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Sample records for c1esterase inhibitor human

  1. Human parvovirus infection: rheumatic manifestations, angioedema, C1 esterase inhibitor deficiency, ANA positivity, and possible onset of systemic lupus erythematosus.

    Science.gov (United States)

    Fawaz-Estrup, F

    1996-07-01

    To investigate the clinical and serological characteristics of parvovirus infection. during 1993-4, 9 adult patients presented with polyarthralgias/polyarthritis. Clinical evaluation and serological studies of antinuclear antibodies, rheumatoid factor (RF), IgM and IgG antibodies to human parvovirus were done in all patients. Other serologies including anti-DNA, serum complement, and C1 esterase inhibitor levels were obtained in some patients. All 9 patients had polyarthralgias/polyarthritis, serological evidence of parvovirus B19 IgM and IgG antibodies, and normal sedimentation rate. All patients were seronegative for RF. Four women had positive ANA titers. In 2 of them the ANA positivity was transient. One developed systemic lupus erythematosus (SLE). Three women had angioedema of the face and tongue; one had transient C1 esterase inhibitor deficiency and another a transient decrease in C4 levels. Parvovirus may be implicated in the development of SLE as well as other chronic arthropathies. This is the first reported case of angioedema and decreased C1 esterase inhibitor levels associated with parvovirus infection.

  2. C1-esterase inhibitor deficiency and elective caesarean section.

    Science.gov (United States)

    Griffiths, R J; O'Sullivan, G

    2005-07-01

    C1-esterase inhibitor deficiency is a rare disorder of the complement system characterised by episodes of cutaneous and mucosal oedema. Life-threatening airway oedema can follow airway instrumentation or minor trauma. We describe the successful management of a 37-year-old primiparous woman with inherited C1-esterase inhibitor deficiency who was admitted at 38 weeks' gestation for elective caesarean section. Whilst undergoing general anaesthesia 18 months previously she had experienced facial and pharyngeal oedema despite prophylaxis (one unit of fresh frozen plasma). On this occasion she underwent elective caesarean section following intrathecal anaesthesia with 0.5% hyperbaric bupivacaine 2 mL and diamorphine 300 microg. Cardiovascular stability was ensured using glycopyrolate and intravenous Hartmann's solution 2 L; a live female infant was delivered successfully. There were no peri- or postoperative complications. Regional anaesthesia is the safest method for providing surgical anaesthesia in the obstetric patient. We believe elective caesarean section under regional anaesthesia should be considered if there are predicted difficulties with vaginal delivery.

  3. Allergenicity and safety of recombinant human C1 esterase inhibitor in patients with allergy to rabbit or cow's milk.

    Science.gov (United States)

    van den Elzen, Mignon T; van Os-Medendorp, Harmieke; Röckmann-Helmbach, Heike; van Hoffen, Els; Lebens, Ans F M; van Doorn, Helma; Klemans, Rob J B; Bruijnzeel-Koomen, Carla A F M; Hack, C Erik; Kaufman, Leonard; Relan, Anurag; Knulst, André C

    2016-08-01

    Recombinant human C1 inhibitor (rhC1INH) for on-demand treatment of hereditary angioedema is purified from milk of transgenic rabbits. It contains low amounts (cow's milk allergy (CMA). This study is an assessment of allergenicity and safety of rhC1INH in patients with RA and/or CMA. Patients with CMA and/or RA underwent skin prick test (SPT), intracutaneous test (ICT), and, when results for both were negative, subcutaneous (SC) challenge with up to 2100U (14 mL) rhC1INH. The negative predictive value of the skin test protocol was calculated, defined as the ratio of patients without systemic symptoms of hypersensitivity following SC challenge, over the number of patients having tested negative for both the SPT and the ICT. Adverse events after exposure to rhC1INH were recorded. Twenty-six patients with RA and/or CMA were enrolled. Twenty-four had negative SPT and ICT results for rhC1INH, whereas 2 had negative SPT result but positive ICT result to rhC1INH (only the highest concentration). Twenty-two patients with negative SPT and ICT results underwent SC challenge. None developed allergic symptoms. Local treatment-emergent adverse events occurred in 7 patients (32%) after SC challenge. In 5 these were considered drug related. All were mild. None of the patients with negative SPT and ICT results for rhC1INH had allergic symptoms during rhC1INH challenge. The negative predictive value of the combination of SPT and ICT for the outcome of the SC challenge was 100% (95% CI, 84.6%-100%). SC administration of rhC1INH was well tolerated. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  4. C1-esterase inhibitor blocks T lymphocyte proliferation and cytotoxic T lymphocyte generation in vitro

    DEFF Research Database (Denmark)

    Nissen, Mogens Holst; Bregenholt, S; Nording, J A

    1998-01-01

    We have previously shown that activated C1s complement and activated T cells cleave beta2-microglobulin (beta2m) in vitro leading to the formation of desLys58 beta2m. This process can specifically be inhibited by C1-esterase inhibitor (C1-inh). Furthermore we showed that exogenously added desLys58...... beta2m in nanomolar amounts to a one-way allogenic mixed lymphocyte culture (MLC) increased the endogenous production of IL-2 and the generation of allo-specific cytotoxic T lymphocytes. C1-inh was purified from fresh human plasma and added to human or murine MLC and mitogen-stimulated lymphocyte...... cultures grown in the presence of complement-inactivated serum. Read-outs were cell proliferation, lymphokine production and development of T cell-mediated cytotoxicity. We found that addition of C1-inh to MLC and mitogen-exposed murine and human lymphocyte cultures inhibited proliferation, the development...

  5. Cost-utility analysis of Ruconest® (conestat alfa) compared to Berinert® P (human C1 esterase inhibitor) in the treatment of acute, life-threatening angioedema attacks in patients with hereditary angioedema

    Science.gov (United States)

    Holko, Przemysław; Paszulewicz, Anna

    2013-01-01

    Introduction Administration of human C1 esterase inhibitor (Berinert® P) from target import is the most widespread treatment strategy for patients with hereditary angioedema (HAE). However, a therapeutic health program including Ruconest® (conestat alfa) could shorten a patient's expectancy for a life-saving treatment. Aim To evaluate the cost-utility of Ruconest® (conestat alfa) financed from public funds within the newly introduced therapeutic health program compared with Berinert® P (human C1 esterase inhibitor) in the treatment of acute angioedema attacks in adults with HAE. Material and methods The cost-utility analysis from the Polish healthcare payer's perspective was performed for 1 year (2012). The costs and health outcomes were simulated for three pairs of eligible HAE patient groups (active treatment and corresponding placebo). The incremental costs of each intervention compared with placebo were listed together (direct or indirect comparisons between options were impossible due to limited clinical data available). Results The incremental cost-utility ratios (ICURs) for the evaluated interventions compared with placebo were as follows: EUR 15,226 per QALY (Ruconest®) and EUR 27,786 per QALY (Berinert® P). The probability of cost-utility (ICUR < EUR 24,279 per QALY) assessed for Ruconest® administered in the case of acute angioedema attack was 61% and 41% for Berinert® P. Conclusions The administration of Ruconest® in acute life-threatening angioedema attacks is economically justified from the Polish healthcare payer's perspective, results in lower costs and is characterized by higher cost-utility probability compared with Berinert® P. PMID:24278067

  6. Nebulized C1-Esterase Inhibitor does not Reduce Pulmonary Complement Activation in Rats with Severe Streptococcus Pneumoniae Pneumonia.

    Science.gov (United States)

    de Beer, Friso; Lagrand, Wim; Glas, Gerie J; Beurskens, Charlotte J P; van Mierlo, Gerard; Wouters, Diana; Zeerleder, Sacha; Roelofs, Joris J T H; Juffermans, Nicole P; Horn, Janneke; Schultz, Marcus J

    2016-12-01

    Complement activation plays an important role in the pathogenesis of pneumonia. We hypothesized that inhibition of the complement system in the lungs by repeated treatment with nebulized plasma-derived human C1-esterase inhibitor reduces pulmonary complement activation and subsequently attenuates lung injury and lung inflammation. This was investigated in a rat model of severe Streptococcus pneumoniae pneumonia. Rats were intra-tracheally challenged with S. pneumoniae to induce pneumonia. Nebulized C1-esterase inhibitor or saline (control animals) was repeatedly administered to rats, 30 min before induction of pneumonia and every 6 h thereafter. Rats were sacrificed 20 or 40 h after inoculation with bacteria. Brochoalveolar lavage fluid and lung tissue were obtained for measuring levels of complement activation (C4b/c), lung injury and inflammation. Induction of pneumonia was associated with pulmonary complement activation (C4b/c at 20 h 1.24 % [0.56-2.59] and at 40 h 2.08 % [0.98-5.12], compared to 0.50 % [0.07-0.59] and 0.03 % [0.03-0.03] in the healthy control animals). The functional fraction of C1-INH was detectable in BALF, but no effect was found on pulmonary complement activation (C4b/c at 20 h 0.73 % [0.16-1.93] and at 40 h 2.38 % [0.54-4.19]). Twenty hours after inoculation, nebulized C1-esterase inhibitor treatment reduced total histology score, but this effect was no longer seen at 40 h. Nebulized C1-esterase inhibitor did not affect other markers of lung injury or lung inflammation. In this negative experimental animal study, severe S. pneumoniae pneumonia in rats is associated with pulmonary complement activation. Repeated treatment with nebulized C1-esterase inhibitor, although successfully delivered to the lungs, does not affect pulmonary complement activation, lung inflammation or lung injury.

  7. Potential therapeutic benefit of C1-esterase inhibitor in neuromyelitis optica evaluated in vitro and in an experimental rat model.

    Directory of Open Access Journals (Sweden)

    Lukmanee Tradtrantip

    Full Text Available Neuromyelitis optica (NMO is an autoimmune demyelinating disease of the central nervous system in which binding of anti-aquaporin-4 (AQP4 autoantibodies (NMO-IgG to astrocytes causes complement-dependent cytotoxicity (CDC and inflammation resulting in oligodendrocyte and neuronal injury. There is compelling evidence for a central role of complement in NMO pathogenesis. Here, we evaluated the potential of C1-esterase inhibitor (C1-inh for complement-targeted therapy of NMO. C1-inh is an anti-inflammatory plasma protein with serine protease inhibition activity that has a broad range of biological activities on the contact (kallikrein, coagulation, fibrinolytic and complement systems. C1-inh is approved for therapy of hereditary angioedema (HAE and has been studied in a small safety trial in acute NMO relapses (NCT 01759602. In vitro assays of NMO-IgG-dependent CDC showed C1-inh inhibition of human and rat complement, but with predicted minimal complement inhibition activity at a dose of 2000 units in humans. Inhibition of complement by C1-inh was potentiated by ∼10-fold by polysulfated macromolecules including heparin and dextran sulfate. In rats, intravenous C1-inh at a dose 30-fold greater than that approved to treat HAE inhibited serum complement activity by <5%, even when supplemented with heparin. Also, high-dose C1-inh did not reduce pathology in a rat model of NMO produced by intracerebral injection of NMO-IgG. Therefore, although C1r and C1s are targets of C1-inh, our in vitro data with human serum and in vivo data in rats suggest that the complement inhibition activity of C1-inh in serum is too low to confer clinical benefit in NMO.

  8. The effect of C1-esterase inhibitor on systemic inflammation in trauma patients with a femur fracture - The CAESAR study: study protocol for a randomized controlled trial

    Directory of Open Access Journals (Sweden)

    Strengers Paul FW

    2011-10-01

    Full Text Available Abstract Background Systemic inflammation in response to a femur fracture and the additional fixation is associated with inflammatory complications, such as acute respiratory distress syndrome and multiple organ dysfunction syndrome. The injury itself, but also the additional procedure of femoral fixation induces a release of pro-inflammatory cytokines such as interleukin-6. This results in an aggravation of the initial systemic inflammatory response, and can cause an increased risk for the development of inflammatory complications. Recent studies have shown that administration of the serum protein C1-esterase inhibitor can significantly reduce the release of circulating pro-inflammatory cytokines in response to acute systemic inflammation. Objective Attenuation of the surgery-induced additional systemic inflammatory response by perioperative treatment with C1-esterase inhibitor of trauma patients with a femur fracture. Methods The study is designed as a double-blind randomized placebo-controlled trial. Trauma patients with a femur fracture, Injury Severity Score ≥ 18 and age 18-80 years are included after obtaining informed consent. They are randomized for administration of 200 U/kg C1-esterase inhibitor intravenously or placebo (saline 0.9% just before the start of the procedure of femoral fixation. The primary endpoint of the study is Δ interleukin-6, measured at t = 0, just before start of the femur fixation surgery and administration of C1-esterase inhibitor, and t = 6, 6 hours after administration of C1-esterase inhibitor and the femur fixation. Conclusion This study intents to identify C1-esterase inhibitor as a safe and potent anti-inflammatory agent, that is capable of suppressing systemic inflammation in trauma patients. This might facilitate early total care procedures by lowering the risk of inflammation in response to the surgical intervention. This could result in increased functional outcomes and reduced health care related

  9. The Use of Plasma-Derived Complement C1-Esterase Inhibitor Concentrate (Berinert®) in the Treatment of Angiotensin Converting Enzyme-Inhibitor Related Angioedema

    DEFF Research Database (Denmark)

    Hermanrud, Thorbjørn; Duus, Nicolaj; Bygum, Anette

    2016-01-01

    concentrate is a well-established treatment option of hereditary and acquired complement C1-esterase inhibitor deficiency, which are also mediated by an increased level of bradykinin resulting in recurrent angioedema. We here present a case of severe angiotensin converting enzyme-inhibitor related angioedema...... (ACEi-AE) of the hypopharynx that completely resolved rapidly after the infusion of plasma-derived C1-inhibitor concentrate adding to the sparse reports in the existing literature.......Angioedema of the upper airways is a severe and potentially life-threatening condition. The incidence has been increasing in the past two decades, primarily due to pharmaceuticals influencing the generation or degradation of the vasoactive molecule bradykinin. Plasma-derived C1-esterase inhibitor...

  10. Safety of C1-Esterase Inhibitor in Acute and Prophylactic Therapy of Hereditary Angioedema

    DEFF Research Database (Denmark)

    Busse, Paula; Bygum, Anette; Edelman, Jonathan

    2014-01-01

    BACKGROUND: The plasma-derived, pasteurized C1-inhibitor (C1-INH) concentrate, Berinert has a 4-decade history of use in hereditary angioedema (HAE), with a substantial literature base that demonstrates safety and efficacy. Thromboembolic events have rarely been reported with C1-INH products, typ...

  11. C1 esterase inhibitor

    Science.gov (United States)

    ... protein called C1, which is part of the complement system. This system is a group of proteins that ... 2013:chap 20. Sullivan KE, Grumach AS. The complement system. In: Adkinson NF, Bochner BS, Burks AW, et ...

  12. Clinical Pattern and Acute and Long-term Management of Hereditary Angioedema Due to C1-Esterase Inhibitor Deficiency.

    Science.gov (United States)

    Gómez-Traseira, C; Pérez-Fernández, E; López-Serrano, M C; García-Ara, M C; Pedrosa, M; López-Trascasa, M; Caballero, T

    2015-01-01

    Hereditary angioedema due to C1-esterase inhibitor deficiency (HAE-C1-INH) is a life-threatening disease. To describe the clinical characteristics and management of patients with HAE-C1-INH during routine clinical practice. An observational, retrospective study was performed in patients with HAE-C1-INH. Demographic, clinical, and analytical data were collected from 2 periods: period A (October 2009-September 2010) and period B (October 2007-September 2009). We studied 112 patients with HAE-C1-INH (57.1% females). Age at onset of symptoms was 14.4 years (lower in patients who had experienced attacks in the previous year). In period B (n=87), 62.1% of patients presented at least 1 edema attack (median, 3.5 attacks/patient/2 years), and 19.1% of attacks were treated. In period A (n=77), 58.4% of patients were on maintenance therapy. Stanozolol was the most widely used drug (48.9%), with a mean weekly dose of 6.7 mg. At least 1 attack was recorded in 72.7% of patients (median, 3.0 attacks/patient/year), and 31.5% of the attacks were treated. Treatment of acute attacks increased by 12.4%. Age at onset of symptoms is associated with clinical expression of disease. The higher age at onset of symptoms, the fewer number of attacks per patient and year, and the lower dose of attenuated androgens necessary to control the disease than in other series lead us to hypothesize that HAE-C1-INH could have a less severe expression in Spain. Acute attacks seem to be treated increasingly often.

  13. Fibulin-1C, C1 Esterase Inhibitor and Glucose Regulated Protein 75 Interact with the CREC Proteins, Calumenin and Reticulocalbin

    DEFF Research Database (Denmark)

    Hansen, G. A. W.; Ludvigsen, M.; Jacobsen, C.

    2015-01-01

    with an affinity of approximately 3-7 nM with reticulocalbin as well as with calumenin. These interactions suggest functional participation of the CREC proteins in chaperone activity, cell proliferation and transformation, cellular aging, haemostasis and thrombosis as well as modulation of the complement system...... in fighting bacterial infection....

  14. Successful C1 inhibitor short-term prophylaxis during redo mitral valve replacement in a patient with hereditary angioedema

    Directory of Open Access Journals (Sweden)

    Coleman Suzanne

    2010-10-01

    Full Text Available Abstract Hereditary angioedema is characterized by sudden episodes of nonpitting edema that cause discomfort and pain. Typically the extremities, genitalia, trunk, gastrointestinal tract, face, and larynx are affected by attacks of swelling. Laryngeal swelling carries significant risk for asphyxiation. The disease results from mutations in the C1 esterase inhibitor gene that cause C1 esterase inhibitor deficiency. Attacks of hereditary angioedema result from contact, complement, and fibrinolytic plasma cascade activation, where C1 esterase inhibitor irreversibly binds substrates. Patients with hereditary angioedema cannot replenish C1 esterase inhibitor levels on pace with its binding. When C1 esterase inhibitor is depleted in these patients, vasoactive plasma cascade products cause swelling attacks. Trauma is a known trigger for hereditary angioedema attacks, and patients have been denied surgical procedures because of this risk. However, uncomplicated surgeries have been reported. Appropriate prophylaxis can reduce peri-operative morbidity in these patients, despite proteolytic cascade and complement activation during surgical trauma. We report a case of successful short-term prophylaxis with C1 esterase inhibitor in a 51-year-old man with hereditary angioedema who underwent redo mitral valve reconstructive surgery.

  15. Functional C1-inhibitor diagnostics in hereditary angioedema: assay evaluation and recommendations

    DEFF Research Database (Denmark)

    Wagenaar-Bos, Ineke G A; Drouet, Christian; Aygören-Pursun, Emel

    2008-01-01

    Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent episodes of potentially life-threatening angioedema. The most widespread underlying genetic deficiency is a heterozygous deficiency of the serine protease inhibitor C1 esterase inhibitor (C1-Inh). In addition ...

  16. 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.

  17. Flavonoids as Inhibitors of Human Butyrylcholinesterase Variants

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    Maja Katalinić

    2014-01-01

    Full Text Available The inhibition of butyrylcholinesterase (BChE, EC 3.1.1.8 appears to be of interest in treating diseases with symptoms of reduced neurotransmitter levels, such as Alzheimer’s disease. However, BCHE gene polymorphism should not be neglected in research since it could have an effect on the expected outcome. Several well-known cholinergic drugs (e.g. galantamine, huperzine and rivastigmine originating from plants, or synthesised as derivatives of plant compounds, have shown that herbs could serve as a source of novel target-directed compounds. We focused our research on flavonoids, biologically active polyphenolic compounds found in many plants and plant-derived products, as BChE inhibitors. All of the tested flavonoids: galangin, quercetin, fisetin and luteolin reversibly inhibited usual, atypical, and fluoride-resistant variants of human BChE. The inhibition potency increased in the following order, identically for all three BChE variants: luteolininhibitor dissociation constants (Ki ranged from 10 to 170 mmol/L. We showed that no significant change in the inhibition potency of selected flavonoids exists in view of BChE polymorphism. Our results suggested that flavonoids could assist the further development of new BChE-targeted drugs for treating symptoms of neurodegenerative diseases and dementia.

  18. Inhibitors

    Science.gov (United States)

    ... and exercise, immune tolerance therapy, and needs of older adults with hemophilia and an inhibitor. For more information, visit https://www.hemophilia.org/Events-Educational-Programs/Inhibitor-Education/Inhibitor-Education-Summits The NHF’s Inhibitor Education Summits ...

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

    Indian Academy of Sciences (India)

    2009-04-01

    Apr 1, 2009 ... helminth (Souza et al. 2002; Oshiro et al. 2004). Previ- ously we reported the results on chromosome aberrations and sister chromatid changes in human lymphocytes exposed to. Ascaris trypsin inhibitor in the absence of metabolic S9 acti- vation (Blaszkowska et al. 2004). In these experimental con- ditions ...

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

    DEFF Research Database (Denmark)

    Christensen, Trine; Skottrup, Peter; 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...... cysteines in TAFI. In this study we have purified and characterized TAFI from human plasma....

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

    DEFF Research Database (Denmark)

    Christensen, Trine; Skottrup, Peter; Valnickova, Zuzana

    2004-01-01

    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...... cysteines in TAFI. In this study we have purified and characterized TAFI from human plasma....

  2. Functional characterization of cholera toxin inhibitors using human intestinal organoids

    NARCIS (Netherlands)

    Zomer-van Ommen, Domenique D.; Pukin, Aliaksei V.; Fu, Ou; Quarles Van Ufford, Linda H C; Janssens, Hettie M.; Beekman, Jeffrey M.|info:eu-repo/dai/nl/27160378X; Pieters, Roland J.

    2016-01-01

    Preclinical drug testing in primary human cell models that recapitulate disease can significantly reduce animal experimentation and time-to-the-clinic. We used intestinal organoids to quantitatively study the potency of multivalent cholera toxin inhibitors. The method enabled the determination of

  3. 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.

  4. 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...... acetylation signatures for 19 different KDACIs, covering all 18 human lysine deacetylases. Most KDACIs increased acetylation of a small, specific subset of the acetylome, including sites on histones and other chromatin-associated proteins. Inhibitor treatment combined with genetic deletion showed...... that the effects of the pan-sirtuin inhibitor nicotinamide are primarily mediated by SIRT1 inhibition. Furthermore, we confirmed that the effects of tubacin and bufexamac on cytoplasmic proteins result from inhibition of HDAC6. Bufexamac also triggered an HDAC6-independent, hypoxia-like response by stabilizing HIF...

