope with enfuvirtide resistance mutations. KRH-3955 inhibits both SDF-1α binding to CXCR4 and Ca2+ signaling through the receptor. KRH-3955 inhibits the binding of anti-CXCR4 monoclonal antibodies that recognize the first, second, or third extracellular loop of CXCR4. The compound shows an oral bioavailability of 25.6% in rats, and its oral administration blocks X4 HIV-1 replication in the human peripheral blood lymphocyte-severe combined immunodeficiency mouse system. Thus, KRH-3955 is a new promising agent for HIV-1 infection and AIDS. PMID:19451305

  4. Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide.

    KAUST Repository

    Nomme, Julian

    2010-08-01

    We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.

  5. Physiologically based pharmacokinetic-pharmacodynamic modeling to predict concentrations and actions of sodium-dependent glucose transporter 2 inhibitor canagliflozin in human intestines and renal tubules.

    Science.gov (United States)

    Mori, Kazumi; Saito, Ryuta; Nakamaru, Yoshinobu; Shimizu, Makiko; Yamazaki, Hiroshi

    2016-11-01

    Canagliflozin is a recently developed sodium-glucose cotransporter (SGLT) 2 inhibitor that promotes renal glucose excretion and is considered to inhibit renal SGLT2 from the luminal side of proximal tubules. Canagliflozin reportedly inhibits SGLT1 weakly and suppresses postprandial plasma glucose, suggesting that it also inhibits intestinal SGLT1. However, it is difficult to measure the drug concentrations of these assumed sites of action directly. The pharmacokinetic-pharmacodynamic (PK/PD) relationships of canagliflozin remain poorly characterized. Therefore, a physiologically based pharmacokinetic (PBPK) model of canagliflozin was developed based on clinical data from healthy volunteers and it was used to simulate luminal concentrations in intestines and renal tubules. In small intestine simulations, the inhibition ratios for SGLT1 were predicted to be 40%-60% after the oral administration of clinical doses (100-300 mg/day). In contrast, inhibition ratios of canagliflozin for renal SGLT2 and SGLT1 were predicted to be approximately 100% and 0.2%-0.4%, respectively. These analyses suggest that canagliflozin only inhibits SGLT2 in the kidney. Using the simulated proximal tubule luminal concentrations of canagliflozin, the urinary glucose excretion rates in canagliflozin-treated diabetic patients were accurately predicted using the renal glucose reabsorption model as a PD model. Because the simulation of canagliflozin pharmacokinetics was successful, this PBPK methodology was further validated by successfully simulating the pharmacokinetics of dapagliflozin, another SGLT2 inhibitor. The present results suggest the utility of this PBPK/PD model for predicting canagliflozin concentrations at target sites and help to elucidate the pharmacological effects of SGLT1/2 inhibition in humans. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  6. Predictive value of pharmacokinetics-adjusted phenotypic susceptibility on response to ritonavir-enhanced protease inhibitors (PIs) in human immunodeficiency virus-infected subjects failing prior PI therapy.

    Science.gov (United States)

    Eron, Joseph J; Park, Jeong-Gun; Haubrich, Richard; Aweeka, Francesca; Bastow, Barbara; Pakes, Gary E; Yu, Song; Wu, Hulin; Richman, Douglas D

    2009-06-01

    The activities of protease inhibitors in vivo may depend on plasma concentrations and viral susceptibility. This nonrandomized, open-label study evaluated the relationship of the inhibitory quotient (IQ [the ratio of drug exposure to viral phenotypic susceptibility]) to the human immunodeficiency virus type 1 (HIV-1) viral load (VL) change for ritonavir-enhanced protease inhibitors (PIs). Subjects on PI-based regimens replaced their PIs with ritonavir-enhanced indinavir (IDV/r) 800/200 mg, fosamprenavir (FPV/r) 700/100 mg, or lopinavir (LPV/r) 400/200 mg twice daily. Pharmacokinetics were assessed at day 14; follow-up lasted 24 weeks. Associations between IQ and VL changes were examined. Fifty-three subjects enrolled, 12 on IDV/r, 33 on FPV/r, and 8 on LPV/r. Median changes (n-fold) (FC) of 50% inhibitory concentrations (IC(50)s) to the study PI were high. Median 2-week VL changes were -0.7, -0.1, and -1.0 log(10) for IDV/r, FPV/r, and LPV/r. With FPV/r, correlations between the IQ and the 2-week change in VL were significant (Spearman's r range, -0.39 to -0.50; P PI-experienced subjects with highly resistant HIV-1, short-term VL responses to RTV-enhanced FPV/r correlated best with baseline susceptibility. The IQ improved correlation in analyses of all arms where a greater range of virologic responses was observed.

  7. Combination treatment of all-trans retinoic acid (ATRA) and γ-secretase inhibitor (DAPT) cause growth inhibition and apoptosis induction in the human gastric cancer cell line.

    Science.gov (United States)

    Patrad, Elham; Niapour, Ali; Farassati, Faris; Amani, Mojtaba

    2018-04-01

    Current medication for gastric cancer patients has a low success rate with resistance and side effects. According to recent studies, γ-secretase inhibitors is used as therapeutic drugs in cancer. Moreover, all-trans retinoic acid (ATRA) is a natural compound proposed for the treatment/chemo-prevention of cancers. The aim of this study was to explore the effects of ATRA in combination with N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT) as γ-secretase inhibitor on viability and apoptosis of the AGS and MKN-45 derived from human gastric cancer. AGS and MKN-45 gastric cancer cell lines were treated with different concentrations of ATRA or DAPT alone or ATRA plus DAPT. The viability, death detection and apoptosis of cells was examined by MTT assay and Ethidium bromide/acridine orange staining. The distribution of cells in different phases of cell cycle was also evaluated through flow cytometry analyses. In addition, caspase 3/7 activity and the expression of caspase-3 and bcl-2 were examined. DAPT and ATRA alone decreased gastric cancer cells viability in a concentration dependent manner. The combination of DAPT and ATRA exhibited significant synergistic inhibitory effects. The greater percentage of cells were accumulated in G0/G1 phase of cell cycle in combination treatment. The combination of DAPT and ATRA effectively increased the proportion of apoptotic cells and the level of caspase 3/7 activities compared to single treatment. Moreover, augmented caspase-3 up-regulation and bcl-2 down-regulation were found following combined application of DAPT and ATRA. The combination of DAPT and ATRA led to more reduction in viability and apoptosis in respect to DAPT or ATRA alone in the investigated cell lines.