  5. Susceptibility of the human retrovirus XMRV to antiretroviral inhibitors

    Directory of Open Access Journals (Sweden)

    Smith Robert A

    2010-08-01

    Full Text Available Abstract Background XMRV (xenotropic murine leukemia virus-related virus is the first known example of an exogenous gammaretrovirus that can infect humans. A limited number of reports suggest that XMRV is intrinsically resistant to many of the antiretroviral drugs used to treat HIV-1 infection, but is sensitive to a small subset of these inhibitors. In the present study, we used a novel marker transfer assay to directly compare the antiviral drug sensitivities of XMRV and HIV-1 under identical conditions in the same host cell type. Results We extend the findings of previous studies by showing that, in addition to AZT and tenofovir, XMRV and HIV-1 are equally sensitive to AZddA (3'-azido-2',3'-dideoxyadenosine, AZddG (3'-azido-2',3'-dideoxyguanosine and adefovir. These results indicate that specific 3'-azido or acyclic nucleoside analog inhibitors of HIV-1 reverse transcriptase (RT also block XMRV infection with comparable efficacy in vitro. Our data confirm that XMRV is highly resistant to the non-nucleoside RT inhibitors nevirapine and efavirenz and to inhibitors of HIV-1 protease. In addition, we show that the integrase inhibitors raltegravir and elvitegravir are active against XMRV, with EC50 values in the nanomolar range. Conclusions Our analysis demonstrates that XMRV exhibits a distinct pattern of nucleoside analog susceptibility that correlates with the structure of the pseudosugar moiety and that XMRV is sensitive to a broader range of antiretroviral drugs than has previously been reported. We suggest that the divergent drug sensitivity profiles of XMRV and HIV-1 are partially explained by specific amino acid differences in their respective protease, RT and integrase sequences. Our data provide a basis for choosing specific antiretroviral drugs for clinical studies in XMRV-infected patients.

  6. 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.

  7. 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.

  8. Pharmacophore-based discovery of new human dihydroorotate dehydrogenase inhibitor.

    Science.gov (United States)

    Lu, Peng; Wang, Yubin; Ma, Bo; She, Jinxiong; Zhang, Qi; He, Mingfang; Liu, Ying

    2014-06-01

    Pharmacophore models of human dihydroorotate dehydrogenase (HsDHODH) have been developed using Discovery Studio V2.1 with a training set of 27 HsDHODH inhibitors. With one hydrogen bond receptor, two hydrophobic, one ring aromatic and one neg ionizable features, Hypo 1 has a correlation coefficient of 0.948, cost difference of 78.894, and RMSD 0.926. This model was validated by test set and Fischer randomization test. Hypo 1 was employed as a 3D query to identify potent molecules from our lab chemical database. Compound 38-C11 had Hypo 1 estimated IC50 of 489 nM. Then 38-C11 was synthesized and evaluated in HsDHODH inhibition assay. The IC50 of 38-C11 was 136.9 nM suggesting that 38-C11 could be proceeded for further evaluation in future study.

  9. Protein C inhibitor may modulate human sperm-oocyte interactions

    NARCIS (Netherlands)

    Elisen, M. G.; van Kooij, R. J.; Nolte, M. A.; Marquart, J. A.; Lock, T. M.; Bouma, B. N.; Meijers, J. C.

    1998-01-01

    Protein C inhibitor (PCI) is a heparin-binding plasma serine protease inhibitor that was originally identified as an inhibitor of activated protein C. PCI has a broad protease specificity, inhibiting several proteases in hemostasis and fibrinolysis by acting as a suicide substrate. Recently it has

  10. The structures of human dihydroorotate dehydrogenase with and without inhibitor reveal conformational flexibility in the inhibitor and substrate binding sites.

    Science.gov (United States)

    Walse, Björn; Dufe, Veronica Tamu; Svensson, Bo; Fritzson, Ingela; Dahlberg, Leif; Khairoullina, Alfia; Wellmar, Ulf; Al-Karadaghi, Salam

    2008-08-26

    Inhibitors of dihydroorotate dehydrogenase (DHODH) have been suggested for the treatment of rheumatoid arthritis, psoriasis, autoimmune diseases, Plasmodium, and bacterial and fungal infections. Here we present the structures of N-terminally truncated (residues Met30-Arg396) DHODH in complex with two inhibitors: a brequinar analogue (6) and a novel inhibitor (a fenamic acid derivative) (7), as well as the first structure of the enzyme to be characterized without any bound inhibitor. It is shown that 7 uses the "standard" brequinar binding mode and, in addition, interacts with Tyr356, a residue conserved in most class 2 DHODH proteins. Compared to the inhibitor-free structure, some of the amino acid side chains in the tunnel in which brequinar binds and which was suggested to be the binding site of ubiquinone undergo changes in conformation upon inhibitor binding. Using our data, the loop regions of residues Leu68-Arg72 and Asn212-Leu224, which were disordered in previously studied human DHODH structures, could be built into the electron density. The first of these loops, which is located at the entrance to the inhibitor-binding pocket, shows different conformations in the three structures, suggesting that it may interfere with inhibitor/cofactor binding. The second loop has been suggested to control the access of dihydroorotate to the active site of the enzyme and may be an important player in the enzymatic reaction. These observations provide new insights into the dynamic features of the DHODH reaction and suggest new approaches to the design of inhibitors against DHODH.

  11. 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.

  12. Insights into the binding mode of MEK type-III inhibitors. A step towards discovering and designing allosteric kinase inhibitors across the human kinome

    OpenAIRE

    Zhao, Zheng; Xie, Lei; Bourne, Philip E.

    2017-01-01

    Protein kinases are critical drug targets for treating a large variety of human diseases. Type-III kinase inhibitors have attracted increasing attention as highly selective therapeutics. Thus, understanding the binding mechanism of existing type-III kinase inhibitors provides useful insights into designing new type-III kinase inhibitors. In this work, we have systematically studied the binding mode of MEK-targeted type-III inhibitors using structural systems pharmacology and molecular dynamic...

  13. 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...

  14. Synthesis and evaluation of tripeptidyl alpha-ketoamides as human rhinovirus 3C protease inhibitors.

    Science.gov (United States)

    Chen, Shu-Hui; Lamar, Jason; Victor, Frantz; Snyder, Nancy; Johnson, Robert; Heinz, Beverly A; Wakulchik, Mark; Wang, Q May

    2003-10-20

    We describe herein the synthesis and biological evaluation of a series of tripeptidyl alpha-ketoamides as human rhinovirus (HRV) 3C protease inhibitors. The most potent inhibitor discussed in this manuscript, 4I, exhibited impressive enzyme inhibitory activity as well as antiviral activity against HRV-14.

  15. 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.

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

    African Journals Online (AJOL)

    (the concentration at which 50 % of the enzyme activity inhibited), which were calculated using. Microsoft Excel 2011 from a dose-response curve obtained using at least five concentrations. (ranging from 1–10 µg/mL) of the inhibitor and carried out in triplicate. Molecular docking. The initial structure of CA-II was obtained ...

  17. 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 ...

  18. Synergistic efficacy in human ovarian cancer cells by histone deacetylase inhibitor TSA and proteasome inhibitor PS-341.

    Science.gov (United States)

    Fang, Yong; Hu, Yi; Wu, Peng; Wang, Beibei; Tian, Yuan; Xia, Xi; Zhang, Qinghua; Chen, Tong; Jiang, Xuefeng; Ma, Quanfu; Xu, Gang; Wang, Shixuan; Zhou, Jianfeng; Ma, Ding; Meng, Li

    2011-05-01

    Histone deacetylase inhibitors and proteasome inhibitor are all emerging as new classes of anticancer agents. We chose TSA and PS-341 to identify whether they have a synergistic efficacy on human ovarian cancer cells. After incubated with 500 nM TSA or/and 40 nM PS-341, we found that combined groups resulted in a striking increase of apoptosis and G2/M blocking rates, no matter in A2780, cisplatin-sensitive ovarian cancer cell line OV2008 or its resistant variant C13*. This demonstrated that TSA interacted synergistically with PS-341, which raised the possibility that combined the two drugs may represent a novel strategy in ovarian cancer.

  19. A human surfactant peptide-elastase inhibitor construct as a treatment for emphysema.

    Science.gov (United States)

    Guarnieri, Frank; Spencer, Jean L; Lucey, Edgar C; Nugent, Matthew A; Stone, Phillip J

    2010-06-08

    Two million Americans suffer from pulmonary emphysema, costing $2.5 billion/year and contributing to 100,000 deaths/year. Emphysema is thought to result from an imbalance between elastase and endogenous inhibitors of elastase, leading to tissue destruction and a loss of alveoli. Decades of research have still not resulted in an effective treatment other than stopping cigarette smoking, a highly addictive behavior. On the basis of our previous work, we hypothesize that small molecule inhibitors of human neutrophil elastase are ineffective because of rapid clearance from the lungs. To develop a long-acting elastase inhibitor with a lung pharmacodynamic profile that has minimal immunogenicity, we covalently linked an elastase inhibitor, similar to a trifluoro inhibitor that was used in clinical trials, to a 25-amino-acid fragment of human surfactant peptide B. We used this construct to prevent human neutrophil elastase-induced emphysema in a rodent model. The elastase inhibitor alone, although in a 70-fold molar excess to elastase in a mixture with target organ with a synthetic small molecule inhibitor is a unique way of endowing an active compound with the pharmacodynamic profile needed to create in vivo efficacy.

  20. Potent Human Telomerase Inhibitors: Molecular Dynamic Simulations, Multiple Pharmacophore-Based Virtual Screening, and Biochemical Assays.

    Science.gov (United States)

    Shirgahi Talari, Faezeh; Bagherzadeh, Kowsar; Golestanian, Sahand; Jarstfer, Michael; Amanlou, Massoud

    2015-12-28

    Telomere maintenance is a universal cancer hallmark, and small molecules that disrupt telomere maintenance generally have anticancer properties. Since the vast majority of cancer cells utilize telomerase activity for telomere maintenance, the enzyme has been considered as an anticancer drug target. Recently, rational design of telomerase inhibitors was made possible by the determination of high resolution structures of the catalytic telomerase subunit from a beetle and subsequent molecular modeling of the human telomerase complex. A hybrid strategy including docking, pharmacophore-based virtual screening, and molecular dynamics simulations (MDS) were used to identify new human telomerase inhibitors. Docking methodology was applied to investigate the ssDNA telomeric sequence and two well-known human telomerase inhibitors' (BIBR1532 and MST-312) modes of interactions with hTERT TEN domain. Subsequently molecular dynamic simulations were performed to monitor and compare hTERT TEN domain, TEN-ssDNA, TEN-BIBR1532, TEN-MST-312, and TEN-ssDNA-BIBR1532 behavior in a dynamic environment. Pharmacophore models were generated considering the inhibitors manner in the TEN domain anchor site. These exploratory studies identified several new potent inhibitors whose IC50 values were generated experimentally in a low micromolar range with the aid of biochemical assays, including both the direct telomerase and the telomeric repeat amplification protocol (TRAP) assays. The results suggest that the current models of human telomerase are useful templates for rational inhibitor design.

  1. Identification by virtual screening and in vitro testing of human DOPA decarboxylase inhibitors.

    Directory of Open Access Journals (Sweden)

    Frederick Daidone

    Full Text Available Dopa decarboxylase (DDC, a pyridoxal 5'-phosphate (PLP enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinson's disease (PD. PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a to use virtual screening to identify potential human DDC inhibitors and (b to evaluate the reliability of our virtual-screening (VS protocol by experimentally testing the "in vitro" activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with K(i values ≥10 µM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with K(i values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a K(i value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery.

  2. Identification by virtual screening and in vitro testing of human DOPA decarboxylase inhibitors.

    Science.gov (United States)

    Daidone, Frederick; Montioli, Riccardo; Paiardini, Alessandro; Cellini, Barbara; Macchiarulo, Antonio; Giardina, Giorgio; Bossa, Francesco; Borri Voltattorni, Carla

    2012-01-01

    Dopa decarboxylase (DDC), a pyridoxal 5'-phosphate (PLP) enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinson's disease (PD). PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide) is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide) are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a) to use virtual screening to identify potential human DDC inhibitors and (b) to evaluate the reliability of our virtual-screening (VS) protocol by experimentally testing the "in vitro" activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with K(i) values ≥10 µM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with K(i) values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a K(i) value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery.

  3. Circulating Metabolites of the Human Immunodeficiency Virus Protease Inhibitor Nelfinavir in Humans: Structural Identification, Levels in Plasma, and Antiviral Activities

    OpenAIRE

    Zhang, Kanyin E.; Wu, Ellen; Amy K Patick; Kerr,Bradley; Zorbas, Mark; Lankford, Angela; Kobayashi, Takuo; Maeda, Yuki; Shetty, Bhasker; Webber, Stephanie

    2001-01-01

    Nelfinavir mesylate (Viracept, formally AG1343) is a potent and orally bioavailable human immunodeficiency virus (HIV) type 1 (HIV-1) protease inhibitor (Ki = 2 nM) and is being widely prescribed in combination with HIV reverse transcriptase inhibitors for the treatment of HIV infection. The current studies evaluated the presence of metabolites circulating in plasma following the oral administration of nelfinavir to healthy volunteers and HIV-infected patients, as well as the levels in plasma...

  4. 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.

  5. Small molecule GSK-3 inhibitors increase neurogenesis of human neural progenitor cells.

    Science.gov (United States)

    Lange, Christian; Mix, Eilhard; Frahm, Jana; Glass, Anne; Müller, Jana; Schmitt, Oliver; Schmöle, Anne-Caroline; Klemm, Kristin; Ortinau, Stefanie; Hübner, Rayk; Frech, Moritz J; Wree, Andreas; Rolfs, Arndt

    2011-01-13

    Human neural progenitor cells provide a source for cell replacement therapy to treat neurodegenerative diseases. Therefore, there is great interest in mechanisms and tools to direct the fate of multipotent progenitor cells during their differentiation to increase the yield of a desired cell type. We tested small molecule inhibitors of glycogen synthase kinase-3 (GSK-3) for their functionality and their influence on neurogenesis using the human neural progenitor cell line ReNcell VM. Here we report the enhancement of neurogenesis of human neural progenitor cells by treatment with GSK-3 inhibitors. We tested different small molecule inhibitors of GSK-3 i.e. LiCl, sodium-valproate, kenpaullone, indirubin-3-monoxime and SB-216763 for their ability to inhibit GSK-3 in human neural progenitor cells. The highest in situ GSK-3 inhibitory effect of the drugs was found for kenpaullone and SB-216763. Accordingly, kenpaullone and SB-216763 were the only drugs tested in this study to stimulate the Wnt/β-catenin pathway that is antagonized by GSK-3. Analysis of human neural progenitor differentiation revealed an augmentation of neurogenesis by SB-216763 and kenpaullone, without changing cell cycle exit or cell survival. Small molecule inhibitors of GSK-3 enhance neurogenesis of human neural progenitor cells and may be used to direct the differentiation of neural stem and progenitor cells in therapeutic applications. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  6. Production of Secretory Leucocyte Protease Inhibitor (SLPI in Human Pancreatic β-Cells

    Directory of Open Access Journals (Sweden)

    Max Nyström

    1999-01-01

    Full Text Available Secretory leucocyte protease inhibitor (SLPI is a potent inhibitor of granulocyte elastase and cathepsin G, and also an inhibitor of pancreatic enzymes like trypsin, chymotrypsin and pancreatic elastase. SLPI has also been shown to inhibit HIV-1 infections by blocking viral DNA synthesis. Since SLPI is an inhibitor of pancreatic proteases we wished to investigate whether SLPI was also actually produced in the pancreas. M-RNA from human pancreatic tissue showed evidence of SLPI production using the reverse transcriptase polymer chain reaction technique (RTPCR. Using immunohistochemical methods SLPI was demonstrated in the β-ce1ls of the islets of Langerhans. The function could be local protease/antiprotease regulation or antiviral/antibacterial defence in the close vicinity of the cell surface, or even inside the β-cell itself.

  7. 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.

  8. Insights from Analysis of Binding Sites of Human Meprins: Screening of Inhibitors by Molecular Dynamics Simulation Study.

    Science.gov (United States)

    Chaudhuri, Ankur; Bera, Asim K; Sarkar, Indrani; Chakraborty, Sibani

    2016-01-01

    Human meprin-α and-β are important regulators of angiogenesis, cancer, inflammation, fibrosis, and neurodegenerative diseases and hence important therapeutic targets. Meprins are the only astacin proteases that are expressed in membrane-bound and secreted form. The cleavage specificity of human meprins is similar in certain cases but differs markedly in others. The inhibitor selectivity of human meprins is controlled by the specific residues involved in binding at the active-site cleft of the proteases. Meprins are inhibited by various small molecular inhibitors as well as macromolecular endogenous inhibitors, making them good drug targets. In the current study, molecular dynamics simulation was performed for 10 ns on ten systems consisting of two apoenzymes of meprin -α/β and eight complexes of human meprin-α and -β complexed to four inhibitors with different metal binding moieties and comparable Ki values. These simulation studies helped to elucidate the molecular details of how several parameters influence protein-inhibitor binding affinity. Analysis of the interaction energies of the protein-inhibitor complexes revealed the diverse binding nature of this series of inhibitors. Several structural segments of human meprins exhibited certain conformational changes during the simulation time course. Among the inhibitors studied captopril had a different disposition in the meprin-bound complexes compared to the other three inhibitors, namely Pro- Leu-Gly-hydroxamate, galardin and EDTA. Comparison of the interaction energies for each system helped us to conclude that the hydroxamic acid-based inhibitors are the most potent inhibitors of meprins.

  9. 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.

  10. Differential inhibitor sensitivity between human kinases VRK1 and VRK2.

    Directory of Open Access Journals (Sweden)

    Marta Vázquez-Cedeira

    Full Text Available Human vaccinia-related kinases (VRK1 and VRK2 are atypical active Ser-Thr kinases implicated in control of cell cycle entry, apoptosis and autophagy, and affect signalling by mitogen activated protein kinases (MAPK. The specific structural differences in VRK catalytic sites make them suitable candidates for development of specific inhibitors. In this work we have determined the sensitivity of VRK1 and VRK2 to kinase inhibitors, currently used in biological assays or in preclinical studies, in order to discriminate between the two proteins as well as with respect to the vaccinia virus B1R kinase. Both VRK proteins and vaccinia B1R are poorly inhibited by inhibitors of different types targeting Src, MEK1, B-Raf, JNK, p38, CK1, ATM, CHK1/2 and DNA-PK, and most of them have no effect even at 100 µM. Despite their low sensitivity, some of these inhibitors in the low micromolar range are able to discriminate between VRK1, VRK2 and B1R. VRK1 is more sensitive to staurosporine, RO-31-8220 and TDZD8. VRK2 is more sensitive to roscovitine, RO 31-8220, Cdk1 inhibitor, AZD7762, and IC261. Vaccinia virus B1R is more sensitive to staurosporine, KU55933, and RO 31-8220, but not to IC261. Thus, the three kinases present a different pattern of sensitivity to kinase inhibitors. This differential response to known inhibitors can provide a structural framework for VRK1 or VRK2 specific inhibitors with low or no cross-inhibition. The development of highly specific VRK1 inhibitors might be of potential clinical use in those cancers where these kinases identify a clinical subtype with a poorer prognosis, as is the case of VRK1 in breast cancer.

  11. 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.

  12. Structure-guided inhibitor design for human FAAH by interspecies active site conversion

    Energy Technology Data Exchange (ETDEWEB)

    Mileni, Mauro; Johnson, Douglas S.; Wang, Zhigang; Everdeen, Daniel S.; Liimatta, Marya; Pabst, Brandon; Bhattacharya, Keshab; Nugent, Richard A.; Kamtekar, Satwik; Cravatt, Benjamin F.; Ahn, Kay; Stevens, Raymond C. (Scripps); (Pfizer)

    2008-11-24

    The integral membrane enzyme fatty acid amide hydrolase (FAAH) hydrolyzes the endocannabinoid anandamide and related amidated signaling lipids. Genetic or pharmacological inactivation of FAAH produces analgesic, anxiolytic, and antiinflammatory phenotypes but not the undesirable side effects of direct cannabinoid receptor agonists, indicating that FAAH may be a promising therapeutic target. Structure-based inhibitor design has, however, been hampered by difficulties in expressing the human FAAH enzyme. Here, we address this problem by interconverting the active sites of rat and human FAAH using site-directed mutagenesis. The resulting humanized rat (h/r) FAAH protein exhibits the inhibitor sensitivity profiles of human FAAH but maintains the high-expression yield of the rat enzyme. We report a 2.75-{angstrom} crystal structure of h/rFAAH complexed with an inhibitor, N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750), that shows strong preference for human FAAH. This structure offers compelling insights to explain the species selectivity of FAAH inhibitors, which should guide future drug design programs.

  13. 5-Lipoxygenase inhibitors attenuate TNF-α-induced inflammation in human synovial fibroblasts.

    Directory of Open Access Journals (Sweden)

    Han-Ching Lin

    Full Text Available The lipoxygenase isoform of 5-lipoxygenase (5-LOX is reported to be overexpressed in human rheumatoid arthritis synovial tissue and involved in the progress of inflammatory arthritis. However, the detailed mechanism of how 5-lipoxygenase regulates the inflammatory response in arthritis synovial tissue is still unclear. The aim of this study was to investigate the involvement of lipoxygenase pathways in TNF-α-induced production of cytokines and chemokines. Human synovial fibroblasts from rheumatoid patients were used in this study. 5-LOX inhibitors and shRNA were used to examine the involvement of 5-LOX in TNF-α-induced cytokines and chemokines expression. The signaling pathways were examined by Western Blotting or immunofluorescence staining. The effect of 5-LOX inhibitor on TNF-α-induced chemokine expression and paw edema was also explored in vivo in C57BL/6 mice. Treatment with 5-LOX inhibitors significantly decreased TNF-α-induced pro-inflammatory mediators including interleukin-6 (IL-6 and monocyte chemo-attractant protein-1 (MCP-1 in human synovial fibroblasts. Knockdown of 5-LOX using shRNA exerted similar inhibitory effects. The abrogation of NF-κB activation was involved in the antagonizing effects of these inhibitors. Furthermore, 5-LOX inhibitor decreased TNF-α-induced up-regulation of serum MCP-1 level and paw edema in mouse model. Our results provide the evidence that the administration of 5-LOX inhibitors is able to ameliorate TNF-α-induced cytokine/chemokine release and paw edema, indicating that 5-LOX inhibitors may be developed for therapeutic treatment of inflammatory arthritis.