  8. In vivo analysis of insulin-like growth factor type 1 receptor humanized monoclonal antibody MK-0646 and small molecule kinase inhibitor OSI-906 in colorectal cancer.

    Science.gov (United States)

    Leiphrakpam, Premila D; Agarwal, Ekta; Mathiesen, Michelle; Haferbier, Katie L; Brattain, Michael G; Chowdhury, Sanjib

    2014-01-01

    The development and characterization of effective anticancer drugs against colorectal cancer (CRC) is of urgent need since it is the second most common cause of cancer death. The study was designed to evaluate the effects of two IGF-1R antagonists, MK-0646, a recombinant fully humanized monoclonal antibody and OSI-906, a small molecule tyrosine kinase inhibitor on CRC cells. Xenograft study was performed on IGF-1R-dependent CRC cell lines for analyzing the antitumor activity of MK-0646 and OSI-906. Tumor proliferation and apoptosis were assessed using Ki67 and TUNEL assays, respectively. We also performed in vitro characterization of MK-0646 and OSI-906 treatment on CRC cells to identify mechanisms associated with drug-induced cell death. Exposure of the GEO and CBS tumor xenografts to MK-0646 or OSI-906 led to a decrease in tumor growth. TUNEL analysis showed an increase of approximately 45-55% in apoptotic cells in both MK-0646 and OSI-906 treated tumor samples. We report the novel finding that treatment with IGF-1R antagonists led to downregulation of X-linked inhibitor of apoptosis (XIAP) protein involved in cell survival and inhibition of cell death. In conclusion, IGF-1R antagonists (MK-0646 and OSI-906) demonstrated single agent inhibition of subcutaneous CRC xenograft growth. This was coupled to pro-apoptotic effects resulting in downregulation of XIAP and inhibition of cell survival. We report a novel mechanism by which MK-0646 and OSI-906 elicits cell death in vivo and in vitro. Moreover, these results indicate that MK-0646 and OSI-906 may be potential anticancer candidates for the treatment of patients with IGF-1R-dependent CRC.

  9. First-in-human trial of multikinase VEGF inhibitor regorafenib and anti-EGFR antibody cetuximab in advanced cancer patients

    Science.gov (United States)

    Khawaja, Muhammad Rizwan; Hong, David S.; Amini, Behrang; Yungfang, Jiang; Liu, Hui; Johnson, Adrienne; Schrock, Alexa B.; Ali, Siraj M.; Sun, James X.; Fabrizio, David; Piha-Paul, Sarina; Fu, Siqing; Tsimberidou, Apostolia M.; Naing, Aung; Janku, Filip; Karp, Daniel D.; Overman, Michael; Eng, Cathy; Meric-Bernstam, Funda; Falchook, Gerald S.

    2017-01-01

    BACKGROUND. The combination of multikinase VEGF inhibitor regorafenib and anti-EGFR antibody cetuximab overcomes intrinsic and acquired resistance in both EGFR-sensitive and EGFR-resistant preclinical models of colorectal cancer (CRC). METHODS. Utilizing a standard 3+3 design, a phase I study was designed to determine safety, maximum tolerated dose (MTD), and dose-limiting toxicities (DLTs) of the regorafenib plus cetuximab combination among patients with advanced cancer including CRC. Comprehensive genomic profiling was performed on the exceptional responder. RESULTS. Among the 27 patients enrolled the median age was 54 years. None of 19 patients treated at dose level 1 (cetuximab i.v. 200 mg/m2 followed by 150 mg/m2 weekly + regorafenib 80 mg daily) experienced a DLT, and 2 of 5 patients treated at dose level 2 (cetuximab i.v. 200 mg/m2 followed by 150 mg/m2 weekly + regorafenib 120 mg daily) experienced a DLT (grade 3 thrombocytopenia [n = 1] and grade 3 intra-abdominal bleed [n = 1]). Most common adverse events were grade 1 or 2 rash (20 patients). Of 24 evaluable patients, 11 (46%) patients had clinical benefit (stable disease > 6 cycles or partial response [PR]) (CRC n = 8, one patient each with head and neck cancer, carcinoma of unknown primary, and glioblastoma). A CRC patient, who progressed on anti-EGFR and regorafenib, achieved a PR (46% decrease per RECIST v1.1) lasting 15 months. Genomic profiling of an exceptional responder with response for over 27 cycles revealed hypermutated genotype with microsatellite instability (MSI). CONCLUSION. Regorafenib 80 mg daily plus cetuximab 200 mg/m2 loading dose, followed by 150 mg/m2 every week is the MTD/recommended phase II dose. The combination demonstrated early signals of activity in wild-type CRC, including 1 exceptional responder with MSI high. TRIAL REGISTRATION. clinicaltrials.gov NCT02095054 FUNDING. The University of Texas MD Anderson Cancer Center is supported by the NIH Cancer Center Support Grant CA

  10. MSH3 mismatch repair protein regulates sensitivity to cytotoxic drugs and a histone deacetylase inhibitor in human colon carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Jae Myung Park

    Full Text Available MSH3 is a DNA mismatch repair (MMR gene that undergoes frequent somatic mutation in colorectal cancers (CRCs with MMR deficiency. MSH3, together with MSH2, forms the MutSβ heteroduplex that interacts with interstrand cross-links induced by drugs such as cisplatin. To date, the impact of MSH3 on chemosensitivity is unknown.We utilized isogenic HCT116 (MLH1-/MSH3- cells where MLH1 is restored by transfer of chromosome 3 (HCT116+ch3 and also MSH3 by chromosome 5 (HCT116+3+5. We generated HCT116+3+5, SW480 (MLH1+/MSH3+ and SW48 (MLH1-/MSH3+ cells with shRNA knockdown of MSH3. Cells were treated with 5-fluorouracil (5-FU, SN-38, oxaliplatin, or the histone deacetylase (HDAC inhibitor PCI-24781 and cell viability, clonogenic survival, DNA damage and apoptosis were analyzed.MSH3-deficient vs proficient CRC cells showed increased sensitivity to the irinotecan metabolite SN-38 and to oxaliplatin, but not 5-FU, as shown in assays for apoptosis and clonogenic survival. In contrast, suppression of MLH1 attenuated the cytotoxic effect of 5-FU, but did not alter sensitivity to SN-38 or oxaliplatin. The impact of MSH3 knockdown on chemosensitivity to SN-38 and oxaliplatin was maintained independent of MLH1 status. In MSH3-deficient vs proficient cells, SN-38 and oxaliplatin induced higher levels of phosphorylated histone H2AX and Chk2, and similar results were found in MLH1-proficient SW480 cells. MSH3-deficient vs proficient cells showed increased 53BP1 nuclear foci after irradiation, suggesting that MSH3 can regulate DNA double strand break (DSB repair. We then utilized PCI-24781 that interferes with homologous recombination (HR indicated by a reduction in Rad51 expression. The addition of PCI-24781 to oxaliplatin enhanced cytotoxicity to a greater extent compared to either drug alone.MSH3 status can regulate the DNA damage response and extent of apoptosis induced by chemotherapy. The ability of MSH3 to regulate chemosensitivity was independent of MLH1

  11. The human complement inhibitor Sushi Domain-Containing Protein 4 (SUSD4) expression in tumor cells and infiltrating T cells is associated with better prognosis of breast cancer patients

    OpenAIRE

    Englund, Emelie; Reitsma, Bart; King, Ben C.; Escudero-Esparza, Astrid; Owen, Sioned; Orimo, Akira; Okroj, Marcin; Anagnostaki, Lola; Jiang, Wen G.; Jirström, Karin; Blom, Anna M.

    2015-01-01

    Background: The human Sushi Domain-Containing Protein 4 (SUSD4) was recently shown to function as a novel inhibitor of the complement system, but its role in tumor progression is unknown. \\ud \\ud Methods: Using immunohistochemistry and quantitative PCR, we investigated SUSD4 expression in breast cancer tissue samples from two cohorts. The effect of SUSD4 expression on cell migration and invasion was studied in vitro using two human breast cancer cell lines overexpressing SUSD4. \\ud \\ud Result...

  12. Age-related increase in Wnt inhibitor causes a senescence-like phenotype in human cardiac stem cells.

    Science.gov (United States)

    Nakamura, Tamami; Hosoyama, Tohru; Murakami, Junichi; Samura, Makoto; Ueno, Koji; Kurazumi, Hiroshi; Suzuki, Ryo; Mikamo, Akihito; Hamano, Kimikazu

    2017-06-03

    Aging of cardiac stem/progenitor cells (CSCs) impairs heart regeneration and leads to unsatisfactory outcomes of cell-based therapies. As the precise mechanisms underlying CSC aging remain unclear, the use of therapeutic strategies for elderly patients with heart failure is severely delayed. In this study, we used human cardiosphere-derived cells (CDCs), a subtype of CSC found in the postnatal heart, to identify secreted factor(s) associated with CSC aging. Human CDCs were isolated from heart failure patients of various ages (2-83 years old). Gene expression of key soluble factors was compared between CDCs derived from young and elderly patients. Among these factors, SFRP1, a gene encoding a Wnt antagonist, was significantly up-regulated in CDCs from elderly patients (≥65 years old). sFRP1 levels was increased significantly also in CDCs, whose senescent phenotype was induced by anti-cancer drug treatment. These results suggest the participation of sFRP1 in CSC aging. We show that the administration of recombinant sFRP1 induced cellular senescence in CDCs derived from young patients, as indicated by increased levels of markers such as p16, and a senescence-associated secretory phenotype. In addition, co-administration of recombinant sFRP1 could abrogate the accelerated CDC proliferation induced by Wnt3A. Taken together, our results suggest that canonical Wnt signaling and its antagonist, sFRP1, regulate proliferation of human CSCs. Furthermore, excess sFRP1 in elderly patients causes CSC aging. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

    Science.gov (United States)

    Li, Jin-Ping; Yang, Yin-Xue; Liu, Qi-Lun; Zhou, Zhi-Wei; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Pan, Si-Yuan; Duan, Wei; He, Shu-Ming; Chen, Xiao-Wu; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition

  14. Competitive fitness of influenza B viruses with neuraminidase inhibitor-resistant substitutions in a coinfection model of the human airway epithelium.