  14. 5-Lipoxygenase Inhibitors Attenuate TNF-α-Induced Inflammation in Human Synovial Fibroblasts

    Science.gov (United States)

    Lin, Han-Ching; Lin, Tzu-Hung; Wu, Ming-Yueh; Chiu, Yung-Cheng; Tang, Chih-Hsin; Hour, Mann-Jen; Liou, Houng-Chi; Tu, Huang-Ju; Yang, Rong-Sen; Fu, Wen-Mei

    2014-01-01

    The lipoxygenase isoform of 5-lipoxygenase (5-LOX) is reported to be overexpressed in human rheumatoid arthritis synovial tissue and involved in the progress of inflammatory arthritis. However, the detailed mechanism of how 5-lipoxygenase regulates the inflammatory response in arthritis synovial tissue is still unclear. The aim of this study was to investigate the involvement of lipoxygenase pathways in TNF-α-induced production of cytokines and chemokines. Human synovial fibroblasts from rheumatoid patients were used in this study. 5-LOX inhibitors and shRNA were used to examine the involvement of 5-LOX in TNF-α-induced cytokines and chemokines expression. The signaling pathways were examined by Western Blotting or immunofluorescence staining. The effect of 5-LOX inhibitor on TNF-α-induced chemokine expression and paw edema was also explored in vivo in C57BL/6 mice. Treatment with 5-LOX inhibitors significantly decreased TNF-α-induced pro-inflammatory mediators including interleukin-6 (IL-6) and monocyte chemo-attractant protein-1 (MCP-1) in human synovial fibroblasts. Knockdown of 5-LOX using shRNA exerted similar inhibitory effects. The abrogation of NF-κB activation was involved in the antagonizing effects of these inhibitors. Furthermore, 5-LOX inhibitor decreased TNF-α-induced up-regulation of serum MCP-1 level and paw edema in mouse model. Our results provide the evidence that the administration of 5-LOX inhibitors is able to ameliorate TNF-α-induced cytokine/chemokine release and paw edema, indicating that 5-LOX inhibitors may be developed for therapeutic treatment of inflammatory arthritis. PMID:25229347

  15. 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 ...

  16. Human neutrophil defensins and secretory leukocyte proteinase inhibitor in squamous metaplastic epithelium of bronchial airways.

    NARCIS (Netherlands)

    Aarbiou, J.; Schadewijk, A. van; Stolk, J.; Sont, J.K.; Boer, W.I.; Rabe, K.F.; Krieken, J.H.J.M. van; Mauad, T.; Hiemstra, P.S.

    2004-01-01

    OBJECTIVE: The aim of this study was to analyze a possible contribution of human neutrophil defensins and secretory leukocyte proteinase inhibitor (SLPI) to the induction of airway epithelial changes such as squamous cell metaplasia. MATERIALS AND METHODS: The presence of these molecules and the

  17. High-performance liquid chromatography of HIV protease inhibitors in human biological matrices

    NARCIS (Netherlands)

    Aarnoutse, Rob E.; verweij-van wissen, CP; Underberg, W.J M; Kleinnijenhuis, Johanneke; Hekster, Yechiel A; Burger, D. M.

    2001-01-01

    Methods for HPLC analysis of protease inhibitors (PIs) in human biological matrices were reviewed. Assays have been developed for analysis of single PIs or for simultaneous measurement of multiple PIs in plasma-serum, saliva, cerebrospinal fluid and semen. Liquid-liquid extraction was most often

  18. High-performance liquid chromatography of HIV protease inhibitors in human biological matrices.

    NARCIS (Netherlands)

    Aarnoutse, R.E.; Verwey-van Wissen, C.P.W.G.M.; Underberg, W.J.M.; Kleinnijenhuis, J.; Hekster, Y.A.; Burger, D.M.

    2001-01-01

    Methods for HPLC analysis of protease inhibitors (PIs) in human biological matrices were reviewed. Assays have been developed for analysis of single PIs or for simultaneous measurement of multiple PIs in plasma-serum, saliva, cerebrospinal fluid and semen. Liquid-liquid extraction was most often

  19. 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...

  20. 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.

  1. Induction of histone deacetylases (HDACs in human abdominal aortic aneurysm: therapeutic potential of HDAC inhibitors

    Directory of Open Access Journals (Sweden)

    María Galán

    2016-05-01

    Full Text Available Clinical management of abdominal aortic aneurysm (AAA is currently limited to elective surgical repair because an effective pharmacotherapy is still awaited. Inhibition of histone deacetylase (HDAC activity could be a promising therapeutic option in cardiovascular diseases. We aimed to characterise HDAC expression in human AAA and to evaluate the therapeutic potential of class I and IIa HDAC inhibitors in the AAA model of angiotensin II (Ang II-infused apolipoprotein-E-deficient (ApoE−/− mice. Real-time PCR, western blot and immunohistochemistry evidenced an increased expression of HDACs 1, 2 (both class I, 4 and 7 (both class IIa in abdominal aorta samples from patients undergoing AAA open repair (n=22 compared with those from donors (n=14. Aortic aneurysms from Ang-II-infused ApoE−/− mice exhibited a similar HDAC expression profile. In these animals, treatment with a class I HDAC inhibitor (MS-275 or a class IIa inhibitor (MC-1568 improved survival, reduced the incidence and severity of AAA and limited aneurysmal expansion evaluated by Doppler ultrasonography. These beneficial effects were more potent in MC-1568-treated mice. The disorganisation of elastin and collagen fibres and lymphocyte and macrophage infiltration were effectively reduced by both inhibitors. Additionally, HDAC inhibition attenuated the exacerbated expression of pro-inflammatory markers and the increase in metalloproteinase-2 and -9 activity induced by Ang II in this model. Therefore, our data evidence that HDAC expression is deregulated in human AAA and that class-selective HDAC inhibitors limit aneurysm expansion in an AAA mouse model. New-generation HDAC inhibitors represent a promising therapeutic approach to overcome human aneurysm progression.

  2. 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.

  3. Factors influencing the specificity of inhibitor binding to the human and malaria parasite dihydroorotate dehydrogenases.

    Science.gov (United States)

    Bedingfield, Paul T P; Cowen, Deborah; Acklam, Paul; Cunningham, Fraser; Parsons, Mark R; McConkey, Glenn A; Fishwick, Colin W G; Johnson, A Peter

    2012-06-28

    The de novo pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase is an emerging drug target for the treatment of malaria. In this context a key property of Plasmodium falciparum DHODH (PfDHODH) is that it can be selectively inhibited over its human homologue (HsDHODH). However, HsDHODH is also a validated drug target for autoimmune diseases such as arthritis. Here a series of novel inhibitors is described that includes compounds that switch specificity between the two enzymes as a result of small alterations in chemical structure. Structure-activity relationship (SAR), crystallography, docking, and mutagenesis studies are used to examine the binding modes of the compounds within the two enzymes and to reveal structural changes induced by inhibitor binding. Within this series, compounds with therapeutically relevant HsDHODH activity are described and their binding modes characterized using X-ray crystallography, which reveals a novel conformational shift within the inhibitor binding site.

  4. Crystal structures of inhibitor complexes of human T-cell leukemia virus (HTLV-1) protease

    Energy Technology Data Exchange (ETDEWEB)

    Satoh, Tadashi; Li, Mi; Nguyen, Jeffrey-Tri; Kiso, Yoshiaki; Gustchina, Alla; Wlodawer, Alexander (NCI); (Kyoto)

    2010-09-28

    Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with several serious diseases, such as adult T-cell leukemia and tropical spastic paraparesis/myelopathy. For a number of years, the protease (PR) encoded by HTLV-1 has been a target for designing antiviral drugs, but that effort was hampered by limited available structural information. We report a high-resolution crystal structure of HTLV-1 PR complexed with a statine-containing inhibitor, a significant improvement over the previously available moderate-resolution structure. We also report crystal structures of the complexes of HTLV-1 PR with five different inhibitors that are more compact and more potent. A detailed study of structure-activity relationships was performed to interpret in detail the influence of the polar and hydrophobic interactions between the inhibitors and the protease.

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

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Lin [Department of Oncology, Zhong-Da Hospital of Southeast University, Nanjing 210009, Jiangsu (China); Chen, Baoan, E-mail: wenyu811@126.com [Department of Oncology, Zhong-Da Hospital of Southeast University, Nanjing 210009, Jiangsu (China); Qin, Shukui [Chinese PLA Cancer Center, The 81st PLA Hospital, Nanjing 210002, Jiangsu (China); Li, Suyi; He, Xiangming [Department of Oncology, Zhong-Da Hospital of Southeast University, Nanjing 210009, Jiangsu (China); Qiu, Shaomin; Zhao, Wei; Zhao, Hong [Department of Internal Medicine, Nanjing Municipal Cancer Hospital, Nanjing 210003, Jiangsu (China)

    2010-02-05

    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.

  6. Exploration of Novel Human Tyrosinase Inhibitors by Molecular Modeling, Docking and Simulation Studies.

    Science.gov (United States)

    Hassan, Mubashir; Ashraf, Zaman; Abbas, Qamar; Raza, Hussain; Seo, Sung-Yum

    2016-04-21

    Research studies on human tyrosinase inhibitors and exploration for better cytotoxic agents remain an important line in drug discovery and development at the present time. Recently, multiple inhibitors are being used to cure melanogenesis by targeting human tyrosinase. A series of coumarin (C1-C9)-, thymol (T1-T8)- and vanillin (V1-V8)-based derivatives have been theoretically analyzed for their inhibitory effects against human tyrosinase. The crystal structure of human tyrosinase is not available in Protein Data Bank. Therefore, homology modeling approach was used to predict three-dimensional (3D) crystal structure of human tyrosinase. The reliability and efficacy of predicted 3D structure were validated by using Ramachandran plots which indicate that 95.01 % residues are present in favored regions. Moreover, multiple computational approaches such as molecular docking and molecular dynamic (MD) simulation along with various online tools were employed to screen the best inhibitor against melanogenesis. The results revealed that V7 and C9 compounds showed significant binding energy values (-7.79 and -7.40 kcal/mol, respectively) compared with the standard drugs such as kojic acid (-4.21 kcal/mol) and arbutin (-4.62 kcal/mol). Moreover, MD simulation results also justified that V7 showed little fluctuations throughout the simulation period as depicted by the root mean square deviation and root mean square fluctuation graphs. Thus, the present in silico study provides a deeper insight into the structural attributes of V7 compound and its overall molecular interactions against human tyrosinase and gives a hypothetical gateway to use this compound as a potential inhibitor against melanogenesis.

  7. 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

  8. 2-Arylbenzo[b]furan derivatives as potent human lipoxygenase inhibitors.

    Science.gov (United States)

    Lang, Li; Dong, Ningning; Wu, Deyan; Yao, Xue; Lu, Weiqiang; Zhang, Chen; Ouyang, Ping; Zhu, Jin; Tang, Yun; Wang, Wei; Li, Jian; Huang, Jin

    2016-01-01

    Human lipoxygenases (LOXs) have been emerging as effective therapeutic targets for inflammatory diseases. In this study, we found that four natural 2-arylbenzo[b]furan derivatives isolated from Artocarpus heterophyllus exhibited potent inhibitory activities against human LOXs, including moracin C (1), artoindonesianin B-1 (2), moracin D (3), moracin M (4). In our in vitro experiments, compound 1 was identified as the most potent LOX inhibitor and the moderate subtype selective inhibitor of 12-LOX. Compounds 1 and 2 act as competitive inhibitors of LOXs. Moreover, 1 significantly inhibits LTB4 production and chemotactic capacity of neutrophils, and is capable of protecting vascular barrier from plasma leakage in vivo. In addition, the preliminary structure-activity relationship analysis was performed based on the above four naturally occurring (1-4) and six additional synthetic 2-arylbenzo[b]furan derivatives. Taken together, these 2-arylbenzo[b]furan derivatives, as LOXs inhibitors, could represent valuable leads for the future development of therapeutic agents for inflammatory diseases.

  9. Reaction of ozone with protease inhibitors from bovine pancreas, egg white, and human serum

    Energy Technology Data Exchange (ETDEWEB)

    Leh, F.; Warr, T.A.; Mudd, J.B.

    1978-07-01

    Ozone inactivates the bovine pancreas trypsin-killikrein inhibitor. Tyrosine and methionine residues are oxidized. When methionine is completely oxidized and 0.5 of the four tyrosine residues is oxidized, 90% of the inhibitor capacity is retained. When both methionine and tyrosine residues are completely oxidized, 37% of the inhibitor capacity is retained. For 50% loss of anti-trypsin activity, 62 moles of O/sub 3//mole of protein were required. Inhibition of the chicken ovomucoid proteinase inhibitor by ozone is accompanied by oxidation of tyrosine, histidine, and methionine residues. For 50% loss of anti-trypsin activity, 32 moles of O/sub 3//mole of protein were required. Anti-trypsin activity of diluted human serum was only slightly inhibited by ozone. Purified ..cap alpha..-l-antiprotease from human serum was 50% inactivated by 88 moles of O/sub 3//mole of protein. The oxidation of free tyrosine by ozone proceeds by way of dihydroxyphenylalanine and dihydroxyphenylalanine quinone, finally yielding colored polymeric material.

  10. 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.

  11. A human urine-derived interleukin 1 inhibitor. Homology with deoxyribonuclease I

    OpenAIRE

    1988-01-01

    We have previously reported that the urine of febrile humans contained large quantities of an inhibitor of IL-1-induced murine thymocyte proliferation that was a glycoprotein between 30 and 40 kD in size. In the present study this factor has been purified to homogeneity using a sequence of eight purification steps (ammonium sulfate precipitation, ion exchange chromatography, molecular sieve chromatography, hydrophobic affinity chromatography, hydroxylapatite chromatography, fast protein liqui...

  12. Anti-leukemic activity of DNA methyltransferase inhibitor procaine targeted on human leukaemia cells

    Directory of Open Access Journals (Sweden)

    Borutinskaite Veronika

    2016-11-01

    Full Text Available Chromatin remodeling in DNA is fundamental to gene expression, DNA replication and repair processes. Methylation of promoter regions of tumor-suppressor genes and histone deacetylation leads to gene silencing and transcriptionally repressive chromatin. For the past few decades DNA methylation agents became very attractive as the targets for cancer therapy. The purpose of this work was to examine the effects of DNMT inhibitor procaine on growth inhibition, apoptosis and differentiation of human leukaemia cells. The changes in expression of genes, proteins and histone modifications caused by procaine were evaluated under different treatments. We demonstrated that procaine arrests growth of human leukaemia cells and in combination with all-trans retinoic acid (ATRA induces cancer cell differentiation. Procaine causes reduction of expression of DNA methyltransferases as well. The treatment of human leukaemia cells with procaine increase the expression of molecules associated with differentiation (CD11b, E-cadherin, G-CSF and apoptosis (PPARγ. Moreover, the examined DNMT inhibitor enhances certain gene transcription activation via chromatin remodelling – the changes in histone H3K4(Me3 and H3K9Ac/S10P modifications were detected. Our results suggest, that DNMT inhibitor – procaine, can be used for further investigations on epigenetic differentiation therapy of leukaemia cells especially when used in combination with retinoic acid.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sasková, Klára Grantz; Koíek, Milan; Rezácová, Pavlína; Brynda, Jirí; Yashina, Tatyana; Kagan, Ron M.; Konvalinka, Jan; (Quest); (Charles U)

    2010-03-04

    Darunavir is the most recently approved human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) and is active against many HIV type 1 PR variants resistant to earlier-generation PIs. Darunavir shows a high genetic barrier to resistance development, and virus strains with lower sensitivity to darunavir have a higher number of PI resistance-associated mutations than viruses resistant to other PIs. In this work, we have enzymologically and structurally characterized a number of highly mutated clinically derived PRs with high levels of phenotypic resistance to darunavir. With 18 to 21 amino acid residue changes, the PR variants studied in this work are the most highly mutated HIV PR species ever studied by means of enzyme kinetics and X-ray crystallography. The recombinant proteins showed major defects in substrate binding, while the substrate turnover was less affected. Remarkably, the overall catalytic efficiency of the recombinant PRs (5% that of the wild-type enzyme) is still sufficient to support polyprotein processing and particle maturation in the corresponding viruses. The X-ray structures of drug-resistant PRs complexed with darunavir suggest that the impaired inhibitor binding could be explained by change in the PR-inhibitor hydrogen bond pattern in the P2 binding pocket due to a substantial shift of the aminophenyl moiety of the inhibitor. Recombinant virus phenotypic characterization, enzyme kinetics, and X-ray structural analysis thus help to explain darunavir resistance development in HIV-positive patients.

  14. Three low molecular weight cysteine proteinase inhibitors of human seminal fluid: purification and enzyme kinetic properties.

    Science.gov (United States)

    Yadav, Vikash Kumar; Chhikara, Nirmal; Gill, Kamaldeep; Dey, Sharmistha; Singh, Sarman; Yadav, Savita

    2013-08-01

    The cystatins form a superfamily of structurally related proteins with highly conserved structural folds. They are all potent, reversible, competitive inhibitors of cysteine proteinases (CPs). Proteins from this group present differences in proteinase inhibition despite their high level of structural similarities. In this study, three cysteine proteinase inhibitors (CPIs) of low molecular weight were isolated from human seminal fluid (HSF) by affinity chromatography on carboxymethyl (CM)-papain-Sepharose column, purified using various chromatographic procedures and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Matrix-assisted laser desorption-ionization-time-of flight-mass spectrometry (MALDI-TOF-MS) identified these proteins as cystatin 9, cystatin SN, and SAP-1 (an N-terminal truncated form of cystatin S). All three CPIs suppressed the activity of papain potentially and showed remarkable heat stability. Interestingly SAP-1 also inhibits the activity of trypsin, chymotrypsin, pepsin, and PSA (prostate specific antigen) and acts as a cross-class protease inhibitor in in vitro studies. Using Surface Plasmon Resonance, we have also observed that SAP-1 shows a significant binding with all these proteases. These studies suggest that SAP-1 is a cross-class inhibitor that may regulate activity of various classes of proteases within the reproductive systems. To our knowledge, this is the first report about purification of CPIs from HSF; the identification of such proteins could provide better insights into the physiological processes and offer intimation for further research. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  15. Computational Approaches for the Discovery of Human Proteasome Inhibitors: An Overview

    Directory of Open Access Journals (Sweden)

    Romina A. Guedes

    2016-07-01

    Full Text Available Proteasome emerged as an important target in recent pharmacological research due to its pivotal role in degrading proteins in the cytoplasm and nucleus of eukaryotic cells, regulating a wide variety of cellular pathways, including cell growth and proliferation, apoptosis, DNA repair, transcription, immune response, and signaling processes. The last two decades witnessed intensive efforts to discover 20S proteasome inhibitors with significant chemical diversity and efficacy. To date, the US FDA approved to market three proteasome inhibitors: bortezomib, carfilzomib, and ixazomib. However new, safer and more efficient drugs are still required. Computer-aided drug discovery has long being used in drug discovery campaigns targeting the human proteasome. The aim of this review is to illustrate selected in silico methods like homology modeling, molecular docking, pharmacophore modeling, virtual screening, and combined methods that have been used in proteasome inhibitors discovery. Applications of these methods to proteasome inhibitors discovery will also be presented and discussed to raise improvements in this particular field.

  16. 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...... 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...

  17. Identification and characterization of a serine protease inhibitor with two trypsin inhibitor-like domains from the human hookworm Ancylostoma duodenale.

    Science.gov (United States)

    Jin, Xian; Deng, Li; Li, Hui; Zhang, Zhenlin; He, Qingfeng; Yang, Chen; Jiang, Hanguo; Zhu, Xing-Quan; Peng, Lifei

    2011-02-01

    Protease inhibitors play important roles in the parasitic nematodes' survival within their host, in the development and reproduction of the parasites. The present study described the isolation, identification, and characterization of a novel member of the Ascaris family of serine protease inhibitors, designated AduTIL-1, from the human hookworm Ancylostoma duodenale. AduTIL-1 is composed of a signal sequence and two trypsin inhibitor-like (TIL) domains, which showed the highest similarity with OdmCRP, a putative serine protease inhibitor with two TIL domains in Oesophagostomum dentatum. Each TIL domain of the AduTIL-1 was expressed in Escherichia coli, and their inhibitory activities against serine proteases from animals and human were characterized, respectively. Both of the two TIL domains inhibited human neutrophil elastase and pancreatic trypsin, but different in effectiveness. Although the first TIL domain of AduTIL-1 inhibited bovine pancreatic chymotrypsin (Ki=18.0 nM), both of the two domains showed no inhibitory activity against the human pancreatic chymotrypsin. Immunohistochemical studies demonstrated that AduTIL-1 was localized in esophagus, intestine, and cuticular surface of the adult worms. These results suggested that AduTIL-1 may be involved in the survival of A. duodenale in host by targeting related digestive enzymes and neutrophil elastase.

  18. Hydrophilic, Potent, and Selective 7-Substituted 2-Aminoquinolines as Improved Human Neuronal Nitric Oxide Synthase Inhibitors.

    Science.gov (United States)

    Pensa, Anthony V; Cinelli, Maris A; Li, Huiying; Chreifi, Georges; Mukherjee, Paramita; Roman, Linda J; Martásek, Pavel; Poulos, Thomas L; Silverman, Richard B

    2017-08-24

    Neuronal nitric oxide synthase (nNOS) is a target for development of antineurodegenerative agents. Most nNOS inhibitors mimic l-arginine and have poor bioavailability. 2-Aminoquinolines showed promise as bioavailable nNOS inhibitors but suffered from low human nNOS inhibition, low selectivity versus human eNOS, and significant binding to other CNS targets. We aimed to improve human nNOS potency and selectivity and reduce off-target binding by (a) truncating the original scaffold or (b) introducing a hydrophilic group to interrupt the lipophilic, promiscuous pharmacophore and promote interaction with human nNOS-specific His342. We synthesized both truncated and polar 2-aminoquinoline derivatives and assayed them against recombinant NOS enzymes. Although aniline and pyridine derivatives interact with His342, benzonitriles conferred the best rat and human nNOS inhibition. Both introduction of a hydrophobic substituent next to the cyano group and aminoquinoline methylation considerably improved isoform selectivity. Most importantly, these modifications preserved Caco-2 permeability and reduced off-target CNS binding.