    Science.gov (United States)

    Burnham, Andrew J; Armstrong, Jianling; Lowen, Anice C; Webster, Robert G; Govorkova, Elena A

    2015-04-01

    Influenza A and B viruses are human pathogens that are regarded to cause almost equally significant disease burdens. Neuraminidase (NA) inhibitors (NAIs) are the only class of drugs available to treat influenza A and B virus infections, so the development of NAI-resistant viruses with superior fitness is a public health concern. The fitness of NAI-resistant influenza B viruses has not been widely studied. Here we examined the replicative capacity and relative fitness in normal human bronchial epithelial (NHBE) cells of recombinant influenza B/Yamanashi/166/1998 viruses containing a single amino acid substitution in NA generated by reverse genetics (rg) that is associated with NAI resistance. The replication in NHBE cells of viruses with reduced inhibition by oseltamivir (recombinant virus with the E119A mutation generated by reverse genetics [rg-E119A], rg-D198E, rg-I222T, rg-H274Y, rg-N294S, and rg-R371K, N2 numbering) or zanamivir (rg-E119A and rg-R371K) failed to be inhibited by the presence of the respective NAI. In a fluorescence-based assay, detection of rg-E119A was easily masked by the presence of NAI-susceptible virus. We coinfected NHBE cells with NAI-susceptible and -resistant viruses and used next-generation deep sequencing to reveal the order of relative fitness compared to that of recombinant wild-type (WT) virus generated by reverse genetics (rg-WT): rg-H274Y > rg-WT > rg-I222T > rg-N294S > rg-D198E > rg-E119A ≫ rg-R371K. Based on the lack of attenuated replication of rg-E119A in NHBE cells in the presence of oseltamivir or zanamivir and the fitness advantage of rg-H274Y over rg-WT, we emphasize the importance of these substitutions in the NA glycoprotein. Human infections with influenza B viruses carrying the E119A or H274Y substitution could limit the therapeutic options for those infected; the emergence of such viruses should be closely monitored. Influenza B viruses are important human respiratory pathogens contributing to a significant portion

  15. The indoleamine-2,3-dioxygenase (IDO inhibitor 1-methyl-D-tryptophan upregulates IDO1 in human cancer cells.

    Directory of Open Access Journals (Sweden)

    Christiane A Opitz

    Full Text Available 1-methyl-D-tryptophan (1-D-MT is currently being used in clinical trials in patients with relapsed or refractory solid tumors with the aim of inhibiting indoleamine-2,3-dioxygenase (IDO-mediated tumor immune escape. IDO is expressed in tumors and tumor-draining lymph nodes and degrades tryptophan (trp to create an immunsuppressive micromilieu both by depleting trp and by accumulating immunosuppressive metabolites of the kynurenine (kyn pathway. Here we show that proliferation of alloreactive T-cells cocultured with IDO1-positive human cancer cells paradoxically was inhibited by 1-D-MT. Surprisingly incubation with 1-D-MT increased kyn production of human cancer cells. Cell-free assays revealed that 1-D-MT did not alter IDO1 enzymatic activity. Instead, 1-D-MT induced IDO1 mRNA and protein expression through pathways involving p38 MAPK and JNK signalling. Treatment of cancer patients with 1-D-MT has transcriptional effects that may promote rather than suppress anti-tumor immune escape by increasing IDO1 in the cancer cells. These off-target effects should be carefully analyzed in the ongoing clinical trials with 1-D-MT.

  16. Metformin Is a Substrate and Inhibitor of the Human Thiamine Transporter, THTR-2 (SLC19A3).

    Science.gov (United States)

    Liang, Xiaomin; Chien, Huan-Chieh; Yee, Sook Wah; Giacomini, Marilyn M; Chen, Eugene C; Piao, Meiling; Hao, Jia; Twelves, Jolyn; Lepist, Eve-Irene; Ray, Adrian S; Giacomini, Kathleen M

    2015-12-07

    The biguanide metformin is widely used as first-line therapy for the treatment of type 2 diabetes. Predominately a cation at physiological pH's, metformin is transported by membrane transporters, which play major roles in its absorption and disposition. Recently, our laboratory demonstrated that organic cation transporter 1, OCT1, the major hepatic uptake transporter for metformin, was also the primary hepatic uptake transporter for thiamine, vitamin B1. In this study, we tested the reverse, i.e., that metformin is a substrate of thiamine transporters (THTR-1, SLC19A2, and THTR-2, SLC19A3). Our study demonstrated that human THTR-2 (hTHTR-2), SLC19A3, which is highly expressed in the small intestine, but not hTHTR-1, transports metformin (Km = 1.15 ± 0.2 mM) and other cationic compounds (MPP(+) and famotidine). The uptake mechanism for hTHTR-2 was pH and electrochemical gradient sensitive. Furthermore, metformin as well as other drugs including phenformin, chloroquine, verapamil, famotidine, and amprolium inhibited hTHTR-2 mediated uptake of both thiamine and metformin. Species differences in the substrate specificity of THTR-2 between human and mouse orthologues were observed. Taken together, our data suggest that hTHTR-2 may play a role in the intestinal absorption and tissue distribution of metformin and other organic cations and that the transporter may be a target for drug-drug and drug-nutrient interactions.

  17. Molecular cloning and chromosomal localization of a pseudogene related to the human Acyl-CoA binding protein/diazepam binding inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Gersuk, V.H. [Virginia Mason Research Center, Seattle, WA (United States); Rose, T.M.; Todaro, G.J. [Univ. of Washington, Seattle, WA (United States)

    1995-01-20

    The acyl-CoA binding protein (ACBP) and the diazepam binding inhibitor (DBI) or endozepine are independent isolates of a single 86-amino-acid, 10-kDa protein. ACBP/DBI is highly conserved between species and has been identified in several diverse organisms, including human, cow, rat, frog, duck, insects, plants, and yeast. Although the genomic locus has not yet been cloned in humans, complementary DNA clones with different 5{prime} ends have been isolated and characterized. These cDNA clones appear to be encoded by a single gene. However, Southern blot analyses, in situ hybridizations, and somatic cell hybrid chromosomal mapping all suggest that there are multiple ACBP/DBI-related sequences in the genome. To identify potential members of this gene family, degenerate oligonucleotides corresponding to highly conserved regions of ACBP/DBI were used to screen a human genomic DNA library using the polymerase chain reaction. A novel gene, DBIP1, that is closely related to ACBP/DBI but is clearly distinct was identified. DBIP1 bears extensive sequence homology to ACBP/DBI but lacks the introns predicted by rat and duck genomic sequence studies. A 1-base deletion in the coding region results in a frameshift and, along with the absence of introns and the lack of a detectable transcript, suggests that DBIP1 is a pseudogene. ACBP/DBI has previously been mapped to chromosome 2, although this was recently disputed, and a chromosome 6 location was suggested. We show that ACBP/DBI is correctly placed on chromosome 2 and that the gene identified on chromosome 6 is DBIP1. 33 refs., 3 figs., 1 tab.

  18. Cystatins--Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells.

    Science.gov (United States)

    Wallin, Hanna; Bjarnadottir, Maria; Vogel, Lotte K; Wassélius, Johan; Ekström, Ulf; Abrahamson, Magnus

    2010-11-01

    Cystatins are present in mammals, birds, fish, insects, plants, fungi and protozoa and constitute a large protein family, with most members sharing a cysteine protease inhibitory function. In humans 12 functional cystatins exist, forming three groups based on molecular organisation and distribution in the organism. The type 1 cystatins (A and B) are known as intracellular, type 2 cystatins (C, D, E/M, F, G, S, SN and SA) extracellular and type 3 cystatins (L- and H-kininogen) intravascular proteins. The present paper is focused on the human cystatins and especially those of type 2, which are directed (with signal peptides) for cellular export following translation. Results indicating existence of systems for significant internalisation of type 2 cystatins from the extracellular to intracellular compartments are reviewed. Data showing that human neuroblastoma cell lines generally secrete high levels, but also contain high amounts of cystatin C are presented. Culturing of these cells in medium containing cystatin C at concentrations found in body fluids resulted in increased intracellular cystatin C, as a result of an uptake process. At immunofluorescence cytochemistry a pronounced vesicular cystatin C staining was observed. The simplistic denotation of the type 2 cystatins as extracellular inhibitors is thus challenged, and possible biological functions of the internalised cystatins are discussed. To illustrate the special case of high cellular cystatin content seen in cells of patients with hereditary cystatin C amyloid angiopathy, expression vectors for wild-type and L68Q mutated cystatin C were used to transfect SK-N-BE(2) cells. Clones overexpressing the two variants showed increased secreted levels of cystatin C. Within the cells the L68Q variant appeared to mainly localise to the endoplasmic reticulum rather than to acidic vesicular organelles, indicating limitations in the transport out from the cell rather than increased uptake as explanation for the

  19. Chitosan-shelled oxygen-loaded nanodroplets abrogate hypoxia dysregulation of human keratinocyte gelatinases and inhibitors: New insights for chronic wound healing

    International Nuclear Information System (INIS)

    Khadjavi, Amina; Magnetto, Chiara; Panariti, Alice; Argenziano, Monica; Gulino, Giulia Rossana; Rivolta, Ilaria; Cavalli, Roberta; Giribaldi, Giuliana; Guiot, Caterina; Prato, Mauro

    2015-01-01

    Background: : In chronic wounds, efficient epithelial tissue repair is hampered by hypoxia, and balances between the molecules involved in matrix turn-over such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are seriously impaired. Intriguingly, new oxygenating nanocarriers such as 2H,3H-decafluoropentane-based oxygen-loaded nanodroplets (OLNs) might effectively target chronic wounds. Objective: : To investigate hypoxia and chitosan-shelled OLN effects on MMP/TIMP production by human keratinocytes. Methods: : HaCaT cells were treated for 24 h with 10% v/v OLNs both in normoxia or hypoxia. Cytotoxicity and cell viability were measured through biochemical assays; cellular uptake by confocal microscopy; and MMP and TIMP production by enzyme-linked immunosorbent assay or gelatin zymography. Results: : Normoxic HaCaT cells constitutively released MMP-2, MMP-9, TIMP-1 and TIMP-2. Hypoxia strongly impaired MMP/TIMP balances by reducing MMP-2, MMP-9, and TIMP-2, without affecting TIMP-1 release. After cellular uptake by keratinocytes, nontoxic OLNs abrogated all hypoxia effects on MMP/TIMP secretion, restoring physiological balances. OLN abilities were specifically dependent on time-sustained oxygen diffusion from OLN core. Conclusion: : Chitosan-shelled OLNs effectively counteract hypoxia-dependent dysregulation of MMP/TIMP balances in human keratinocytes. Therefore, topical administration of exogenous oxygen, properly encapsulated in nanodroplet formulations, might be a promising adjuvant approach to promote healing processes in hypoxic wounds. - Highlights: • Hypoxia impairs MMP9/TIMP1 and MMP2/TIMP2 balances in HaCaT human keratinocytes. • Chitosan-shelled oxygen-loaded nanodroplets (OLNs) are internalised by HaCaT cells. • OLNs are not toxic to HaCaT cells. • OLNs effectively counteract hypoxia effects on MMP/TIMP balances in HaCaT cells. • OLNs appear as promising and cost-effective therapeutic tools for hypoxic

  20. Chitosan-shelled oxygen-loaded nanodroplets abrogate hypoxia dysregulation of human keratinocyte gelatinases and inhibitors: New insights for chronic wound healing