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

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    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.

  20. Secretory leukocyte protease inhibitor in punch biopsies from human colonic mucosa

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    Max Nyström

    2001-01-01

    Full Text Available Secretory leukocyte protease inhibitor (SLPI is a wellknown protease inhibitor. Its function is thought to be protease/protease-inhibitor balance. Free proteolytic activity, mainly pancreatic elastase, anionic trypsin and granulocytic elastase, has been demonstrated in faecal extracts from patients with ulcerative colitis. We wanted to verify that SLPI is actually secreted from normal human colonic mucosa. Also, we wanted to ascertain whether studies of SLPI secretion based on punch biopsies were dependent on biopsy area or on biopsy circumference. Normal colonic mucosa was sampled during surgery for colonic cancer. A total of 36 samples from four patients were used. Mucosa preparation was carried out using a punch biopsy technique, and samples of 3, 4 and 6 mm diameter were used. All media contained SLPI at varying concentrations. When expressed in terms of the sample area, the secretion per millimetre-squared seemed to decrease with increasing area. When calculated as secretion per circumference, secretion seemed to be constant. In conclusion, SLPI was secreted from normal human colonic mucosa. The SLPI secretion seemed dependent on the circumference of the biopsy rather than on the area of the biopsy.

  1. The effects of DPP-4 inhibitor on hypoxia-induced apoptosis in human umbilical vein endothelial cells

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    Akari Nagamine

    2017-01-01

    Full Text Available Dipeptidyl peptidase-4 (DPP-4 inhibitors are a new class of oral hypoglycemic agents for patients with type 2 diabetes mellitus and have potential antiatherosclerotic properties. Meanwhile, it is unclear how DPP-4 inhibitors have protective effects on atherosclerosis. Our aim was to determine the effects and its mechanisms of DPP-4 inhibitors on cultured endothelial cells. Human umbilical vein endothelial cells (HUVECs were cultured in hypoxic condition. To evaluate the protective effects of DPP-4 inhibitor on HUVECs, DPP-4 inhibitor was added in the cell culture medium and the cell viability was assessed by TUNEL assay. And we examined the intracellular signaling pathways in relation to the effects of DPP-4 inhibitor. DPP-4 inhibition had beneficial effects by inhibiting the apoptosis under hypoxic conditions in HUVECs. The antiapoptotic effects of DPP-4 inhibitor were abolished by the pretreatment with a CXCR4 antagonist or a Stat3 inhibitor. DPP-4 inhibition has beneficial effects on HUVECs by inhibiting the apoptosis under hypoxic conditions. SDF-1α/CXCR4/Stat3 pathways might be involved in the mechanisms of the cytoprotective effects of DPP-4 inhibitor. These results suggested that DPP-4 inhibitor has a potential for protecting vessels.

  2. The MEK Inhibitors Trametinib and Cobimetinib Induce a Type I Interferon Response in Human Keratinocytes

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    Daniela Lulli

    2017-10-01

    Full Text Available Mitogen-activated protein kinase kinases (MEK 1 and 2 have crucial roles in tumorigenesis, cell proliferation, and protection from apoptosis, and their inhibition is therefore an attractive therapeutic strategy in cancer. Orally available and highly selective MEK inhibitors have been developed and assessed in numerous clinical trials, either alone or in combination with cytotoxic chemotherapy and/or other targeted agents. Of note, a complex picture of class-specific adverse effects associates with these drugs, frequently including inflammatory skin rash. Here, we investigated the response of normal human keratinocytes to the MEK inhibitors trametinib and cobimetinib, alone and in combination with the v-Raf murine sarcoma viral oncogene homolog B (BRAF inhibitors dabrafenib and vemurafenib, in terms of signal transduction and de novo gene expression. MEK inhibitors triggered enhanced expression of interferon regulatory factor 1 (IRF1 and phosphorylation of signal transducer and activator of transcription 1 (STAT1, and up-regulated the keratinocyte-specific type I interferon κ (IFN-κ, the anti-viral effectors interferon-induced tetratricopeptide repeats (IFIT 1 and 2, and the pro-inflammatory chemokine (C-C motif ligand 2 (CCL2 and the C-X-C motif chemokine 10 (CXCL10, both at the mRNA and protein level. Impairment of IRF1 expression, or abrogation of STAT1 phosphorylation due to IFN-κ gene silencing, suppressed anti-viral and pro-inflammatory gene expression. These data suggest that, similar to what we observed for epidermal growth factor receptor (EGFR blockade, MEK inhibition activates a type I interferon response, which is now recognized as an effective anti-cancer response, in human epidermal keratinocytes.

  3. A new class of synthetic peptide inhibitors blocks attachment and entry of human pathogenic viruses.

    Science.gov (United States)

    Krepstakies, Marcel; Lucifora, Julie; Nagel, Claus-Henning; Zeisel, Mirjam B; Holstermann, Barbara; Hohenberg, Heinrich; Kowalski, Ina; Gutsmann, Thomas; Baumert, Thomas F; Brandenburg, Klaus; Hauber, Joachim; Protzer, Ulrike

    2012-06-01

    Many enveloped viruses, including herpes viruses, hepatitis B virus (HBV), and hepatitis C virus (HCV), and human immunodeficiency virus (HIV), are among the most important human pathogens and are often responsible for coinfections involving ≥2 types of viruses. However, therapies that are effective against multiple virus classes are rare. Here we present a new class of synthetic anti-lipopolysaccharide peptides (SALPs) that bind to heparan sulfate moieties on the cell surface and inhibit infection with a variety of enveloped viruses. We demonstrate that SALPs inhibit entry of human immunodeficiency virus type 1 (HIV-1), herpes simplex virus (HSV) 1 and 2, HBV, and HCV to their respective host cells. Despite their high antiviral efficiency, SALPs were well tolerated, and neither toxicity nor measurable inhibitor-induced adverse effects were observed. Since these broad-spectrum antiviral peptides target a host cell rather than a viral component, they may also be useful for suppression of viruses that are resistant to antiviral drugs.

  4. Molecular docking analysis of curcumin analogues as human neutrophil elastase inhibitors

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    Radhakrishnan Narayanaswamy

    2014-03-01

    Full Text Available In the present study, we aimed to dock 17 different ligands of curcumin analogues with that of human neutrophil elastase. Molecular descriptors analysis using Molinspiration online tool was carried out including investigation on human neutrophil elastase putative binding sites using Discovery Studio. The molecular physicochemical analysis revealed that all of the curcumin analogues complied well with the five rules of thumb. With regard to bioact-ivity score, compound 17 has exhibited least score towards nuclear receptor ligand (0.05 and enzyme inhibitor (0.10 compared to all other ligands. Compounds 2, 4 and 13 exhibited the maximum interaction energy (-40 kcal/mol. Interestingly, seven compounds namely 3, 11-14, 16 and 17 interacted well with Arg147 amino acid residue. The present study outcomes therefore might provide new insight in understanding these 17 curcumin analogues as potential candidates for human neutrophil elastase inhibitory agents.

  5. Pyrazolone-quinazolone hybrids: a novel class of human 4-hydroxyphenylpyruvate dioxygenase inhibitors.

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    Xu, Yu-Ling; Lin, Hong-Yan; Cao, Run-Jie; Ming, Ze-Zhong; Yang, Wen-Chao; Yang, Guang-Fu

    2014-10-01

    4-Hydroxyphenylpyruvate dioxygenase (HPPD), converting 4-hydroxyphenylpyruvate acid to homogentisate, is an important target for treating type I tyrosinemia and alkaptonuria due to its significant role in tyrosine catabolism. However, only one commercial drug, NTBC, also known as nitisinone, has been available for clinical use so far. Herein, we have elucidated the structure-based design of a series of pyrazolone-quinazolone hybrids that are novel potent human HPPD inhibitors through the successful integration of various techniques including computational simulations, organic synthesis, and biochemical characterization. Most of the new compounds displayed potent inhibitory activity against the recombinant human HPPD in nanomolar range. Compounds 3h and 3u were identified as the most potent candidates with Ki values of around 10 nM against human HPPD, about three-fold more potent than NTBC. Molecular modeling indicated that the interaction between the pyrazolone ring and ferrous ion, and the hydrophobic interaction of quinazolone with its surrounding residues, such as Phe347 and Phe364, contributed greatly to the high potency of these inhibitors. Therefore, compounds 3h and 3u could be potentially useful for the treatment of type I tyrosinemia and other diseases with defects in tyrosine degradation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. A new synthetic matrix metalloproteinase inhibitor reduces human mesenchymal stem cell adipogenesis.

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    Dale B Bosco

    Full Text Available Development of adipose tissue requires the differentiation of less specialized cells, such as human mesenchymal stem cells (hMSCs, into adipocytes. Since matrix metalloproteinases (MMPs play critical roles in the cell differentiation process, we conducted investigations to determine if a novel mercaptosulfonamide-based MMP inhibitor (MMPI, YHJ-7-52, could affect hMSC adipogenic differentiation and lipid accumulation. Enzyme inhibition assays, adipogenic differentiation experiments, and quantitative PCR methods were employed to characterize this inhibitor and determine its effect upon adipogenesis. YHJ-7-52 reduced lipid accumulation in differentiated cells by comparable amounts as a potent hydroxamate MMPI, GM6001. However, YHJ-7-82, a non-inhibitory structural analog of YHJ-7-52, in which the zinc-binding thiol group is replaced by a hydroxyl group, had no effect on adipogenesis. The two MMPIs (YHJ-7-52 and GM6001 were also as effective in reducing lipid accumulation in differentiated cells as T0070907, an antagonist of peroxisome-proliferator activated receptor gamma (PPAR-gamma, at a similar concentration. PPAR-gamma is a typical adipogenic marker and a key regulatory protein for the transition of preadiopocyte to adipocyte. Moreover, MMP inhibition was able to suppress lipid accumulation in cells co-treated with Troglitazone, a PPAR-gamma agonist. Our results indicate that MMP inhibitors may be used as molecular tools for adipogenesis and obesity treatment research.

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

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    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.

  8. RO0504985 is an inhibitor of CMGC kinase proteins and has anti-human cytomegalovirus activity.

    Science.gov (United States)

    Strang, Blair L

    2017-08-01

    Public-private partnerships allow many previously unavailable compounds to be screened for antiviral activity. Here a screening method was used to identify an oxindole compound, RO0504985, from a Roche kinase inhibitor library that inhibited human cytomegalovirus (HCMV) protein production. RO0504985 was previously described as an inhibitor of cyclin-dependent kinase 2 (CDK2). However, using kinase selectivity assays it was found that RO0504985 was an inhibitor of several CMGC group kinase proteins, including CDK2. Using virus yield reduction assays it was observed that RO0504985 inhibited replication of different HCMV strains at low micromolar concentrations. Western blotting was used to investigate how RO0504985 inhibited HCMV replication. Treatment of HCMV infected cells with RO0504985 inhibited production of the immediate early viral IE2 proteins and the late viral protein pp28. Thus, RO0504985 inhibited HCMV replication by preventing production of specific HCMV proteins necessary for virus replication. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  9. Structural characterization of human heme oxygenase-1 in complex with azole-based inhibitors.

    Science.gov (United States)

    Rahman, Mona N; Vlahakis, Jason Z; Roman, Gheorghe; Vukomanovic, Dragic; Szarek, Walter A; Nakatsu, Kanji; Jia, Zongchao

    2010-03-01

    The development of inhibitors specific for heme oxygenases (HO) aims to provide powerful tools in understanding the HO system. Based on the lead structure (2S, 4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[((4-aminophenyl)thio)methyl]-1,3-dioxolane (azalanstat, QC-1) we have synthesized structural modifications to develop novel and selective HO inhibitors. The structural study of human HO-1 (hHO-1) in complex with a select group of the inhibitors was initiated using X-ray crystallographic techniques. Comparison of the structures of four such compounds each in complex with hHO-1 revealed a common binding mode, despite having different structural fragments. The compounds bind to the distal side of heme through an azole "anchor" which coordinates with the heme iron. An expansion of the distal pocket, mainly due to distal helix flexibility, allows accommodation of the compounds without displacing heme or the critical Asp140 residue. Rather, binding displaces a catalytically critical water molecule and disrupts an ordered hydrogen-bond network involving Asp140. The presence of a triazole "anchor" may provide further stability via a hydrogen bond with the protein. A hydrophobic pocket acts to stabilize the region occupied by the phenyl or adamantanyl moieties of these compounds. Further, a secondary hydrophobic pocket is formed via "induced fit" to accommodate bulky substituents at the 4-position of the dioxolane ring. Copyright 2009 Elsevier Inc. All rights reserved.

  10. 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.

  11. Ordering of the N-terminus of human MDM2 by small molecule inhibitors.

    Science.gov (United States)

    Michelsen, Klaus; Jordan, John B; Lewis, Jeffrey; Long, Alexander M; Yang, Evelyn; Rew, Yosup; Zhou, Jing; Yakowec, Peter; Schnier, Paul D; Huang, Xin; Poppe, Leszek

    2012-10-17

    Restoration of p53 function through the disruption of the MDM2-p53 protein complex is a promising strategy for the treatment of various types of cancer. Here, we present kinetic, thermodynamic, and structural rationale for the remarkable potency of a new class of MDM2 inhibitors, the piperidinones. While these compounds bind to the same site as previously reported for small molecule inhibitors, such as the Nutlins, data presented here demonstrate that the piperidinones also engage the N-terminal region (residues 10-16) of human MDM2, in particular, Val14 and Thr16. This portion of MDM2 is unstructured in both the apo form of the protein and in MDM2 complexes with p53 or Nutlin, but adopts a novel β-strand structure when complexed with the piperidinones. The ordering of the N-terminus upon binding of the piperidinones extends the current model of MDM2-p53 interaction and provides a new route to rational design of superior inhibitors.

  12. Structure of the human autophagy initiating kinase ULK1 in complex with potent inhibitors.

    Science.gov (United States)

    Lazarus, Michael B; Novotny, Chris J; Shokat, Kevan M

    2015-01-16

    Autophagy is a conserved cellular process that involves the degradation of cellular components for energy maintenance and cytoplasmic quality control that has recently gained interest as a novel target for a variety of human diseases, including cancer. A prime candidate to determine the potential therapeutic benefit of targeting autophagy is the kinase ULK1, whose activation initiates autophagy. Here, we report the first structures of ULK1, in complex with multiple potent inhibitors. These structures show features unique to the enzyme and will provide a path for the rational design of selective compounds as cellular probes and potential therapeutics.

  13. Structure-Activity Relationships of Pentacyclic Triterpenoids as Potent and Selective Inhibitors against Human Carboxylesterase 1

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    Li-Wei Zou

    2017-06-01

    Full Text Available Human carboxylesterase 1 (hCE1, one of the most important serine hydrolases distributed in liver and adipocytes, plays key roles in endobiotic homeostasis and xenobiotic metabolism. This study aimed to find potent and selective inhibitors against hCE1 from phytochemicals and their derivatives. To this end, a series of natural triterpenoids were collected and their inhibitory effects against human carboxylesterases (hCEs were assayed using D-Luciferin methyl ester (DME and 6,8-dichloro-9,9-dimethyl-7-oxo-7,9-dihydroacridin-2-yl benzoate (DDAB as specific optical substrate for hCE1, and hCE2, respectively. Following screening of a series of natural triterpenoids, oleanolic acid (OA, and ursolic acid (UA were found with strong inhibitory effects on hCE1 and relative high selectivity over hCE2. In order to get the highly selective and potent inhibitors of hCE1, a series of OA and UA derivatives were synthesized from OA and UA by chemical modifications including oxidation, reduction, esterification, and amidation. The inhibitory effects of these derivatives on hCEs were assayed and the structure-activity relationships of tested triterpenoids as hCE1 inhibitors were carefully investigated. The results demonstrated that the carbonyl group at the C-28 site is essential for hCE1 inhibition, the modifications of OA or UA at this site including esters, amides and alcohols are unbeneficial for hCE1 inhibition. In contrast, the structural modifications on OA and UA at other sites, such as converting the C-3 hydroxy group to 3-O-β-carboxypropionyl (compounds 20 and 22, led to a dramatically increase of the inhibitory effects against hCE1 and very high selectivity over hCE2. 3D-QSAR analysis of all tested triterpenoids including OA and UA derivatives provide new insights into the fine relationships linking between the inhibitory effects on hCE1 and the steric-electrostatic properties of triterpenoids. Furthermore, both inhibition kinetic analyses and docking

  14. Direct comparisons of inhibitor sensitivities of reverse transcriptases from feline and human immunodeficiency viruses.

    Science.gov (United States)

    North, T W; Cronn, R C; Remington, K M; Tandberg, R T

    1990-08-01

    The sensitivities of reverse transcriptases (RTs) from feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (HIV) were directly compared. The two enzymes had similar sensitivities to three analogs of dTTP, namely, 3'-azido-3'-deoxythymidine 5'-triphosphate, 2',3'-dideoxythymidine 5'-triphosphate, and 2',3'-dideoxy-2',3'-didehydrothymidine 5'-triphosphate. Each of these analogs demonstrated competitive inhibition of both enzymes. Ki values for inhibition of FIV RT by these three inhibitors were 3.3, 6.7, and 1.8 nM, respectively; Ki values for inhibition of the HIV enzyme were 6.5, 5.9, and 8.3 nM, respectively. Ratios of the Ki for the inhibitor to the Km for the substrate were also determined for each inhibitor, and no differences between the two enzymes greater than threefold were observed. Inhibition constants for 3'-amino-3'-deoxythymidine 5'-triphosphate and 3'-fluoro-3'-deoxythymidine 5'-triphosphate were determined for FIV RT, and these were similar to published values for HIV RT. The activities of three dideoxynucleoside 5'-triphosphates against FIV RT were determined; ddGTP was slightly more potent than ddTTP, whereas both were much more effective than ddCTP. The activity of a noncompetitive inhibitor, phosphonoformate, was also examined with the FIV enzyme; it was much more active with poly(rA)-oligo(dT) as the template-primer than with poly(rC)-oligo(dG) or poly(rI)-oligo(dC).

  15. Oncolytic virotherapy synergism with signaling inhibitors: Rapamycin increases myxoma virus tropism for human tumor cells.

    Science.gov (United States)

    Stanford, Marianne M; Barrett, John W; Nazarian, Steven H; Werden, Steven; McFadden, Grant

    2007-02-01

    Myxoma virus is a rabbit-specific poxvirus pathogen that also exhibits a unique tropism for human tumor cells and is dramatically oncolytic for human cancer xenografts. Most tumor cell lines tested are permissive for myxoma infection in a fashion intimately tied to the activation state of Akt kinase. A host range factor of myxoma virus, M-T5, directly interacts with Akt and mediates myxoma virus tumor cell tropism. mTOR is a regulator of cell growth and metabolism downstream of Akt and is specifically inhibited by rapamycin. We report that treatment of nonpermissive human tumor cell lines, which normally restrict myxoma virus replication, with rapamycin dramatically increased virus tropism and spread in vitro. This increased myxoma replication is concomitant with global effects on mTOR signaling, specifically, an increase in Akt kinase. In contrast to the effects on human cancer cells, rapamycin does not increase myxoma virus replication in rabbit cell lines or permissive human tumor cell lines with constitutively active Akt. This indicates that rapamycin increases the oncolytic capacity of myxoma virus for human cancer cells by reconfiguring the internal cell signaling environment to one that is optimal for productive virus replication and suggests the possibility of a potentially therapeutic synergism between kinase signaling inhibitors and oncolytic poxviruses for cancer treatment.

  16. Cyclic guanosine monophosphate modulates accumulation of phosphodiesterase 5 inhibitors in human platelets.

    Science.gov (United States)

    Bajraktari, Gzona; Burhenne, Jürgen; Bugert, Peter; Haefeli, Walter Emil; Weiss, Johanna

    2017-12-01

    Sildenafil and tadalafil are widely-used phosphodiesterase 5 (PDE5) inhibitors for which no clear dose-response relationship could be established. Using isolated and/or recombinant PDE5, it has been demonstrated that cGMP can increase the affinity of this enzyme for sildenafil and tadalafil. We thus hypothesized that in cells expressing the nitric oxide - soluble guanylyl cyclase - cyclic guanosine monophosphate - PDE5 (NO-sGC-cGMP-PDE5) pathway such as platelets, the presence of NO increases the intracellular cGMP content and thus promotes the intracellular accumulation of sildenafil or tadalafil. As a cell model, isolated and washed human platelets were used. Platelet suspensions were incubated with sildenafil or tadalafil at different concentrations and for various time intervals with or without an NO donor to increase intraplatelet cGMP concentrations. Intracellular sildenafil or tadalafil was quantified by ultra-performance liquid chromatography tandem mass spectrometry and intracellular cGMP by an enzyme-linked immunosorbent assay. Sildenafil accumulated in platelets with an up to 4-fold higher accumulation when platelets were pretreated with an NO donor (p < .0001). Accumulation of tadalafil in platelets was even higher, whereas the increase was 2-fold when an NO donor was present (p < .001). This accumulation was time-dependent and happened concomitantly with a rise in intracellular cGMP. Our data demonstrate that intracellular cGMP increases intracellular PDE5 inhibitor concentrations most likely by raising the affinity of these compounds for PDE5. These findings suggest that PDE5 inhibitor action in humans is critically influenced by modulators of the activity of the NO pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Nicotine-related alkaloids and metabolites as inhibitors of human cytochrome P-450 2A6.

    Science.gov (United States)

    Denton, Travis T; Zhang, Xiaodong; Cashman, John R

    2004-02-15

    S-(-)-Nicotine and 13 of the most prevalent nicotine-related alkaloids and metabolites (i.e., S-(-)-nornicotine, myosmine, beta-nicotyrine, S-cotinine, S-norcotinine, S-(-)-nicotine N-1'-oxide, S-(-)-nicotine Delta1'-5'-iminium ion, S-(-)-anabasine, S-(-)-N-methylanabasine, anabaseine, S-(-)-anatabine, nicotelline, and 2,3'-bipyridyl) were evaluated as inhibitors of human cDNA-expressed cytochrome P-450 2A6 (CYP2A6) mediated coumarin 7-hydroxylation. Tobacco alkaloids myosmine, S-(-)-nornicotine, S-cotinine, S-norcotinine, S-(-)-nicotine N-1'-oxide, S-(-)-nicotine Delta1'-5'-iminium ion, S-(-)-N-methylanabasine, anabaseine, and nicotelline had Ki values for inhibition of coumarin 7-hydroxylation ranging from 20 microM to more than 300 microM whereas nicotine and S-(-)-anatabine were much more potent (i.e. 4.4 and 3.8 microM, respectively). The tobacco alkaloids 2,3'-bipyridyl (7.7 microM) and S-(-)-anabasine (5.4 microM), were somewhat less potent compared with S-(-)-nicotine or S-(-)-anatabine in inhibition of human CYP2A6. beta-Nicotyrine, in which the N-methylpyrrolidino moiety of nicotine was replaced by the aromatic N-methylpyrrole ring, was shown to inhibit human CYP2A6 with much greater potency (Ki=0.37 microM) compared with S-(-)-nicotine. Among the compounds examined, only nicotine and beta-nicotyrine were mechanism-based inhibitors of human CYP2A6. The potency of the mechanism-based CYP2A6 inhibitors suggests that, for smokers, modulation of CYP2A6 may be greater than that predicted on the basis of serum concentration of these alkaloids. Our results indicate that the prominent nicotine-related alkaloid beta-nicotyrine present after smoking potently inhibits human CYP2A6.

  18. Robotic inhibition assay for determination of HMG-CoA reductase inhibitors in human plasma.

    Science.gov (United States)

    Fang, Wei; Liu, Lida; Hsieh, John Y-K; Zhao, Jamie; Matuszewski, Bogdan K; Rogers, John D; Dobrinska, Michael R

    2002-01-01

    The cholesterol-lowering drug simvastatin (SIMV, Zocor reduced heart attacks by 42% in patients who had high cholesterol levels and suffered from heart disease. Upon oral administration, SIMV is quickly hydrolyzed to its beta-hydroxyacid and other acid metabolites, which are potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase. A Tecan-based enzyme inhibition assay has been developed to improve the existing Zymark-based assay for the determination of both active and total concentrations of HMG-CoA reductase inhibitors in human plasma. A Tecan Genesis 200 robotic workstation equipped with eight probes and customized hardware was utilized to achieve higher sample throughput and improve assay reproducibility and mechanical stability. The developed enzyme inhibition assay was validated over two concentration ranges of 0.4-20 ng equivalent/mL, and 2-50 ng equivalent/mL. Intra- and interday precision data (coefficient of variation (CV)) for both concentration ranges were less than 9%, with an accuracy of 93-107%. The interday precision for the determination of quality control (QC) samples was less than 2% and 8%, respectively. The respective interday QC accuracy values were 93-103% and 97-104%. Good linearity across the two concentration ranges was observed, with acceptable reproducibility. This improved enzyme inhibition assay has been utilized to analyze human plasma samples from several clinical studies. Copyright 2002 Wiley-Liss, Inc.

  19. Identification of human telomerase assembly inhibitors enabled by a novel method to produce hTERT

    Science.gov (United States)

    Kellermann, Guillaume; Kaiser, Markus; Dingli, Florent; Lahuna, Olivier; Naud-Martin, Delphine; Mahuteau-Betzer, Florence; Loew, Damarys; Ségal-Bendirdjian, Evelyne; Teulade-Fichou, Marie-Paule; Bombard, Sophie

    2015-01-01

    Telomerase is the enzyme that maintains the length of telomeres. It is minimally constituted of two components: a core reverse transcriptase protein (hTERT) and an RNA (hTR). Despite its significance as an almost universal cancer target, the understanding of the structure of telomerase and the optimization of specific inhibitors have been hampered by the limited amount of enzyme available. Here, we present a breakthrough method to produce unprecedented amounts of recombinant hTERT and to reconstitute human telomerase with purified components. This system provides a decisive tool to identify regulators of the assembly of this ribonucleoprotein complex. It also enables the large-scale screening of small-molecules capable to interfere with telomerase assembly. Indeed, it has allowed us to identify a compound that inhibits telomerase activity when added prior to the assembly of the enzyme, while it has no effect on an already assembled telomerase. Therefore, the novel system presented here may accelerate the understanding of human telomerase assembly and facilitate the discovery of potent and mechanistically unique inhibitors. PMID:25958399

  20. 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.

  1. 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.

  2. 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.

  3. Identification of indole inhibitors of human hematopoietic prostaglandin D2 synthase (hH-PGDS).

    Science.gov (United States)

    Edfeldt, Fredrik; Evenäs, Johan; Lepistö, Matti; Ward, Alison; Petersen, Jens; Wissler, Lisa; Rohman, Mattias; Sivars, Ulf; Svensson, Karin; Perry, Matthew; Feierberg, Isabella; Zhou, Xiao-Hong; Hansson, Thomas; Narjes, Frank

    2015-06-15

    Human H-PGDS has shown promise as a potential target for anti-allergic and anti-inflammatory drugs. Here we describe the discovery of a novel class of indole inhibitors, identified through focused screening of 42,000 compounds and evaluated using a series of hit validation assays that included fluorescence polarization binding, 1D NMR, ITC and chromogenic enzymatic assays. Compounds with low nanomolar potency, favorable physico-chemical properties and inhibitory activity in human mast cells have been identified. In addition, our studies suggest that the active site of hH-PGDS can accommodate larger structural diversity than previously thought, such as the introduction of polar groups in the inner part of the binding pocket. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. 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

    of cIAP2 caused a substantial impairment of the IEC regeneration through inhibition of migration (P migrating IECs and upregulation of expression of RhoA and Rac1 as well as GTP-activation of Rac1. Transforming growth factor-β1 enhanced the expression......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...... of cIAP2 but was not upregulated in wounds in vivo and in vitro. NF-κB and MAPK pathways did not affect cIAP2 expression. cIAP2 is in conclusion a regulator of human intestinal wound healing through enhanced migration along with activation of Rac1, and the findings suggest that cIAP2 could be a future...

  5. 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

    of ciclosporin and tacrolimus. We document that both drugs acutely increase insulin sensitivity, while first phase and pulsatile insulin secretion remain unaffected. This study demonstrates that ciclosporin and tacrolimus have similar acute effects on glucose metabolism in healthy humans. AIM The introduction...... and tacrolimus has been attributed to both beta cell dysfunction and impaired insulin sensitivity. WHAT THIS STUDY ADDS: This is the first trial to investigate beta cell function and insulin sensitivity using gold standard methodology in healthy human volunteers treated with clinically relevant doses...... of calcineurin inhibitors (CNIs) ciclosporin (CsA) and tacrolimus (Tac) has improved the outcome of organ transplants, but complications such as new onset diabetes mellitus after transplantation (NODAT) cause impairment of survival rates. The relative contribution of each CNI to the pathogenesis and development...

  6. Phospholipase A2 Inhibitor from Crotalus durissus terrificus rattlesnake: Effects on human peripheral blood mononuclear cells and human neutrophils cells.

    Science.gov (United States)

    Xavier, Caroline V; da S Setúbal, Sulamita; Lacouth-Silva, Fabianne; Pontes, Adriana S; Nery, Neriane M; de Castro, Onassis Boeri; Fernandes, Carla F C; Soares, Andreimar M; Fortes-Dias, Consuelo L; Zuliani, Juliana P

    2017-12-01

    Crotalus Neutralizing Factor (CNF) is an inhibitor of phospholipase A2 (PLA2), present in the blood plasma of Crotalus durissus terrificus snake. This inhibitor neutralizes the lethal and enzymatic activity of crotoxin, the main neurotoxin from this venom. In this study, we investigated the effects of CNF on the functionality of human peripheral blood mononuclear cells (PBMCs) and human neutrophils. The following parameters were evaluated: viability and proliferation, chemotaxis, cytokines and LTB4 production, cytosolic PLA2s activity, myeloperoxidase (MPO) and superoxide anion (O2-) production. CNF showed no toxicity on PBMCs or neutrophils, and acts by stimulating the release of TNF-α and LTB4, but neither stimulates IL-10 and IL-2 nor affects PBMCs proliferation and O2- release. In neutrophils, CNF induces chemotaxis but does not induce the release of both MPO and O2-. However, it induces LTB4 and IL-8 production. These data show the influence of CNF on PBMCs' function by inducing TNF-α and LTB4 production, and on neutrophils, by stimulating chemotaxis and LTB4 production, via cytosolic PLA2 activity, and IL-8 release. The inflammatory profile produced by CNF is shown for the first time. Our present results suggest that CNF has a role in activation of leukocytes and exert proinflammatory effects on these cell. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Effect of oncogene activating mutations and kinase inhibitors on amino acid metabolism of human isogenic breast cancer cells.

    Science.gov (United States)

    Kim, Eung-Sam; Samanta, Animesh; Cheng, Hui Shan; Ding, Zhaobing; Han, Weiping; Toschi, Luisella; Chang, Young Tae

    2015-12-01

    We investigated the changes in amino acid (AA) metabolism induced in MCF10A, a human mammary epithelial cell line, by the sequential knock-in of K-Ras and PI3K mutant oncogenes. Differentially regulated genes associated to AA pathways were identified on comparing gene expression patterns in the isogenic cell lines. Additionally, we monitored the changes in the levels of AAs and transcripts in the cell lines treated with kinase inhibitors (REGO: a multi-kinase inhibitor, PI3K-i: a PI3K inhibitor, and MEK-i: a MEK inhibitor). In total, 19 AAs and 58 AA-associated transcripts were found to be differentially regulated by oncogene knock-in and by drug treatment. In particular, the multi-kinase and MEK inhibitor affected pathways in K-Ras mutant cells, whereas the PI3K inhibitor showed a major impact in the K-Ras/PI3K double mutant cells. These findings may indicate the dependency of AA metabolism on the oncogene mutation pattern in human cancer. Thus, future therapy might include combinations of kinase inhibitors and drug targeting enzymes of AA pathways.

  8. Tissue factor pathway inhibitor attenuates ER stress-induced inflammation in human M2-polarized macrophages.

    Science.gov (United States)

    Espada, Sandra; Stavik, Benedicte; Holm, Sverre; Sagen, Ellen Lund; Bjerkeli, Vigdis; Skjelland, Mona; Dahl, Tuva B; Espevik, Terje; Kanse, Sandip; Sandset, Per Morten; Skretting, Grethe; Halvorsen, Bente

    2017-09-16

    Endoplasmic reticulum (ER) stress has been shown to play a key role during the initiation and clinical progression of the cardiovascular diseases, such as atherosclerosis. We have recently shown that expression of tissue factor pathway inhibitor (TFPI) in human monocyte-derived macrophages (MDMs) was induced by cholesterol crystals (CC). In the present study we aimed to determine the role of TFPI under ER stress conditions using human MDMs. qRT-PCR and immunohistochemistry analysis were performed to determine the presence of the ER stress marker CCAAT/enhancer binding protein homologous protein (CHOP) and TFPI in human carotid plaque material and also in human MDMs polarized into pro-inflammatory M1 or anti-inflammatory M2 populations. CHOP mRNA levels were upregulated in the plaques compared to healthy vessels, and CHOP protein was localized in the same area as TFPI in the plaques. Both CHOP and TFPI mRNA levels were upregulated after CC treatment, especially in the M2 phenotype, and the ER stress inhibitor 4-phenylbutyric acid (PBA) reversed this effect. Furthermore, CC treatment increased the levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-8, which for TNF-α and IL-8 was inhibited by PBA, and reduced the levels of the anti-inflammatory cytokine IL-10 in M2-polarized macrophages. Knockdown of TFPI prior to CC treatment exacerbated TNF-α and IL-6 levels, but reduced IL-8 and IL-10 levels. Our results show that CC induce TFPI and cytokine expression in M2-polarized macrophages through activation of the ER stress pathway and that TFPI has a protective effect against TNF-α and IL-6 mediated inflammation. These mechanisms may have implications for the pathogenesis of atherosclerosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Different effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors on sterol synthesis in various human cell types

    NARCIS (Netherlands)

    Vliet, A.K. van; Thiel, G.C.F. van; Huisman, R.H.; Moshage, H.; Yap, S.H.; Cohen, L.H.

    1995-01-01

    The three vastatins examined, lovastatin, simvastatin and pravastatin, are equally strong inhibitors of the sterol synthesis in human hepatocytes in culture with IC50-values of 4.1, 8.0 and 2.0 nM, respectively. However, in the human extrahepatic cells: umbilical vascular endothelial cells, retinal

  10. Expression of plasminogen activator inhibitor-1 in human adipose tissue: a role for TNF-alpha?

    Science.gov (United States)

    Cigolini, M; Tonoli, M; Borgato, L; Frigotto, L; Manzato, F; Zeminian, S; Cardinale, C; Camin, M; Chiaramonte, E; De Sandre, G; Lunardi, C

    1999-03-01

    Elevated plasminogen activator inhibitor-1 (PAI-1) plasma levels, responsible for reduced fibrinolysis, are associated with animal and human obesity and with increased cardiovascular disease. The expression of PAI-1 has been found recently in animal and human adipose tissue. Factors and mechanisms regulating such an expression remain to be elucidated. In omental and/or subcutaneous biopsies from obese non-diabetic patients, incubated in Medium 199, we have confirmed that human adipose tissue expresses PAI-1 protein and mRNA; furthermore we have demonstrated that such an expression is clearly evident also in collagenase isolated human adipocytes and that it is stimulated by incubation itself and enhanced by exogenous human tumor necrosis factor-alpha (h-TNF-alpha). Since human adipose tissue produces TNF-alpha, to further characterize the relationship of PAI-1 to TNF-alpha, human fat biopsies were also incubated with Pentoxifylline (PTX) or Genistein, both known to inhibit endogenous TNF-alpha through different mechanisms. PTX caused a dose-dependent decrease of basal PAI-1 protein release, reaching 80% maximal inhibitory effect at 10(-3)M, the same inhibitory effect caused by Genistein at 100 microg/ml. This was associated to a marked inhibition of PAI-1 mRNA and of endogenous TNF-alpha production. Furthermore, when human fat biopsies were incubated in the presence of polyclonal rabbit neutralizing anti-human TNF-alpha antibody (at a concentration able to inhibit 100 UI/ml human TNF-alpha activity), a modest but significant decrease of the incubation induced expression of PAI-1 mRNA was observed (19.8+/-19.0% decrease, P = 0.04, n = 7). In conclusion, the results of this study demonstrate that PAI-I expression is present in human isolated adipocytes and that it is enhanced in human adipose tissue in vitro by exogenous TNF-alpha. Furthermore our data support the possibility of a main role of endogenous TNF-alpha on human adipose tissue PAI-1 expression. This

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

    Directory of Open Access Journals (Sweden)

    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.

  12. The HSP90 inhibitor alvespimycin enhances the potency of telomerase inhibition by imetelstat in human osteosarcoma.

    Science.gov (United States)

    Hu, Yafang; Bobb, Daniel; He, Jianping; Hill, D Ashley; Dome, Jeffrey S

    2015-01-01

    The unsatisfactory outcomes for osteosarcoma necessitate novel therapeutic strategies. This study evaluated the effect of the telomerase inhibitor imetelstat in pre-clinical models of human osteosarcoma. Because the chaperone molecule HSP90 facilitates the assembly of telomerase protein, the ability of the HSP90 inhibitor alvespimycin to potentiate the effect of the telomerase inhibitor was assessed. The effect of single or combined treatment with imetelstat and alvespimycin on long-term growth was assessed in osteosarcoma cell lines (143B, HOS and MG-63) and xenografts derived from 143B cells. Results indicated that imetelstat as a single agent inhibited telomerase activity, induced telomere shortening, and inhibited growth in all 3 osteosarcoma cell lines, though the bulk cell cultures did not undergo growth arrest. Combined treatment with imetelstat and alvespimycin resulted in diminished telomerase activity and shorter telomeres compared to either agent alone as well as higher levels of γH2AX and cleaved caspase-3, indicative of increased DNA damage and apoptosis. With dual telomerase and HSP90 inhibition, complete growth arrest of bulk cell cultures was achieved. In xenograft models, all 3 treatment groups significantly inhibited tumor growth compared with the placebo-treated control group, with the greatest effect seen in the combined treatment group (imetelstat, p = 0.045, alvespimycin, p = 0.034; combined treatment, p = 0.004). In conclusion, HSP90 inhibition enhanced the effect of telomerase inhibition in pre-clinical models of osteosarcoma. Dual targeting of telomerase and HSP90 warrants further investigation as a therapeutic strategy.

  13. N-glycans of human protein C inhibitor: tissue-specific expression and function.

    Directory of Open Access Journals (Sweden)

    Wei Sun

    Full Text Available Protein C inhibitor (PCI is a serpin type of serine protease inhibitor that is found in many tissues and fluids in human, including blood plasma, seminal plasma and urine. This inhibitor displays an unusually broad protease specificity compared with other serpins. Previous studies have shown that the N-glycan(s and the NH₂-terminus affect some blood-related functions of PCI. In this study, we have for the first time determined the N-glycan profile of seminal plasma PCI, by mass spectrometry. The N-glycan structures differed markedly compared with those of both blood-derived and urinary PCI, providing evidence that the N-glycans of PCI are expressed in a tissue-specific manner. The most abundant structure (m/z 2592.9 had a composition of Fuc₃Hex₅HexNAc₄, consistent with a core fucosylated bi-antennary glycan with terminal Lewis(x. A major serine protease in semen, prostate specific antigen (PSA, was used to evaluate the effects of N-glycans and the NH₂-terminus on a PCI function related to the reproductive tract. Second-order rate constants for PSA inhibition by PCI were 4.3±0.2 and 4.1±0.5 M⁻¹ s⁻¹ for the natural full-length PCI and a form lacking six amino acids at the NH₂-terminus, respectively, whereas these constants were 4.8±0.1 and 29±7 M⁻¹ s⁻¹ for the corresponding PNGase F-treated forms. The 7-8-fold higher rate constants obtained when both the N-glycans and the NH₂-terminus had been removed suggest that these structures jointly affect the rate of PSA inhibition, presumably by together hindering conformational changes of PCI required to bind to the catalytic pocket of PSA.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  16. An isocratic liquid chromatography method for determining HIV non-nucleoside reverse transcriptase inhibitor and protease inhibitor concentrations in human plasma.

    Science.gov (United States)

    Weller, Dennis R; Brundage, Richard C; Balfour, Henry H; Vezina, Heather E

    2007-04-01

    An efficient, isocratic high performance liquid chromatography (HPLC) method for determining human immunodeficiency virus (HIV) non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) in plasma is advantageous for laboratories participating in clinical trials and therapeutic drug monitoring (TDM) programs, or conducting small animal research. The combination of isocratic reversed phase chromatography using an S-3, 3.0 mm x 150 mm column along with low plasma volume (200 microl), rapid liquid-liquid extraction, and detection at a single wavelength (212 nm) over a short run time makes this method valuable. Within and between assay variability ranges from 0.8 to 3.5% and 1.2-6.2%, respectively. Accuracy ranges from 91.0 to 112.8% for four quality controls (50, 100, 1000, and 10,000 ng/ml) for all drugs measured (efavirenz, nevirapine, amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir).

  17. Identification and Structure-Activity Relationships of Diarylhydrazides as Novel Potent and Selective Human Enterovirus Inhibitors.

    Science.gov (United States)

    Han, Xin; Sun, Ningyuan; Wu, Haoming; Guo, Deyin; Tien, Po; Dong, Chune; Wu, Shuwen; Zhou, Hai-Bing

    2016-03-10

    Enterovirus 71 (EV71) plays an important role in hand-foot-and-mouth disease. In this study, a series of diarylhydrazide analogues was synthesized, and the systematic exploration of SAR led to potent enterovirus inhibitors, of which compound 15 exhibits significant improvements in inhibition potency with an EC50 value of 0.02 μM against EV71. It is very interesting that this class of diarylhydrazides exhibits activities against a series of human enteroviruses at the picomolar level, including EV71 and Coxsackieviruses B1 (CVB1), CVB2, CVB3, CVB4, CVB5, and CVB6 (EC50 as low as 0.5 nM). Compared with the reference antienterovirus drug 1 (enviroxime) and known inhibitor 5 (WIN 51711), the four highly selective compounds 15, 27, 41 and 47 inhibited EV71 replication with EC50 values of 0.17-0.02 μM and SI values in a range of 978.4-12338. A preliminary mechanistic study indicated that VP1 might be the target site for this type of compound.

  18. Defined human pluripotent stem cell culture enables highly efficient neuroepithelium derivation without small molecule inhibitors.

    Science.gov (United States)

    Lippmann, Ethan Scott; Estevez-Silva, Maria Carolina; Ashton, Randolph Scott

    2014-04-01

    The embryonic neuroepithelium gives rise to the entire central nervous system in vivo, making it an important tissue for developmental studies and a prospective cell source for regenerative applications. Current protocols for deriving homogenous neuroepithelial cultures from human pluripotent stem cells (hPSCs) consist of either embryoid body-mediated neuralization followed by a manual isolation step or adherent differentiation using small molecule inhibitors. Here, we report that hPSCs maintained under chemically defined, feeder-independent, and xeno-free conditions can be directly differentiated into pure neuroepithelial cultures ([mt]90% Pax6(+)/N-cadherin(+) with widespread rosette formation) within 6 days under adherent conditions, without small molecule inhibitors, and using only minimalistic medium consisting of Dulbecco's modified Eagle's medium/F-12, sodium bicarbonate, selenium, ascorbic acid, transferrin, and insulin (i.e., E6 medium). Furthermore, we provide evidence that the defined culture conditions enable this high level of neural conversion in contrast to hPSCs maintained on mouse embryonic fibroblasts (MEFs). In addition, hPSCs previously maintained on MEFs could be rapidly converted to a neural compliant state upon transfer to these defined conditions while still maintaining their ability to generate all three germ layers. Overall, this fully defined and scalable protocol should be broadly useful for generating therapeutic neural cells for regenerative applications. © 2013 AlphaMed Press.