    Energy Technology Data Exchange (ETDEWEB)

    Khadjavi, Amina [Dipartimento di Neuroscienze, Università di Torino, Torino (Italy); Magnetto, Chiara [Istituto Nazionale di Ricerca Metrologica (INRIM), Torino (Italy); Panariti, Alice [Dipartimento di Scienze della Salute, Università di Milano Bicocca, Monza (Italy); Argenziano, Monica [Dipartimento di Scienza e Tecnologia del Farmaco, Università di Torino, Torino (Italy); Gulino, Giulia Rossana [Dipartimento di Oncologia, Università di Torino, Torino (Italy); Rivolta, Ilaria [Dipartimento di Scienze della Salute, Università di Milano Bicocca, Monza (Italy); Cavalli, Roberta [Dipartimento di Scienza e Tecnologia del Farmaco, Università di Torino, Torino (Italy); Giribaldi, Giuliana [Dipartimento di Oncologia, Università di Torino, Torino (Italy); Guiot, Caterina [Dipartimento di Neuroscienze, Università di Torino, Torino (Italy); Prato, Mauro, E-mail: mauro.prato@unito.it [Dipartimento di Neuroscienze, Università di Torino, Torino (Italy); Dipartimento di Scienze della Sanità Pubblica e Pediatriche, Università di Torino, Torino (Italy)

    2015-08-01

    Background: : In chronic wounds, efficient epithelial tissue repair is hampered by hypoxia, and balances between the molecules involved in matrix turn-over such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are seriously impaired. Intriguingly, new oxygenating nanocarriers such as 2H,3H-decafluoropentane-based oxygen-loaded nanodroplets (OLNs) might effectively target chronic wounds. Objective: : To investigate hypoxia and chitosan-shelled OLN effects on MMP/TIMP production by human keratinocytes. Methods: : HaCaT cells were treated for 24 h with 10% v/v OLNs both in normoxia or hypoxia. Cytotoxicity and cell viability were measured through biochemical assays; cellular uptake by confocal microscopy; and MMP and TIMP production by enzyme-linked immunosorbent assay or gelatin zymography. Results: : Normoxic HaCaT cells constitutively released MMP-2, MMP-9, TIMP-1 and TIMP-2. Hypoxia strongly impaired MMP/TIMP balances by reducing MMP-2, MMP-9, and TIMP-2, without affecting TIMP-1 release. After cellular uptake by keratinocytes, nontoxic OLNs abrogated all hypoxia effects on MMP/TIMP secretion, restoring physiological balances. OLN abilities were specifically dependent on time-sustained oxygen diffusion from OLN core. Conclusion: : Chitosan-shelled OLNs effectively counteract hypoxia-dependent dysregulation of MMP/TIMP balances in human keratinocytes. Therefore, topical administration of exogenous oxygen, properly encapsulated in nanodroplet formulations, might be a promising adjuvant approach to promote healing processes in hypoxic wounds. - Highlights: • Hypoxia impairs MMP9/TIMP1 and MMP2/TIMP2 balances in HaCaT human keratinocytes. • Chitosan-shelled oxygen-loaded nanodroplets (OLNs) are internalised by HaCaT cells. • OLNs are not toxic to HaCaT cells. • OLNs effectively counteract hypoxia effects on MMP/TIMP balances in HaCaT cells. • OLNs appear as promising and cost-effective therapeutic tools for hypoxic

  1. Selective Inhibitors of Protein Methyltransferases

    Science.gov (United States)

    2015-01-01

    Mounting evidence suggests that protein methyltransferases (PMTs), which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and human diseases. In particular, PMTs have been recognized as major players in regulating gene expression and chromatin state. PMTs are divided into two categories: protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs). There has been a steadily growing interest in these enzymes as potential therapeutic targets and therefore discovery of PMT inhibitors has also been pursued increasingly over the past decade. Here, we present a perspective on selective, small-molecule inhibitors of PMTs with an emphasis on their discovery, characterization, and applicability as chemical tools for deciphering the target PMTs’ physiological functions and involvement in human diseases. We highlight the current state of PMT inhibitors and discuss future directions and opportunities for PMT inhibitor discovery. PMID:25406853

  2. Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS

    Science.gov (United States)

    Savarino, Andrea; Pistello, Mauro; D'Ostilio, Daniela; Zabogli, Elisa; Taglia, Fabiana; Mancini, Fabiola; Ferro, Stefania; Matteucci, Donatella; De Luca, Laura; Barreca, Maria Letizia; Ciervo, Alessandra; Chimirri, Alba; Ciccozzi, Massimo; Bendinelli, Mauro

    2007-01-01

    Background Treatment of feline immunodeficiency virus (FIV) infection has been hampered by the absence of a specific combination antiretroviral treatment (ART). Integrase strand transfer inhibitors (INSTIs) are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs. Methods Phylogenetic analysis of lentiviral integrase (IN) sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD) was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD). Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR. Results The calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810. Conclusion We report a drug class (other than nucleosidic reverse transcriptase inhibitors) that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound successfully tested in

  3. Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS

    Directory of Open Access Journals (Sweden)

    Ciervo Alessandra

    2007-10-01

    Full Text Available Abstract Background Treatment of feline immunodeficiency virus (FIV infection has been hampered by the absence of a specific combination antiretroviral treatment (ART. Integrase strand transfer inhibitors (INSTIs are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs. Methods Phylogenetic analysis of lentiviral integrase (IN sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD. Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR. Results The calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810. Conclusion We report a drug class (other than nucleosidic reverse transcriptase inhibitors that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound

  4. Expression and assembly of largest foreign protein in chloroplasts: oral delivery of human FVIII made in lettuce chloroplasts robustly suppresses inhibitor formation in haemophilia A mice.