  19. 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.

  20. Selectivity of substrate (trifluoperazine) and inhibitor (amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone) "probes" for human udp-glucuronosyltransferases

    National Research Council Canada - National Science Library

    Uchaipichat, Verawan; Mackenzie, Peter I; Elliot, David J; Miners, John O

    2006-01-01

    ...). This work investigated the selectivity of trifluoperazine (TFP), as a substrate, and amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone, as inhibitors, for human UGTs...

  1. Novel virtual screening approach for the discovery of human tyrosinase inhibitors.

    Science.gov (United States)

    Ai, Ni; Welsh, William J; Santhanam, Uma; Hu, Hong; Lyga, John

    2014-01-01

    Tyrosinase is the key enzyme involved in the human pigmentation process, as well as the undesired browning of fruits and vegetables. Compounds inhibiting tyrosinase catalytic activity are an important class of cosmetic and dermatological agents which show high potential as depigmentation agents used for skin lightening. The multi-step protocol employed for the identification of novel tyrosinase inhibitors incorporated the Shape Signatures computational algorithm for rapid screening of chemical libraries. This algorithm converts the size and shape of a molecule, as well its surface charge distribution and other bio-relevant properties, into compact histograms (signatures) that lend themselves to rapid comparison between molecules. Shape Signatures excels at scaffold hopping across different chemical families, which enables identification of new actives whose molecular structure is distinct from other known actives. Using this approach, we identified a novel class of depigmentation agents that demonstrated promise for skin lightening product development.

  2. Novel virtual screening approach for the discovery of human tyrosinase inhibitors.

    Directory of Open Access Journals (Sweden)

    Ni Ai

    Full Text Available Tyrosinase is the key enzyme involved in the human pigmentation process, as well as the undesired browning of fruits and vegetables. Compounds inhibiting tyrosinase catalytic activity are an important class of cosmetic and dermatological agents which show high potential as depigmentation agents used for skin lightening. The multi-step protocol employed for the identification of novel tyrosinase inhibitors incorporated the Shape Signatures computational algorithm for rapid screening of chemical libraries. This algorithm converts the size and shape of a molecule, as well its surface charge distribution and other bio-relevant properties, into compact histograms (signatures that lend themselves to rapid comparison between molecules. Shape Signatures excels at scaffold hopping across different chemical families, which enables identification of new actives whose molecular structure is distinct from other known actives. Using this approach, we identified a novel class of depigmentation agents that demonstrated promise for skin lightening product development.

  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. Oximes: Inhibitors of Human Recombinant Acetylcholinesterase. A Structure-Activity Relationship (SAR) Study

    Science.gov (United States)

    Sepsova, Vendula; Karasova, Jana Zdarova; Korabecny, Jan; Dolezal, Rafael; Zemek, Filip; Bennion, Brian J.; Kuca, Kamil

    2013-01-01

    Acetylcholinesterase (AChE) reactivators were developed for the treatment of organophosphate intoxication. Standard care involves the use of anticonvulsants (e.g., diazepam), parasympatolytics (e.g., atropine) and oximes that restore AChE activity. However, oximes also bind to the active site of AChE, simultaneously acting as reversible inhibitors. The goal of the present study is to determine how oxime structure influences the inhibition of human recombinant AChE (hrAChE). Therefore, 24 structurally different oximes were tested and the results compared to the previous eel AChE (EeAChE) experiments. Structural factors that were tested included the number of pyridinium rings, the length and structural features of the linker, and the number and position of the oxime group on the pyridinium ring. PMID:23959117

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    Mahajan, Tushar R; Ytre-Arne, Mari Eknes; Strøm-Andersen, Pernille; Dalhus, Bjørn; Gundersen, Lise-Lotte

    2015-09-02

    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.

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

    Science.gov (United States)

    Rajabi, Nima; Auth, Marina; Troelsen, Kathrin R; Pannek, Martin; Bhatt, Dhaval P; Fontenas, Martin; Hirschey, Matthew D; Steegborn, Clemens; Madsen, Andreas S; Olsen, Christian A

    2017-11-20

    The sirtuin enzymes are important regulatory deacylases in a variety of biochemical contexts and may therefore be potential therapeutic targets through either activation or inhibition by small molecules. Here, we describe the discovery of the most potent inhibitor of sirtuin 5 (SIRT5) reported 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 a slow, tight-binding mechanism of inhibition, which is unprecedented for SIRT5. This is important information when applying inhibitors to probe mechanisms in biology. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Phosphatase inhibitor 2 promotes acetylation of tubulin in the primary cilium of human retinal epithelial cells

    Directory of Open Access Journals (Sweden)

    Wang Weiping

    2008-11-01

    Full Text Available Abstract Background Primary cilia are flagella-like projections from the centriole of mammalian cells that have a key role in cell signaling. Human diseases are linked to defects in primary cilia. Microtubules make up the axoneme of cilia and are selectively acetylated and this is thought to contribute to the stability of the structure. However, mechanisms to regulate tubulin acetylation in cilia are poorly understood. Results Endogenous phosphatase inhibitor-2 (I-2 was found concentrated in cilia of human epithelial cells, and was localized to cilia early in the process of formation, prior to the full acetylation of microtubules. Knockdown of I-2 by siRNA significantly reduced the acetylation of microtubules in cilia, without a net decrease in whole cell tubulin acetylation. There was a reduction in the percentage of I-2 knockdown cells with a primary cilium, but no apparent alteration in the cilium length, suggesting no change in microtubule-based transport processes. Inhibition of either histone deacetylases with trichostatin A, or protein phosphatase-1 with calyculin A in I-2 knockdown cells partially rescued the acetylation of microtubules in cilia and the percentage of cells with a primary cilium. Conclusion The regulatory protein I-2 localizes to the primary cilium where it affects both Ser/Thr phosphorylation and is required for full tubulin acetylation. Rescue of tubulin acetylation in I-2 knockdown cells by different chemical inhibitors shows that deacetylases and phosphatases are functionally interconnected to regulate microtubules. As a multifunctional protein, I-2 may link cell cycle progression to structure and stability of the primary cilium.

  9. Thermostabilisation of human serum butyrylcholinesterase for detection of its inhibitors in water and biological fluids

    Directory of Open Access Journals (Sweden)

    Lakshmanan Jaganathan

    1999-01-01

    Full Text Available The ability of gelatine-trehalose to convert the normally fragile, dry human serum BChE into a thermostable enzyme and its use in the detection of cholinesterase inhibitors in water and biological fluids is described. Gelatine or trehalose alone is unable to protect the dry enzyme against exposure to high temperature, while a combination of gelatine and trehalose were able to protect the enzyme activity against prolonged exposure to temperature as high as +50°C. A method for rapid, simple and inexpensive means of screening for cholinesterase inhibitors such as carbamates and organophosphates in water, vegetables and human blood has been developed.A capacidade da gelatina-trehalose em converter a frágil BChE do soro humano em uma enzima termoestável e seu uso na descoberta de inibidores de colinesterase em água e fluidos biológicos é apresentado. A Gelatina ou trehalose são incapazes de proteger a enzima seca BchE do soro humano contra exposição a elevadas temperaturas, enquanto que uma combinação de gelatina e trehalose são capazes de proteger a atividade de enzima contra exposição prolongada a temperaturas elevadas e da ordem de 50° C. Um método barato, simples e rápido de screening para inibidores de colinesterase tal como carbamatos e organofosfatos em água, verduras e sangue humano foi desenvolvido.

  10. Integrin-mediated cell migration is blocked by inhibitors of human neuraminidase.

    Science.gov (United States)

    Jia, Feng; Howlader, Md Amran; Cairo, Christopher W

    2016-09-01

    Integrins are critical receptors in cell migration and adhesion. A number of mechanisms are known to regulate the function of integrins, including phosphorylation, conformational change, and cytoskeletal anchoring. We investigated whether native neuraminidase (Neu, or sialidase) enzymes which modify glycolipids could play a role in regulating integrin-mediated cell migration. Using a scratch assay, we found that exogenously added Neu3 and Neu4 activity altered rates of cell migration. We observed that Neu4 increased the rate of migration in two cell lines (HeLa, A549); while Neu3 only increased migration in HeLa cells. A bacterial neuraminidase was able to increase the rate of migration in HeLa, but not in A549 cells. Treatment of cells with complex gangliosides (GM1, GD1a, GD1b, and GT1b) resulted in decreased cell migration rates, while LacCer was able to increase rates of migration in both lines. Importantly, our results show that treatment of cells with inhibitors of native Neu enzymes had a dramatic effect on the rates of cell migration. The most potent compound tested targeted the human Neu4 isoenzyme, and was able to substantially reduce the rate of cell migration. We found that the lateral mobility of integrins was reduced by treatment of cells with Neu3, suggesting that Neu3 enzyme activity resulted in changes to integrin-co-receptor or integrin-cytoskeleton interactions. Finally, our results support the hypothesis that inhibitors of human Neu can be used to investigate mechanisms of cell migration and for the development of anti-adhesive therapies. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Blebbistatin, a myosin inhibitor, is phototoxic to human cancer cells under exposure to blue light.

    Science.gov (United States)

    Mikulich, Aliaksandr; Kavaliauskiene, Simona; Juzenas, Petras

    2012-07-01

    Blebbistatin is a new inhibitor of cell motility. It is used to study dynamics of cytokinesis machinery in cells. However, the potential of this inhibitor as an anticancer agent has not been studied so far. Cytotoxicity of blebbistatin was evaluated in five human cell lines, FEMX-I melanoma, U87 glioma, androgen independent Du145 and androgen sensitive LNCaP prostate adenocarcinoma, and F11-hTERT immortalized fibroblasts. Phototoxicity of blebbistatin was assessed in these cell lines after their exposure to a blue light (390-470 nm). Photostability of blebbistatin and its reactive oxygen species (ROS) generating properties were measured during irradiation with the blue light. Blebbistatin at a concentration range of 10-200 μmol/L was toxic to all studied cells. Toxic concentrations (TC) were about 10-25 μmol/L corresponding to TC10, 50-100 μmol/L to TC50 and 140-190 μmol/L to TC90. Only for the U87 glioma cells TC90 could not be measured as the highest studied concentration of 200 μmol/L gave around 70% toxicity. However, after exposure to the blue light blebbistatin exhibited phototoxicity on the cells, with a cytotoxicity enhancement ratio that was greatest for the FEMX-I cells (about 9) followed by LNCaP (5), Du145 (3), U87 (2) and F11-hTERT (1.7) cells. Blebbistatin inhibits cell motility and viability. Under exposure to the blue light blebbistatin exhibits photodynamic action on human cancer cells. During the irradiation blebbistatin oxidizes dihydrorhodamine 123 but not Singlet Oxygen Sensor Green. Our findings offer new possibilities for blebbistatin as a potential anticancer and photodynamic agent. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Crystal structure of human AKT1 with an allosteric inhibitor reveals a new mode of kinase inhibition.

    Directory of Open Access Journals (Sweden)

    Wen-I Wu

    Full Text Available AKT1 (NP_005154.2 is a member of the serine/threonine AGC protein kinase family involved in cellular metabolism, growth, proliferation and survival. The three human AKT isozymes are highly homologous multi-domain proteins with both overlapping and distinct cellular functions. Dysregulation of the AKT pathway has been identified in multiple human cancers. Several clinical trials are in progress to test the efficacy of AKT pathway inhibitors in treating cancer. Recently, a series of AKT isozyme-selective allosteric inhibitors have been reported. They require the presence of both the pleckstrin-homology (PH and kinase domains of AKT, but their binding mode has not yet been elucidated. We present here a 2.7 Å resolution co-crystal structure of human AKT1 containing both the PH and kinase domains with a selective allosteric inhibitor bound in the interface. The structure reveals the interactions between the PH and kinase domains, as well as the critical amino residues that mediate binding of the inhibitor to AKT1. Our work also reveals an intricate balance in the enzymatic regulation of AKT, where the PH domain appears to lock the kinase in an inactive conformation and the kinase domain disrupts the phospholipid binding site of the PH domain. This information advances our knowledge in AKT1 structure and regulation, thereby providing a structural foundation for interpreting the effects of different classes of AKT inhibitors and designing selective ones.

  13. New applications for known drugs: Human glycogen synthase kinase 3 inhibitors as modulators of Aspergillus fumigatus growth.

    Science.gov (United States)

    Sebastián-Pérez, Víctor; Manoli, Maria-Tsampika; Pérez, Daniel I; Gil, Carmen; Mellado, Emilia; Martínez, Ana; Espeso, Eduardo A; Campillo, Nuria E

    2016-06-30

    Invasive aspergillosis (IA) is one of the most severe forms of fungi infection. IA disease is mainly due to Aspergillus fumigatus, an air-borne opportunistic pathogen. Mortality rate caused by IA is still very high (50-95%), because of difficulty in early diagnostics and reduced antifungal treatment options, thus new and efficient drugs are necessary. The aim of this work is, using Aspergillus nidulans as non-pathogen model, to develop efficient drugs to treat IA. The recent discovered role of glycogen synthase kinase-3 homologue, GskA, in A. fumigatus human infection and our previous experience on human GSK-3 inhibitors focus our attention on this kinase as a target for the development of antifungal drugs. With the aim to identify effective inhibitors of colonial growth of A. fumigatus we use A. nidulans as an accurate model for in vivo and in silico studies. Several well-known human GSK-3β inhibitors were tested for inhibition of A. nidulans colony growth. Computational tools as docking studies and binding site prediction was used to explain the different biological profile of the tested inhibitors. Three of the five tested hGSK3β inhibitors are able to reduce completely the colonial growth by covalent bind to the enzyme. Therefore these compounds may be useful in different applications to eradicate IA. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  14. Pharmacokinetics, metabolism, and excretion of torcetrapib, a cholesteryl ester transfer protein inhibitor, in humans.

    Science.gov (United States)

    Dalvie, Deepak; Chen, Weichao; Zhang, Chenghong; Vaz, Alfin D; Smolarek, Teresa A; Cox, Loretta M; Lin, Jian; Obach, R Scott

    2008-11-01

    The pharmacokinetics, metabolism, and excretion of torcetrapib, a selective inhibitor of human cholesteryl ester transfer protein, were investigated in healthy human male volunteers after oral administration of [(14)C]torcetrapib (120-mg dose). The total mean recovery of radiolabeled dose after 21 days was 75.7%, and most of the dose (63%) was excreted in the urine. The total circulating radioactivity and unchanged torcetrapib plasma concentrations increased over the first 6 h and then declined slowly with mean terminal elimination half-lives of 373 and 211 h. Metabolism of torcetrapib was extensive in humans. Only 5.2% of the total dose constituted unchanged torcetrapib in the feces, whereas no parent was excreted unchanged in the urine. Similarly, pharmacokinetic analysis of total radioactivity and unchanged torcetrapib revealed that the area under the concentration versus time curve from zero to infinity of torcetrapib accounted for approximately 7.0% of the circulating radioactivity. Torcetrapib was metabolized to numerous metabolites via oxidation. The primary metabolic pathway involved initial oxidative decarbamoylation followed by extensive further oxidation, resulting in the formation of bistrifluoromethylbenzoic acid (M1) and quinaldic acid (M4) metabolites. A mean 40% of the total dose was excreted in the urine as M4 (and its glucuronide and urea conjugates), whereas 7.0% of the total dose was excreted as M1. In vitro studies using human subcellular fractions suggested that the initial metabolism of torcetrapib proceeds via CYP3A-mediated decarbamoylation. Subsequent oxidations lead to the major circulating and excretory metabolites M1 and M4.

  15. Ribonucleotide reductase inhibitors hydroxyurea, didox, and trimidox inhibit human cytomegalovirus replication in vitro and synergize with ganciclovir.

    Science.gov (United States)

    Bhave, Sukhada; Elford, Howard; McVoy, Michael A

    2013-10-01

    Ganciclovir (GCV) is a deoxyguanosine analog that is effective in inhibiting human cytomegalovirus (HCMV) replication. In infected cells GCV is converted to GCV-triphosphate which competes with dGTP for incorporation into the growing DNA strand by the viral DNA polymerase. Incorporated GCV promotes chain termination as it is an inefficient substrate for elongation. Because viral DNA synthesis also relies on cellular ribonucleotide reductase (RR) to synthesize deoxynucleotides, RR inhibitors are predicted to inhibit HCMV replication. Moreover, as dGTP competes with GCV-triphosphate for incorporation, RR inhibitors may also synergize with GCV by reducing intracellular dGTP levels and there by promoting increased GCV-triphosphate utilization by DNA polymerase. To investigate potential of RR inhibitors as anti-HCMV agents both alone and in combination with GCV, HCMV-inhibitory activities of three RR inhibitors, hydroxyurea, didox, and trimidox, were determined. In both spread inhibition and yield reduction assays RR inhibitors had modest anti-HCMV activity with 50% inhibitory concentrations ranging from 36±1.7 to 221±52μM. However, all three showed significant synergy with GCV at concentrations below their 50% inhibitory and 50% toxic concentrations. These results suggest that combining GCV with relatively low doses of RR inhibitors could significantly potentiate the anti-HCMV activity of GCV in vivo and could improve clinical response to therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Preclinical Metabolism and Disposition of SB939 (Pracinostat), an Orally Active Histone Deacetylase Inhibitor, and Prediction of Human Pharmacokinetics

    NARCIS (Netherlands)

    Jayaraman, Ramesh; Reddy, Venkatesh Pilla; Pasha, Mohammed Khalid; Wang, Haishan; Sangthongpitag, Kanda; Yeo, Pauline; Hu, Chang Yong; Wu, Xiaofeng; Xin, Liu; Goh, Evelyn; New, Lee Sun; Ethirajulu, Kantharaj

    2011-01-01

    The preclinical absorption, distribution, metabolism, and excretion (ADME) properties of Pracinostat [(2E)-3-[2-butyl-1-[2-(diethylamino)ethyl]-1H-benzimidazol-5-yl]-N-hydroxyarylamide hydrochloride; SB939], an orally active histone deacetylase inhibitor, were characterized and its human

  17. 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...

  18. Circulating Metabolites of the Human Immunodeficiency Virus Protease Inhibitor Nelfinavir in Humans: Structural Identification, Levels in Plasma, and Antiviral Activities

    Science.gov (United States)

    Zhang, Kanyin E.; Wu, Ellen; Patick, Amy K.; Kerr, Bradley; Zorbas, Mark; Lankford, Angela; Kobayashi, Takuo; Maeda, Yuki; Shetty, Bhasker; Webber, Stephanie

    2001-01-01

    Nelfinavir mesylate (Viracept, formally AG1343) is a potent and orally bioavailable human immunodeficiency virus (HIV) type 1 (HIV-1) protease inhibitor (Ki = 2 nM) and is being widely prescribed in combination with HIV reverse transcriptase inhibitors for the treatment of HIV infection. The current studies evaluated the presence of metabolites circulating in plasma following the oral administration of nelfinavir to healthy volunteers and HIV-infected patients, as well as the levels in plasma and antiviral activities of these metabolites. The results showed that the parent drug was the major circulating chemical species, followed in decreasing abundance by its hydroxy-t-butylamide metabolite (M8) and 3′-methoxy-4′-hydroxynelfinavir (M1). Antiviral assays with HIV-1 strain RF-infected CEM-SS cells showed that the 50% effective concentrations (EC50) of nelfinavir, M8, and M1 were 30, 34, and 151 nM, respectively, and that the corresponding EC50 against another HIV-1 strain, IIIB, in MT-2 cells were 60, 86, and 653 nM. Therefore, apparently similar in vitro antiviral activities were demonstrated for nelfinavir and M8, whereas an approximately 5- to 11-fold-lower level of antiviral activity was observed for M1. The active metabolite, M8, showed a degree of binding to human plasma proteins similar to that of nelfinavir (ca. 98%). Concentrations in plasma of nelfinavir and its metabolites in 10 HIV-positive patients receiving nelfinavir therapy (750 mg three times per day) were determined by a liquid chromatography tandem mass spectrometry assay. At steady state (day 28), the mean plasma nelfinavir concentrations ranged from 1.73 to 4.96 μM and the M8 concentrations ranged from 0.55 to 1.96 μM, whereas the M1 concentrations were low and ranged from 0.09 to 0.19 μM. In conclusion, the findings from the current studies suggest that, in humans, nelfinavir forms an active metabolite circulating at appreciable levels in plasma. The active metabolite M8 may account for

  19. 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.

  20. 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.

  1. The Antiproliferative and Colony-suppressive Activities of STAT3 Inhibitors in Human Cancer Cells Is Compromised Under Hypoxic Conditions.

    Science.gov (United States)

    Tian, Jilai; Xiao, Hui; Wu, Ruohan; Cao, Yang; Li, Chenglong; Xu, Ronald; Pierson, Christopher R; Finlay, Jonathan L; Yang, Fang; Gu, Ning; Lin, Jiayuh

    2017-02-01

    Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been indicated as a novel cancer drug target, since it plays an important role in diverse oncogenic processes including survival, cell proliferation and migration. Emerging STAT3 inhibitors have demonstrated efficacy in cancer cells and animal tumor models. It is well known that most solid tumors are characterized by hypoxia, but it is not clear if hypoxic conditions affect activity of STAT3 inhibitors. To examine this, two STAT3 inhibitors were tested to investigate their inhibitory efficacy in cancer cells grown under hypoxic conditions compared with those without hypoxia. Cell proliferation, colony formation and western blot assays were performed to examine the differences in the cell viability, proliferation and proteins in the STAT3 pathway. Under hypoxic conditions, the half-maximal inhibitory concentration values for both STAT3 inhibitors were increased compared to normoxic conditions in human pancreatic cancer, medulloblastoma and sarcoma cell lines. In addition, the ability of both STAT3 inhibitors to inhibit colony formation in pancreatic cancer, medulloblastoma and sarcoma cell lines was reduced under hypoxic conditions when compared to cells under normoxic conditions. Furthermore, there was an increase in phosphorylated STAT3 levels in cancer cells under hypoxic conditions, suggesting this may be one of the mechanisms of resistance. In summary, the results presented here provide a novel finding of STAT3 inhibitor activity under hypoxic conditions and indicate that under such low oxygen conditions, the anticancer efficacy of STAT3 inhibitors was indeed hampered. These results highlight the need to develop new therapeutic strategies to overcome the resistance of cancer cells to STAT3 inhibitors under hypoxic conditions. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  2. 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.

  3. Thermodynamic Analysis of Transition State Features in Picomolar Inhibitors of Human 5′ Methylthioadenosine Phosphorylase

    Science.gov (United States)

    Guan, Rong; Tyler, Peter C.; Evans, Gary B.; Schramm, Vern L.

    2013-01-01

    Human 5′-methylthioadenosine phosphorylase (MTAP) is solely responsible for 5′-methylthioadenosine (MTA) metabolism to permit S-adenosylmethionine salvage. Transition state (TS) analogues of MTAP are in development as anticancer candidates. TS analogues of MTAP incorporate a cationic nitrogen and a protonated 9-deazaadenine leaving group, mimics of the ribocation transition state. MT-ImmA and MT-DADMe-ImmA are two examples of these TS analogues. Thermodynamic analysis of MTA, inhibitor and phosphate binding reveals the cationic nitrogen to provide −2.6 and −3.6 kcal/mol binding free energy for MT-ImmA and MT-DADMe-ImmA, respectively. The protonated deazaadenine provides an additional −1.3 (MT-ImmA ) to −1.7 kcal/mol (MT-DADMe-ImmA). MT-DADMe-ImmA is a better match in TS geometry than MT-ImmA and is thermodynamically favored. Binding of TS analogues to the MTAP:phosphate complex is fully entropic, in contrast to TS analogue binding to the related human purine nucleoside phosphorylase:phosphate complex, which is fully enthalpic. The binding thermodynamics of phosphate or TS analogues alone to MTAP are fully dominated by enthalpy. Phosphate anchored in the catalytic site forms an ion pair with the cationic TS analogue to cause stabilization of the enzyme structure in the ternary complex. The ternary-induced conformational changes convert the individual enthalpic binding energies to entropy, a presumed shift of the protein architecture toward the transition state. Formation of the ternary TS analogue complex with MTAP induces a remarkable increase in thermal stability (ΔTm 35 °C). The enthalpic, entropic and protein stability features of TS analogue binding to human MTAP are resolved in these studies. PMID:24148083

  4. 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.

  5. 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...... in on-demand treatment, increasing the inhibitor risk. Aim To compare the antibody response to treatment with recombinant human FVIII (rhFVIII) in relation to induced knee joint bleeds and treatment without concurrent bleeds in a HA rat model. Method HA rats were divided into two groups: one group (n...

  6. Thrombin-activatable fibrinolysis inhibitor in human abdominal aortic aneurysm disease.

    Science.gov (United States)

    Bridge, K I; Bollen, L; Zhong, J; Hesketh, M; Macrae, F L; Johnson, A; Philippou, H; Scott, D J; Gils, A; Ariёns, R A S

    2017-08-21

    Essentials Patients with abdominal aortic aneurysms (AAA) develop dense clots that are resistant to lysis. This study explores the role of thrombin-activatable fibrinolysis inhibitor (TAFI) in human AAA. There is evidence of chronically increased TAFI activation in patients with AAA. TAFI may represent a pharmacological target for cardiovascular risk reduction in AAA. Background Intra-luminal thrombosis is a key factor in growth of abdominal aortic aneurysms (AAAs). Patients with AAA form dense clots that are resistant to fibrinolysis. Thrombin-activatable fibrinolysis inhibitor (TAFI) has been shown to influence AAA development in murine models. Objective The aim of this study is to characterize the role of TAFI in human AAA. Methods Plasma levels of TAFI, TAFI activation peptide (TAFI-AP), activated/inactivated TAFI (TAFIa/ai) and plasmin-α2-antiplasmin complex were measured by ELISAs in patients with AAA (n = 202) and controls (n = 188). Results TAFIa/ai and TAFI-AP levels were higher in patients than controls (median [IQR], 20.3 [14.6-32.8] ng mL(-1) vs. 14.2 [11.2-19.3] ng mL(-1) and 355.0 [232.4-528.1] ng mL(-1) vs. 248.6 [197.1-328.1] ng mL(-1) ). TAFIa/ai was positively correlated with TAFI-AP (r = 0.164). Intact TAFI levels were not different between patients and controls (13.4 [11.2-16.1] μg mL(-1) vs. 12.8 [10.6-15.4] μg mL(-1) ). Plasmin-α2-antiplasmin was higher in AAA patients than controls (690.0 [489.1-924.3] ng mL(-1) vs. 480.7 [392.6-555.3] ng mL(-1) ). Conclusions The increase in TAFIa/ai and TAFI-AP suggests an increased TAFI activation in patients with AAA. Prospective studies are required to further elucidate the role of TAFI and fibrinolysis in AAA pathogenesis. © 2017 International Society on Thrombosis and Haemostasis.

  7. Original 2-(3-Alkoxy-1H-pyrazol-1-yl)azines Inhibitors of Human Dihydroorotate Dehydrogenase (DHODH)

    Science.gov (United States)

    2016-01-01

    Following our discovery of human dihydroorotate dehydrogenase (DHODH) inhibition by 2-(3-alkoxy-1H-pyrazol-1-yl)pyrimidine derivatives as well as 2-(4-benzyl-3-ethoxy-5-methyl-1H-pyrazol-1-yl)-5-methylpyridine, we describe here the syntheses and evaluation of an array of azine-bearing analogues. As in our previous report, the structure–activity study of this series of human DHODH inhibitors was based on a phenotypic assay measuring measles virus replication. Among other inhibitors, this round of syntheses and biological evaluation iteration led to the highly active 5-cyclopropyl-2-(4-(2,6-difluorophenoxy)-3-isopropoxy-5-methyl-1H-pyrazol-1-yl)-3-fluoropyridine. Inhibition of DHODH by this compound was confirmed in an array of in vitro assays, including enzymatic tests and cell-based assays for viral replication and cellular growth. This molecule was found to be more active than the known inhibitors of DHODH, brequinar and teriflunomide, thus opening perspectives for its use as a tool or for the design of an original series of immunosuppressive agent. Moreover, because other series of inhibitors of human DHODH have been found to also affect Plasmodium falciparum DHODH, all the compounds were assayed for their effect on P. falciparum growth. However, the modest in vitro inhibition solely observed for two compounds did not correlate with their inhibition of P. falciparum DHODH. PMID:26079043

  8. 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.

  9. The Microtubule Inhibitor Podofilox Inhibits an Early Entry Step of Human Cytomegalovirus

    Directory of Open Access Journals (Sweden)

    Tobias Cohen

    2016-10-01

    Full Text Available Human cytomegalovirus is a ubiquitous β-herpesvirus that infects many different cell types through an initial binding to cell surface receptors followed by a fusion event at the cell membrane or endocytic vesicle. A recent high-throughput screen to identify compounds that block a step prior to viral gene expression identified podofilox as a potent and nontoxic inhibitor. Time-of-addition studies in combination with quantitative-PCR analysis demonstrated that podofilox limits an early step of virus entry at the cell surface. Podofilox was also able to drastically reduce infection by herpes simplex 1, an α-herpesvirus with a very similar entry process to CMV. Podofilox caused a reduced maximal plateau inhibition of infection by viruses with single step binding processes prior to fusion-like Newcastle disease virus, Sendai virus, and influenza A virus or viruses that enter via endocytosis like vesicular stomatitis virus and a clinical-like strain of CMV. These results indicate that microtubules appear to be participating in the post-binding step of virus entry including the pre- and post-penetration events. Modulation of the plasma membrane is required to promote virus entry for herpesviruses, and that podofilox, unlike colchicine or nocodazole, is able to preferentially target microtubule networks at the plasma membrane.

  10. 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.

  11. Potential human cholesterol esterase inhibitor design: benefits from the molecular dynamics simulations and pharmacophore modeling studies.

    Science.gov (United States)

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

    2012-01-01

    Human pancreatic cholesterol esterase (hCEase) is one of the lipases found to involve in the digestion of large and broad spectrum of substrates including triglycerides, phospholipids, cholesteryl esters, etc. The presence of bile salts is found to be very important for the activation of hCEase. Molecular dynamic simulations were performed for the apoform and bile salt complexed form of hCEase using the co-ordinates of two bile salts from bovine CEase. The stability of the systems throughout the simulation time was checked and two representative structures from the highly populated regions were selected using cluster analysis. These two representative structures were used in pharmacophore model generation. The generated pharmacophore models were validated and used in database screening. The screened hits were refined for their drug-like properties based on Lipinski's rule of five and ADMET properties. The drug-like compounds were further refined by molecular docking simulation using GOLD program based on the GOLD fitness score, mode of binding, and molecular interactions with the active site amino acids. Finally, three hits of novel scaffolds were selected as potential leads to be used in novel and potent hCEase inhibitor design. The stability of binding modes and molecular interactions of these final hits were re-assured by molecular dynamics simulations.

  12. 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.

  13. Selective Serotonin Reuptake Inhibitors in Human Pregnancy: To Treat or Not to Treat?

    Directory of Open Access Journals (Sweden)

    Orna Diav-Citrin

    2012-01-01

    Full Text Available Selective serotonin reuptake inhibitors (SSRIs are increasingly prescribed during pregnancy. The purpose of the present paper is to summarize and evaluate the current evidence for the risk/benefit analysis of SSRI use in human pregnancy. The literature has been inconsistent. Although most studies have not shown an increase in the overall risk of major malformations, several studies have suggested that SSRIs may be associated with a small increased risk for cardiovascular malformations. Others have noted associations between SSRIs and specific types of rare major malformations. In some studies, there appears to be a small increased risk for miscarriages, which may be associated with the underlying maternal condition. Neonatal effects have been described in up to 30% of neonates exposed to SSRIs late in pregnancy. Persistent pulmonary hypertension of the newborn has also been described with an absolute risk of <1%. The risk associated with treatment discontinuation, for example, higher frequency of relapse and increased risk of preterm delivery, should also be considered. The overall benefit of treatment seems to outweigh the potential risks.

  14. Histone deacetylase inhibitor AR42 regulates telomerase activity in human glioma cells via an Akt-dependent mechanism.

    Science.gov (United States)

    Yang, Ya-Luen; Huang, Po-Hsien; Chiu, Hao-Chieh; Kulp, Samuel K; Chen, Ching-Shih; Kuo, Cheng-Ju; Chen, Huan-Da; Chen, Chang-Shi

    2013-05-24

    Epigenetic regulation via abnormal activation of histone deacetylases (HDACs) is a mechanism that leads to cancer initiation and promotion. Activation of HDACs results in transcriptional upregulation of human telomerase reverse transcriptase (hTERT) and increases telomerase activity during cellular immortalization and tumorigenesis. However, the effects of HDAC inhibitors on the transcription of hTERT vary in different cancer cells. Here, we studied the effects of a novel HDAC inhibitor, AR42, on telomerase activity in a PTEN-null U87MG glioma cell line. AR42 increased hTERT mRNA in U87MG glioma cells, but suppressed total telomerase activity in a dose-dependent manner. Further analyses suggested that AR42 decreases the phosphorylation of hTERT via an Akt-dependent mechanism. Suppression of Akt phosphorylation and telomerase activity was also observed with PI3K inhibitor LY294002 further supporting the hypothesis that Akt signaling is involved in suppression of AR42-induced inhibition of telomerase activity. Finally, ectopic expression of a constitutive active form of Akt restored telomerase activity in AR42-treated cells. Taken together, our results demonstrate that the novel HDAC inhibitor AR42 can suppress telomerase activity by inhibiting Akt-mediated hTERT phosphorylation, indicating that the PI3K/Akt pathway plays an important role in the regulation of telomerase activity in response to this HDAC inhibitor. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Establishment of a selective evaluation method for DPP4 inhibitors based on recombinant human DPP8 and DPP9 proteins

    Directory of Open Access Journals (Sweden)

    Jinglong Liu

    2014-04-01

    Full Text Available Dipeptidyl peptidase 4 (DPP4 is recognised as an attractive anti-diabetic drug target, and several DPP4 inhibitors are already on the market. As members of the same gene family, dipeptidyl peptidase 8 (DPP8 and dipeptidyl peptidase 9 (DPP9 share high sequence and structural homology as well as functional activity with DPP4. However, the inhibition of their activities was reported to cause severe toxicities. Thus, the development of DPP4 inhibitors that do not have DPP8 and DPP9 inhibitory activity is critical for safe anti-diabetic therapy. To achieve this goal, we established a selective evaluation method for DPP4 inhibitors based on recombinant human DPP8 and DPP9 proteins expressed by Rosetta cells. In this method, we used purified recombinant 120 kDa DPP8 or DPP9 protein from the Rosetta expression system. The optimum concentrations of the recombinant DPP8 and DPP9 proteins were 30 ng/mL and 20 ng/mL, respectively, and the corresponding concentrations of their substrates were both 0.2 mmol/L. This method was highly reproducible and reliable for the evaluation of the DPP8 and DPP9 selectivity for DPP4 inhibitor candidates, which would provide valuable guidance in the development of safe DPP4 inhibitors.

  16. 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.

  17. 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.

  18. The hederagenin saponin SMG-1 is a natural FMLP receptor inhibitor that suppresses human neutrophil activation.

    Science.gov (United States)

    Hwang, Tsong-Long; Wang, Chien-Chiao; Kuo, Yao-Haur; Huang, Hui-Chi; Wu, Yang-Chang; Kuo, Liang-Mou; Wu, Yi-Hsiu

    2010-10-15

    The pericarp of Sapindus mukorossi Gaertn is traditionally used as an expectorant in Japan, China, and Taiwan. Activated neutrophils produce high concentrations of the superoxide anion (O(2)(-)) and elastase known to be involved in airway mucus hypersecretion. In the present study, the anti-inflammatory functions of hederagenin 3-O-(3,4-O-di-acetyl-alpha-L-arabinopyranoside)-(1-->3)-alpha-l-rhamnopyranosyl-(1-->2)-alpha-l-arabinopyranoside (SMG-1), a saponin isolated from S. mukorossi, and its underlying mechanisms were investigated in human neutrophils. SMG-1 potently and concentration-dependently inhibited O(2)(*-) generation and elastase release in N-Formyl-Met-Leu-Phe (FMLP)-activated human neutrophils. Furthermore, SMG-1 reduced membrane-associated p47(phox) expression in FMLP-induced intact neutrophils, but did not alter subcellular NADPH oxidase activity in reconstituted systems. SMG-1 attenuated FMLP-induced increase of cytosolic calcium concentration and phosphorylation of p38 MAPK, ERK, JNK, and AKT. However, SMG-1 displayed no effect on cellular cAMP levels and activity of adenylate cyclase and phosphodiesterase. Significantly, receptor-binding analysis showed that SMG-1 inhibited FMLP binding to its receptor in a concentration-dependent manner. In contrast, neither phorbol myristate acetate-induced O(2)(*-) generation and MAPKs activation nor thapsigargin-caused calcium mobilization was altered by SMG-1. Taken together, our results demonstrate that SMG-1 is a natural inhibitor of the FMLP receptor, which may have the potential to be developed into a useful new therapeutic agent for treating neutrophilic inflammatory diseases. Copyright 2010 Elsevier Inc. All rights reserved.

  19. 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.

  20. Identification and characterization of a potent and selective inhibitor of human urate transporter 1.

    Science.gov (United States)

    Wu, Ting; Chen, Jiasheng; Dong, Shuai; Li, Haixin; Cao, Ying; Tian, Yuanxin; Fu, Weimin; Zhou, Pingzheng; Xi, Baomin; Pang, Jianxin

    2017-05-06

    Selective inhibitors of human urate transporter 1 (hURAT1) are considered to be effective treatment for hyperuricemia and gout, which can reduce the reabsorption of more than 90% of uric acid in the proximal tubule of the kidney. We aimed to design and synthesize a more potent hURAT1 based on the structure of Lesinurad (LU), which was reported to lower uric acid levels with IC50 value of hURAT1 (about 60μM). A cell model was conducted and characterized via Real-time qRCR and Western blot. We synthesized and identified a new midazole analogue of LU. Cells stably-expressing hURAT1 or human organic anion transporter 1 (hOAT1) were used in the [(14)C] urate or 6-carboxyfluorescein (6-CF) uptake assays to test the activities of the newly synthesized compound. The uric acid lowering effects of LU and LUM and their effects on urea nitrogen and creatinine in potassium oxonate-induced hyperuricemic rats were analyzed. The [(14)C] Urate uptake assay using hURAT1 stably transfected MDCK cells indicated that LUM was more potent than LU against hURAT1, with IC50 values of 3.22μM and 65.47μM, respectively. LU and LUM also effectively suppressed hOAT1-mediated 6-CF uptake, and the IC50 hURAT1/IC50 hOAT1 of LU and LUM was1.49 and 0.35 respectively, indicating a better selectivity for LUM than LU. In vivo, LUM-Na (40mg/kg) showed more potent activity in reducing serum uric acid levels in potassium oxonate-induced hyperuricemic rats, compared to similar doses of LU-Na. LUM was demonstrated to be as potent a uricosuric drug as LU. Copyright © 2017. Published by Elsevier Urban & Partner Sp. z o.o.

  1. Differential diagnosis of human ascites: inhibitors of the contact system and total proteins.

    Science.gov (United States)

    Buø, L; Karlsrud, T S; Dyrhaug, G; Jacobsen, M B; Bell, H; Johansen, H T; Aasen, A O

    1993-09-01

    To assess their accuracies as markers for malignancy, we assayed alpha 2-macroglobulin, C1-inhibitor, alpha 1-protease inhibitor, and total proteins in ascites and plasma from patients with gastrointestinal cancer (n = 15) and non-malignant liver disease (n = 13), using functional and immunologic assays. For all inhibitors and total proteins determined in ascites, the values in the cancer group were significantly higher than the corresponding values in the group with non-malignant liver disease. The diagnostic accuracy for differentiating malignancy-related from non-malignant ascites was 93% for a alpha 1-protease inhibitor value > or = 50% of the pool plasma value and 90% for alpha 2-macroglobulin > or = 16%, C1-inhibitor > or = 40% (all functional assays), and total proteins > or = 20 g/l (biuret). In conclusion, functional assays for alpha 2-macroglobulin, C1-inhibitor, and alpha 1-protease inhibitor and determination of total proteins in ascites appeared to be very informative tests for the differential diagnosis of ascites. The test for alpha 1-protease inhibitor gave higher specificity (92% versus 77%) and likelihood ratio for a positive test (12 versus 4) compared with the other tests.

  2. 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)....

  3. Characterization of inhibitor binding to human kinesin spindle protein by site-directed mutagenesis.

    Science.gov (United States)

    Marshall, C Gary; Torrent, Maricel; Williams, Olusegun; Hamilton, Kelly A; Buser, Carolyn A

    2009-04-01

    A number of inhibitors of kinesin spindle protein (KSP) have been described, which are known from X-ray crystallography studies to bind to an induced fit pocket defined by the L5 loop. We describe the characterization of eight mutant forms of KSP in which six residues that line this pocket have been altered. Mutants were analyzed by measuring rates of enzyme catalysis, in the presence and absence of six KSP inhibitors of four diverse structural classes and of varied ATP-competition status. Our analysis was in agreement with the model of binding established by the structural studies and suggests that binding energy is well distributed across functional groups in these molecules. The majority of the mutants retained significant enzymatic activity while diminishing inhibitor binding, indicating potential for the development of drug resistance. These data provide detailed information on interactions between inhibitor and binding pocket at the functional group level and enable the development of novel KSP inhibitors.

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

    DEFF Research Database (Denmark)

    Sonne-Schmidt, Carsten Scavenius; Sanggaard, Kristian W; Nikolajsen, Camilla L

    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...... that inter-α-inhibitor is cross-linked to the fibrin clot in a 1:20 ratio relative to the known factor XIIIa substrate α2-antiplasmin. This interaction may protect fibrin or other Lys-donating proteins from adventitious proteolysis by increasing the local concentration of bikunin. In addition, the reaction....... These findings suggest that transglutaminases catalyze the covalent conjugation of inter-α-inhibitor to other proteins. This was demonstrated by the cross-linking between inter-α-inhibitor and fibrinogen by either factor XIIIa or tissue transglutaminase. Finally, using quantitative mass spectrometry, we show...

  5. 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

    study was to outline a simple procedure for isolating and identifying this inhibitor. Purification was done as follows: precipitation of the major proteins (albumin and uromucoid) with trichloroacetic acid, followed by anion exchange chromatography, hydroxyapatite chromatography, anion exchange......, an unidentified protein rich in uronic acid, and uropontin have all been described as possessing such activity. We have recently isolated an unknown inhibitor of calcium oxalate crystal growth that co-eluted with trypsin inhibitor in several separation steps, which suggested its identity. The aim of the present...... chromatography, negative affinity chromatography, and twice reversed phase chromatographies of the supernatant. By this procedure, the inhibitor was identified as being a fragment of osteopontin; urinary trypsin inhibitor and nucleic acids were excluded as being responsible for inhibitory action....

  6. 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

  7. 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.