    Science.gov (United States)

    Kwon, Kwang-Chul; Sherman, Alexandra; Chang, Wan-Jung; Kamesh, Aditya; Biswas, Moanaro; Herzog, Roland W; Daniell, Henry

    2017-11-06

    Inhibitor formation is a serious complication of factor VIII (FVIII) replacement therapy for the X-linked bleeding disorder haemophilia A and occurs in 20%-30% of patients. No prophylactic tolerance protocol currently exists. Although we reported oral tolerance induction using FVIII domains expressed in tobacco chloroplasts, significant challenges in clinical advancement include expression of the full-length CTB-FVIII sequence to cover the entire patient population, regardless of individual CD4 + T-cell epitope responses. Codon optimization of FVIII heavy chain (HC) and light chain (LC) increased expression 15- to 42-fold higher than the native human genes. Homoplasmic lettuce lines expressed CTB fusion proteins of FVIII-HC (99.3 kDa), LC (91.8 kDa), C2 (31 kDa) or single chain (SC, 178.2 kDa) up to 3622, 263, 3321 and 852 μg/g in lyophilized plant cells, when grown in a cGMP hydroponic facility (Fraunhofer). CTB-FVIII-SC is the largest foreign protein expressed in chloroplasts; despite a large pentamer size (891 kDa), assembly, folding and disulphide bonds were maintained upon lyophilization and long-term storage as revealed by GM1-ganglioside receptor binding assays. Repeated oral gavages (twice/week for 2 months) of CTB-FVIII-HC/CTB-FVIII-LC reduced inhibitor titres ~10-fold (average 44 BU/mL to 4.7 BU/mL) in haemophilia A mice. Most importantly, increase in the frequency of circulating LAP-expressing CD4 + CD25 + FoxP3 + Treg in tolerized mice could be used as an important cellular biomarker in human clinical trials for plant-based oral tolerance induction. In conclusion, this study reports the first clinical candidate for oral tolerance induction that is urgently needed to protect haemophilia A patients receiving FVIII injections. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  5. The Protease Inhibitor Monotherapy Versus Ongoing Triple Therapy (PIVOT) trial: a randomised controlled trial of a protease inhibitor monotherapy strategy for long-term management of human immunodeficiency virus infection.

    Science.gov (United States)

    Paton, Nicholas I; Stöhr, Wolfgang; Oddershede, Lars; Arenas-Pinto, Alejandro; Walker, Simon; Sculpher, Mark; Dunn, David T

    2016-03-01

    Standard-of-care antiretroviral therapy (ART) for human immunodeficiency virus (HIV) infection uses a combination of drugs, until now considered essential to minimise treatment failure and development of drug resistance. Protease inhibitors (PIs) are potent with a high genetic barrier to resistance and have the potential for use as monotherapy after viral load (VL) suppression achieved on combination therapy. However, longer-term resistance and toxicity risks are uncertain. To compare the effectiveness, toxicity profile and cost-effectiveness of PI monotherapy with those of standard-of-care triple therapy in a pragmatic long-term clinical trial. Open-label, parallel-group, randomised controlled trial. Forty-three HIV clinical centres in the UK NHS. HIV-positive adults taking standard combination ART with a suppressed VL for ≥ 6 months. Patients were randomised to maintain ongoing triple therapy (OT) or switch to a strategy of physician-selected ritonavir-boosted PI monotherapy (PI-mono), with prompt return to combination therapy in the event of VL rebound. The primary outcome was reduction of future drug options, defined as new intermediate-/high-level resistance to one or more drugs to which the patient's virus was considered to be sensitive at trial entry (non-inferiority comparison, 10% margin). Secondary outcomes included confirmed virological rebound, serious drug- or disease-related complications, total grade 3 or 4 adverse events (AEs), neurocognitive function change, cluster of differentiation 4 (CD4) cell count change, change in health-related quality of life, cardiovascular risk change, health-care costs and health economic analysis. In total, 587 participants were randomised (77% male, 68% white) to OT (n = 291) or PI-mono (n = 296) and followed for a median of 44 months, of whom 2.7% withdrew/were lost to follow-up. One or more episodes of confirmed VL rebound were observed in eight patients (Kaplan-Meier estimate 3.2%) in the OT group and

  6. Kaempferol as Selective Human MAO-A Inhibitor: Analytical Detection in Calabrian Red Wines, Biological and Molecular Modeling Studies.

    Science.gov (United States)

    Gidaro, Maria Concetta; Astorino, Christian; Petzer, Anél; Carradori, Simone; Alcaro, Francesca; Costa, Giosuè; Artese, Anna; Rafele, Giancarlo; Russo, Francesco M; Petzer, Jacobus P; Alcaro, Stefano

    2016-02-17

    The purpose of this work was to determine the kaempferol content in three red wines of Calabria, a southern Italian region with a great number of certified food products. Considering that wine cultivar, climate, and soil influence the qualitative and quantitative composition in flavonoids of Vitis vinifera L. berries, the three analyzed samples were taken from the 2013 vintage. Moreover, the Gaglioppo samples, with assigned Controlled Origin Denomination (DOC), were also investigated in the production of years 2008, 2010, and 2011. In addition to the analysis of kaempferol, which is present in higher concentration than in other Italian wines, in vitro assays were performed to evaluate, for the first time, the inhibition of the human monoamine oxidases (hMAO-A and hMAO-B). Molecular recognition studies were also carried out to provide insight into the binding mode of kaempferol and selectivity of inhibition of the hMAO-A isoform.

  7. Inhibition of Human Steroid 5-Reductase (AKR1D1) by Finasteride and Structure of the Enzyme-Inhibitor Complex

    Energy Technology Data Exchange (ETDEWEB)

    Drury, J.; Di Costanzo, L; Penning, T; Christianson, D

    2009-01-01

    The {Delta}{sup 4}-3-ketosteroid functionality is present in nearly all steroid hormones apart from estrogens. The first step in functionalization of the A-ring is mediated in humans by steroid 5{alpha}- or 5{beta}-reductase. Finasteride is a mechanism-based inactivator of 5{alpha}-reductase type 2 with subnanomolar affinity and is widely used as a therapeutic for the treatment of benign prostatic hyperplasia. It is also used for androgen deprivation in hormone-dependent prostate carcinoma, and it has been examined as a chemopreventive agent in prostate cancer. The effect of finasteride on steroid 5{beta}-reductase (AKR1D1) has not been previously reported. We show that finasteride competitively inhibits AKR1D1 with low micromolar affinity but does not act as a mechanism-based inactivator. The structure of the AKR1D1 {center_dot} NADP{sup +} {center_dot} finasteride complex determined at 1.7 {angstrom} resolution shows that it is not possible for NADPH to reduce the {Delta}{sup 1-2}-ene of finasteride because the cofactor and steroid are not proximal to each other. The C3-ketone of finasteride accepts hydrogen bonds from the catalytic residues Tyr-58 and Glu-120 in the active site of AKR1D1, providing an explanation for the competitive inhibition observed. This is the first reported structure of finasteride bound to an enzyme involved in steroid hormone metabolism.

  8. NO Metabolites Levels in Human Red Blood Cells are Affected by Palytoxin, an Inhibitor of Na(+)/K(+)-ATPase Pump.

    Science.gov (United States)

    Carelli-Alinovi, Cristiana; Tellone, Ester; Russo, Anna Maria; Ficarra, Silvana; Pirolli, Davide; Galtieri, Antonio; Giardina, Bruno; Misiti, Francesco

    2014-01-01

    Palytoxin (PTX), a marine toxin, represents an increasing hazard for human health. Despite its high toxicity for biological systems, the mechanisms triggered by PTX, are not well understood. The high affinity of PTX for erythrocyte Na(+)/K(+)-ATPase pump is largely known, and it indicates PTX as a sensitive tool to characterize the signal transducer role for Na(+)/K(+)-ATPase pump. Previously, it has been reported that in red blood cells (RBC), probably via a signal transduction generated by the formation of a PTX-Na(+)/K(+)-ATPase complex, PTX alters band 3 functions and glucose metabolism. The present study addresses the question of which other signaling pathways are regulated by Na(+)/K(+)-ATPase in RBC. Here it has been evidenced that PTX following its interaction with Na(+)/K(+)-ATPase pump, alters RBC morphology and this event is correlated to decreases by 30% in nitrites and nitrates levels, known as markers of plasma membrane eNOS activity. Orthovanadate (OV), an antagonist of PTX binding to Na(+)/K(+)-ATPase pump, was able to reverse the effects elicited by PTX. Finally, current investigation firstly suggests that Na(+)/K(+)-ATPase pump, following its interaction with PTX, triggers a signal transduction involved in NO metabolism regulation.

  9. Differential effects of inhibitors and detergents on the Ca2+-ATPase and Mg2+-ATPase activities of the plasma membrane of a human oat cell carcinoma

    International Nuclear Information System (INIS)

    Knowles, A.F.; Lawrence, C.M.

    1986-01-01

    Plasma membranes of human oat cell carcinoma possess Mg 2+ - and Ca 2+ -dependent ATPase activities of similar magnitude. These activities exhibit the unusual characteristic of being inactiviated by prolonged incubation of the membrane with 1-2 mM dithiothreitol (DTT). Inactivation by DTT was prevented by lowering the incubation temperature, elevation of the membrane protein concentration, and addition of ATP. Fluorosulfonylbenzoyl adenosine (FSBA), an affinity ATP analog, also inactivates these activities. The Ca 2+ -ATPase activity appears to be more sensitive to both DTT and FSBA. The Ca 2+ -ATPase activity is more easily inactivated by Triton X-100, while the Mg 2+ -ATPase is preferentially activated by digitonin. These differential effects of inhibitors and detergents suggest that the Ca 2+ -ATPase and Mg 2+ -ATPase are separate enzymes. Incubation of oat cell carcinoma plasma membrane with [ 3 H]FSBA resulted in the labeling of several proteins. A labelled 35,000 dalton protein corresponds to the molecular weight of the oat cell carcinoma plasma membrane Ca 2+ -ATPase previously purified in this laboratory. The identity of one or more of the other labelled proteins with the Mg 2+ -ATPase has not been demonstrated, but is presently under investigation

  10. Pretreatment with a γ-Secretase Inhibitor Prevents Tumor-like Overgrowth in Human iPSC-Derived Transplants for Spinal Cord Injury

    Directory of Open Access Journals (Sweden)

    Toshiki Okubo

    2016-10-01

    Full Text Available Neural stem/progenitor cells (NS/PCs derived from human induced pluripotent stem cells (hiPSCs are considered to be a promising cell source for cell-based interventions that target CNS disorders. We previously reported that transplanting certain hiPSC-NS/PCs in the spinal cord results in tumor-like overgrowth of hiPSC-NS/PCs and subsequent deterioration of motor function. Remnant immature cells should be removed or induced into more mature cell types to avoid adverse effects of hiPSC-NS/PC transplantation. Because Notch signaling plays a role in maintaining NS/PCs, we evaluated the effects of γ-secretase inhibitor (GSI and found that pretreating hiPSC-NS/PCs with GSI promoted neuronal differentiation and maturation i