  8. Synergistic inhibition of human melanoma proliferation by combination treatment with B-Raf inhibitor BAY43-9006 and mTOR inhibitor Rapamycin

    Directory of Open Access Journals (Sweden)

    Slingluff Craig L

    2005-10-01

    Full Text Available Abstract Background Targeted inhibition of protein kinases is now acknowledged as an effective approach for cancer therapy. However, targeted therapies probably have limited success because cancer cells have alternate pathways for survival and proliferation thereby avoiding inhibition. We tested the hypothesis that combination of targeted agents would be more effective than single agents in arresting melanoma cell proliferation. Methods We evaluated whether BAY43-9006, an inhibitor of the B-Raf kinase, and rapamycin, an inhibitor of the mTOR kinase, would inhibit serum-stimulated proliferation of human melanoma cell lines, either alone or in combination. Proliferation was measured by quantitating melanoma cell numbers with a luciferase for ATP. Phosphorylation of proteins downstream of targeted kinase(s was assayed by immunoblots. Statistical significance was determined with the Student-T test. Isobologram analysis was performed to distinguish additive versus synergistic effects of combinations of drugs. Results Serum-stimulated proliferation of multiple human melanoma cell lines was inhibited by BAY43-9006 and by rapamycin. Melanoma cells containing the B-Raf mutation V599E were more sensitive than cells with wild-type B-raf to 10 nM doses of both BAY43-9006 and rapamycin. Regardless of B-Raf mutational status, the combination of low dose rapamycin and BAY43-9006 synergistically inhibited melanoma cell proliferation. As expected, rapamycin inhibited the phosphorylation of mTOR substrates, p70S6K and 4EBP1, and BAY43-9006 inhibited phosphorylation of ERK, which is dependent on B-Raf activity. We also observed unexpected rapamycin inhibition of the phosphorylation of ERK, as well as BAY43-9006 inhibition of the phosphorylation of mTOR substrates, p70S6K and 4EBP1. Conclusion There was synergistic inhibition of melanoma cell proliferation by the combination of rapamycin and BAY 43-9006, and unexpected inhibition of two signaling pathways by agents

  9. A Highly Selective Hydantoin Inhibitor of Aggrecanase-1 and Aggrecanase-2 with a Low Projected Human Dose.

    Science.gov (United States)

    Durham, Timothy B; Marimuthu, Jothirajah; Toth, James L; Liu, Chin; Adams, Lisa; Mudra, Daniel R; Swearingen, Craig; Lin, Chaohua; Chambers, Mark G; Thirunavukkarasu, Kannan; Wiley, Michael R

    2017-07-13

    Aggrecanase-1 and -2 (ADAMTS-4 and ADAMTS-5) are zinc metalloproteases involved in the degradation of aggrecan in cartilage. Inhibitors could provide a means of altering the progression of osteoarthritis. We report the identification of 7 which had good oral pharmacokinetics in rats and showed efficacy in a rat chemical model of osteoarthritis. The projected human dose required to achieve sustained plasma levels ≥10 times the hADAMTS-5 IC50 is 5 mg q.d.

  10. 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.

  11. Reduced human herpesvirus-8 oropharyngeal shedding associated with protease inhibitor-based antiretroviral therapy.

    Science.gov (United States)

    Gantt, Soren; Cattamanchi, Ashok; Krantz, Elizabeth; Magaret, Amalia; Selke, Stacy; Kuntz, Steven R; Huang, Meei-Li; Corey, Lawrence; Wald, Anna; Casper, Corey

    2014-06-01

    Human herpesvirus 8 (HHV-8) replication increases the risk of Kaposi sarcoma (KS). Highly-active antiretroviral therapy (HAART) reduces the incidence of KS, and regimens that contain protease inhibitors (PIs) may be particularly effective. To determine whether PI-based HAART regimens may more effectively inhibit HHV-8 shedding compared to regimens without PIs. Prospective, observational study of 142 HIV-1 and HHV-8 co-infected men conducted in Seattle, Washington. Quantitative HHV-8 PCR testing was performed on daily swabs of the oropharynx, the primary site of HHV-8 replication. Associations between antiretroviral regimen and detection of HHV-8 DNA in swabs were evaluated using generalized estimating equations. HHV-8 DNA was detected in 3016 (26%) of 11,608 specimens collected. PI-based HAART was associated with a statistically significantly lower frequency of detection (RR 0.2; 95% CI 0.1-0.5) compared to ART-naïve persons, whereas HAART without a PI was not (RR 0.7; 95% CI 0.4-1.3). Compared to ART-naïve persons, there was also a trend toward lower quantities of HHV-8 detected during treatment with HAART regimens that contained a PI. These associations between PIs and measures of HHV-8 shedding could not be attributed to use of nelfinavir, which inhibits HHV-8 replication in vitro, and were independent of CD4 count and HIV plasma viral load (VL). HAART regimens that contain PIs appear to decrease HHV-8 shedding compared to NNRTIs. Further study of PI-based HAART is warranted to determine the optimal regimens for prevention and treatment of KS. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. 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

    2015-01-01

    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. Leu406 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. PMID:25903124

  13. Limited efficacy of COX-2 inhibitors on nerve growth factor and metalloproteinases expressions in human synovial fibroblasts.

    Science.gov (United States)

    Yorifuji, Makiko; Sawaji, Yasunobu; Endo, Kenji; Kosaka, Taiichi; Yamamoto, Kengo

    2016-05-01

    Nerve growth factor (NGF) is associated with arthritic pain and metalloproteinases are implicated in collagen and aggrecan degradation. Although selective COX-2 inhibitors are recommended for the treatment of arthritic diseases, their effects on NGF and metalloproteinases remain unclear. This study investigated the regulations of NGF and metalloproteinases by selective COX-2 inhibitors in isolated human synovial cells. The isolated human synovial cells were stimulated with IL-1β in the presence of selective COX-2 inhibitors (NS-398 or celecoxib) with or without exogenous PGE2 or its receptor (EP1-4) agonists. The expressions of NGF, MMP-1, -3, -13, ADAMTS-4, and -5 were quantified by real-time PCR and their proteins were determined by Western blotting. The amount of PGE2 released was measured by enzyme-linked immunosorbent assay (ELISA). The IL-1β inductions of NGF and MMP-1 and MMP-13 were augmented by the COX-2 inhibitors, whereas the inductions of ADAMTS-4 and ADAMTS-5 were inhibited. These actions were reversed by supplementing PGE2 or the EP4 agonist exogenously. Our comprehensive analysis revealed that COX-2 inhibitors may be beneficial for suppressing aggrecan degradation and for reducing inflammatory pain by inhibiting PGE2 release, although they may have limited efficacy in suppressing collagen degradation and nerve growth. This study suggests the feedback roles of PGE2 in the negative regulation of NGF and MMP-1 and MMP-13 and the positive regulation of ADAMTS-4 and ADAMTS-5. Copyright © 2016 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.

  14. Human dihydroorotate dehydrogenase inhibitors, a novel approach for the treatment of autoimmune and inflammatory diseases.

    Science.gov (United States)

    Leban, Johann; Vitt, Daniel

    2011-01-01

    Dihydroorotate dehydrogenase (DHODH), a novel and recently discovered enzyme, is involved in the biosynthesis of uridine. Leflunomide (CAS 75706-12-6), a drug approved for the treatment of treat rheumatoid arthritis (RA), was identified as an inhibitor of DHODH. Structure based drug design using the leflunomide/DHODH X-ray structure yielded novel inhibitors with improved pharmacological properties. Such drug candidates are in clinical trials against various autoimmune diseases.

  15. Combinatorial optimization of cystine-knot peptides towards high-affinity inhibitors of human matriptase-1.

    Directory of Open Access Journals (Sweden)

    Bernhard Glotzbach

    Full Text Available Cystine-knot miniproteins define a class of bioactive molecules with several thousand natural members. Their eponymous motif comprises a rigid structured core formed by six disulfide-connected cysteine residues, which accounts for its exceptional stability towards thermic or proteolytic degradation. Since they display a remarkable sequence tolerance within their disulfide-connected loops, these molecules are considered promising frameworks for peptide-based pharmaceuticals. Natural open-chain cystine-knot trypsin inhibitors of the MCoTI (Momordica cochinchinensis trypsin inhibitor and SOTI (Spinacia oleracea trypsin inhibitor families served as starting points for the generation of inhibitors of matriptase-1, a type II transmembrane serine protease with possible clinical relevance in cancer and arthritic therapy. Yeast surface-displayed libraries of miniproteins were used to select unique and potent matriptase-1 inhibitors. To this end, a knowledge-based library design was applied that makes use of detailed information on binding and folding behavior of cystine-knot peptides. Five inhibitor variants, four of the MCoTI family and one of the SOTI family, were identified, chemically synthesized and oxidatively folded towards the bioactive conformation. Enzyme assays revealed inhibition constants in the low nanomolar range for all candidates. One subnanomolar binder (Ki = 0.83 nM with an inverted selectivity towards trypsin and matriptase-1 was identified.

  16. 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.

  17. X-ray Crystallographic Analysis of [alpha]-Ketoheterocycle Inhibitors Bound to a Humanized Variant of Fatty Acid Amide Hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Mileni, Mauro; Garfunkle, Joie; Ezzili, Cyrine; Kimball, F.Scott; Cravatt, Benjamin F.; Stevens, Raymond C.; Boger, Dale L. (Scripps)

    2010-11-03

    Three cocrystal X-ray structures of the {alpha}-ketoheterocycle inhibitors 3-5 bound to a humanized variant of fatty acid amide hydrolase (FAAH) are disclosed and comparatively discussed alongside those of 1 (OL-135) and its isomer 2. These five X-ray structures systematically probe each of the three active site regions key to substrate or inhibitor binding: (1) the conformationally mobile acyl chain-binding pocket and membrane access channel responsible for fatty acid amide substrate and inhibitor acyl chain binding, (2) the atypical active site catalytic residues and surrounding oxyanion hole that covalently binds the core of the {alpha}-ketoheterocycle inhibitors captured as deprotonated hemiketals mimicking the tetrahedral intermediate of the enzyme-catalyzed reaction, and (3) the cytosolic port and its uniquely important imbedded ordered water molecules and a newly identified anion binding site. The detailed analysis of their key active site interactions and their implications on the interpretation of the available structure-activity relationships are discussed providing important insights for future design.

  18. High-performance liquid chromatography assay for the quantification of HIV protease inhibitors and non-nucleoside reverse transcriptase inhibitors in human plasma.

    Science.gov (United States)

    Rezk, Naser L; Tidwell, Richard R; Kashuba, Angela D M

    2004-06-15

    An accurate, sensitive, and specific reverse-phase high-performance liquid chromatography (HPLC) assay for the simultaneous quantitative determination of HIV-protease inhibitors (PIs) (indinavir, IDV; amprenavir, APV; saquinavir, SQV; nelfinavir, NFV; ritonavir, RTV; and lopinavir, LPV) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) (nevirapine, NVP; delavirdine, DLV; and efavirenz, EFV) in human blood plasma is described. The method provides excellent resolution and peak shape for nine analytes through a linear gradient (36-86%) of 25% phosphate buffer (pH 4.5), 60% acetonitrile, 15% methanol, and 0.75 ml TFA, with a gradient mobile phase flow rate (0.9-1.1 ml) over 30 min run time. The optimized solid phase extraction (SPE) extraction method using (1.0 ml, 100mg BOND ELUT-C18 Varian) column provides a clean base line and high extraction efficiency using a 550 microl plasma sample. The method was validated over the range of 10-10,000 ng/ml for NVP, IDV, and SQV; 10-5000 ng/ml for EFV; 25-10000 ng/ml for APV; and 25-5000 ng/ml for DLV, NFV, RTV, and LPV. This method is accurate (average accuracies of three different concentrations ranged from 91 to 112%), and precise (within- and between-day precision measures ranged from 0.2 to 5.7% and 0.1 to 5.4%, respectively). This method is suitable for use in clinical pharmacokinetic studies as well as in therapeutic drug monitoring (TDM).

  19. Antitumor activity of sorafenib in human cancer cell lines with acquired resistance to EGFR and VEGFR tyrosine kinase inhibitors.

    Directory of Open Access Journals (Sweden)

    Floriana Morgillo

    Full Text Available Treatment of non small cell lung cancer (NSCLC and colorectal cancer (CRC have substantially changed in the last years with the introduction of epidermal growth factor receptor (EGFR inhibitors in the clinical practice. The understanding of mechanisms which regulate cells sensitivity to these drugs is necessary for their optimal use.An in vitro model of acquired resistance to two tyrosine kinase inhibitors (TKI targeting the EGFR, erlotinib and gefitinib, and to a TKI targeting EGFR and VEGFR, vandetanib, was developed by continuously treating the human NSCLC cell line CALU-3 and the human CRC cell line HCT116 with escalating doses of each drug. MTT, western blot analysis, migration, invasion and anchorage-independent colony forming assays were conducted in vitro and experiments with established xenografts in athymic nude mice were performed in vivo in sensitive, wild type (WT and TKI-resistant CALU-3 and HCT116 cell lines.As compared to WT CALU-3 and HCT116 human cancer cells, TKI-resistant cell lines showed a significant increase in the levels of activated, phosphorylated AKT, MAPK, and of survivin. Considering the role of RAS and RAF as downstream signals of both the EGFR and VEGFR pathways, we treated resistant cells with sorafenib, an inhibitor of C-RAF, B-RAF, c-KIT, FLT-3, RET, VEGFR-2, VEGFR-3, and PDGFR-β. Sorafenib reduced the activation of MEK and MAPK and caused an inhibition of cell proliferation, invasion, migration, anchorage-independent growth in vitro and of tumor growth in vivo of all TKI-resistant CALU-3 and HCT116 cell lines.These data suggest that resistance to EGFR inhibitors is predominantly driven by the RAS/RAF/MAPK pathway and can be overcame by treatment with sorafenib.

  20. Histone deacetylase inhibitor valproic acid promotes the differentiation of human induced pluripotent stem cells into hepatocyte-like cells.

    Directory of Open Access Journals (Sweden)

    Yuki Kondo

    Full Text Available In this study, we aimed to elucidate the effects and mechanism of action of valproic acid on hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells. Human induced pluripotent stem cells were differentiated into endodermal cells in the presence of activin A and then into hepatic progenitor cells using dimethyl sulfoxide. Hepatic progenitor cells were matured in the presence of hepatocyte growth factor, oncostatin M, and dexamethasone with valproic acid that was added during the maturation process. After 25 days of differentiation, cells expressed hepatic marker genes and drug-metabolizing enzymes and exhibited drug-metabolizing enzyme activities. These expression levels and activities were increased by treatment with valproic acid, the timing and duration of which were important parameters to promote differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells into hepatocytes. Valproic acid inhibited histone deacetylase activity during differentiation of human induced pluripotent stem cells, and other histone deacetylase inhibitors also enhanced differentiation into hepatocytes. In conclusion, histone deacetylase inhibitors such as valproic acid can be used to promote hepatic differentiation from human induced pluripotent stem cell-derived hepatic progenitor cells.

  1. Engineering Archeal Surrogate Systems for the Development of Protein-Protein Interaction Inhibitors against Human RAD51.

    Science.gov (United States)

    Moschetti, Tommaso; Sharpe, Timothy; Fischer, Gerhard; Marsh, May E; Ng, Hong Kin; Morgan, Matthew; Scott, Duncan E; Blundell, Tom L; R Venkitaraman, Ashok; Skidmore, John; Abell, Chris; Hyvönen, Marko

    2016-11-20

    Protein-protein interactions (PPIs) are increasingly important targets for drug discovery. Efficient fragment-based drug discovery approaches to tackle PPIs are often stymied by difficulties in the production of stable, unliganded target proteins. Here, we report an approach that exploits protein engineering to "humanise" thermophilic archeal surrogate proteins as targets for small-molecule inhibitor discovery and to exemplify this approach in the development of inhibitors against the PPI between the recombinase RAD51 and tumour suppressor BRCA2. As human RAD51 has proved impossible to produce in a form that is compatible with the requirements of fragment-based drug discovery, we have developed a surrogate protein system using RadA from Pyrococcus furiosus. Using a monomerised RadA as our starting point, we have adopted two parallel and mutually instructive approaches to mimic the human enzyme: firstly by mutating RadA to increase sequence identity with RAD51 in the BRC repeat binding sites, and secondly by generating a chimeric archaeal human protein. Both approaches generate proteins that interact with a fourth BRC repeat with affinity and stoichiometry comparable to human RAD51. Stepwise humanisation has also allowed us to elucidate the determinants of RAD51 binding to BRC repeats and the contributions of key interacting residues to this interaction. These surrogate proteins have enabled the development of biochemical and biophysical assays in our ongoing fragment-based small-molecule inhibitor programme and they have allowed us to determine hundreds of liganded structures in support of our structure-guided design process, demonstrating the feasibility and advantages of using archeal surrogates to overcome difficulties in handling human proteins. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. Discovery and biochemical characterization of selective ATP competitive inhibitors of the human mitotic kinesin KSP.

    Science.gov (United States)

    Rickert, Keith W; Schaber, Michael; Torrent, Maricel; Neilson, Lou Anne; Tasber, Edward S; Garbaccio, Robert; Coleman, Paul J; Harvey, Diane; Zhang, Yun; Yang, Yi; Marshall, Gary; Lee, Ling; Walsh, Eileen S; Hamilton, Kelly; Buser, Carolyn A

    2008-01-15

    The kinesin spindle protein (KSP, also known as Eg5) is essential for the proper separation of spindle poles during mitosis, and inhibition results in mitotic arrest and the formation of characteristic monoaster spindles. Several distinct classes of KSP inhibitors have been described previously in the public and patent literature. However, most appear to share a common induced-fit allosteric binding site, suggesting a common mechanism of inhibition. In a high-throughput screen for inhibitors of KSP, a novel class of thiazole-containing inhibitors was identified. Unlike the previously described allosteric KSP inhibitors, the thiazoles described here show ATP competitive kinetic behavior, consistent with binding within the nucleotide binding pocket. Although they bind to a pocket that is highly conserved across kinesins, these molecules exhibit significant selectivity for KSP over other kinesins and other ATP-utilizing enzymes. Several of these compounds are active in cells and produce a phenotype similar to that observed with previously published allosteric inhibitors of KSP.

  3. Angiogenesis Inhibitors

    Science.gov (United States)

    ... blood vessels “feed” growing tumors with oxygen and nutrients , allowing the cancer cells to invade nearby tissue , to move throughout ... any angiogenesis inhibitors currently being used to treat cancer in humans? Yes. The U.S. Food and Drug Administration (FDA) has approved bevacizumab to ...

  4. The enhancement of radiosensitivity by celecoxib, selective cyclooxygenase-2 inhibitor, on human cancer cells expressing differential levels of cyclooxygenase-2

    Energy Technology Data Exchange (ETDEWEB)

    Pyo, Hong Ryull; Shin, You Keun; Kim, Hyun Seok [National Cancer Center, Seoul (Korea, Republic of); Seong, Jin Sil; Suh, Chang Ok; Kim, Gwi Eon [College of Medicine, Yonsei Univ., Seoul (Korea, Republic of)

    2003-09-01

    To investigate the modulation of radiosensitivity by celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, on cancer cells over- and under-expressing COX-2. A clonogenic radiation survival analysis was performed on A549 human lung and MCF-7 human breast cancer cell lines incubated in both 1 and 10% fetal bovine serum (FBS) containing media. The apoptosis in both cell lines was measured after treatment with radiation and/or celecoxib. Celecoxib enhanced the radiation sensitivity of the A549 cells in the medium containing the 10% FBS, with radiation enhancement ratios of 1.58 and 1.81 respectively, at surviving fractions of 0.1, with 30 {mu} M and 50 {mu} M celecoxib. This enhanced radiosensitivity disappeared in the medium containing the 1% FBS. Celecoxib did not change the radiation sensitivity of the MCF-7 cells in either media. The induction of apoptosis by celecoxib and radiation was not synergistic in either cell line. Celecoxib, a selective COX-2 inhibitor, preferentially enhanced the effect of radiation on COX-2 over-expressing cancer cells compared to the cells with a low expression, and this effect disappeared on incubation of the cells during drug treatment in the medium with suboptimal serum concentration. Apoptosis did not appear to be the underlying mechanism of this radiation enhancement effect due to celecoxib on the A549 cells. These findings suggest radiosensitization by a selective COX-2 inhibitor is COX-2 dependent.

  5. 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.

  6. Gibberellin Biosynthetic Inhibitors Make Human Malaria Parasite Plasmodium falciparum Cells Swell and Rupture to Death

    Science.gov (United States)

    Toyama, Tomoko; Tahara, Michiru; Nagamune, Kisaburo; Arimitsu, Kenji; Hamashima, Yoshio; Palacpac, Nirianne M. Q.; Kawaide, Hiroshi; Horii, Toshihiro; Tanabe, Kazuyuki

    2012-01-01

    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. PMID:22412858

  7. Ferrocene labelings as inhibitors and dual electrochemical sensors of human glutathione S-transferase P1-1.

    Science.gov (United States)

    Martos-Maldonado, Manuel C; Quesada-Soriano, Indalecio; García-Maroto, Federico; Vargas-Berenguel, Antonio; García-Fuentes, Luís

    2012-12-01

    The inhibitory and sensor properties of two ferrocene conjugates, in which the ferrocene and glutathione are linked through a spacer arm of different length and chemical structure, on human Pi glutathione S-transferase, were examined by activity assays, ITC, fluorescence spectroscopy and voltammetry. Such ferrocene conjugates are strong competitive inhibitors of this enzyme with an enhanced binding affinity, the one bearing the longest spacer arm being the most potent inhibitor. Voltammetric measurements showed a strong decrease of the peak current intensity and an increase of the oxidation potential upon binding of ferrocene-glutathione conjugates to GST P1-1 showing that both conjugates can be used as dual electrochemical sensors for GST P1-1. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. 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

    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......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...

  9. Direct inactivation of human immunodeficiency virus type 1 by a novel small-molecule entry inhibitor, DCM205.

    Science.gov (United States)

    Duong, Yen T; Meadows, D Christopher; Srivastava, Indresh K; Gervay-Hague, Jacquelyn; North, Thomas W

    2007-05-01

    With more than 40 million people living with human immunodeficiency virus (HIV), there is an urgent need to develop drugs that can be used in the form of a topical microbicide to prevent infection through sexual transmission. DCM205 is a recently discovered small-molecule inhibitor of HIV type 1 (HIV-1) that is able to directly inactivate HIV-1 in the absence of a cellular target. DCM205 is active against CXCR4-, CCR5-, and dual-tropic laboratory-adapted and primary strains of HIV-1. DCM205 binds to the HIV-1 envelope glycoprotein, and competition studies map the DCM205 binding at or near the V3 loop of gp120. Binding to this site interferes with the soluble CD4 interaction. With its ability to disable the virus particle, DCM205 represents a promising new class of HIV entry inhibitor that can be used as a strategy in the prevention of HIV-1/AIDS.

  10. Histone deacetylase inhibitor MGCD0103 synergizes with gemcitabine in human pancreatic cells.

    Science.gov (United States)

    Sung, Victoria; Richard, Normand; Brady, Helen; Maier, Armin; Kelter, Gerhard; Heise, Carla

    2011-06-01

    Histone deacetylase inhibitors are a group of recently developed compounds that modulate cell growth and survival. We evaluated the effects of the histone deacetylase inhibitor MGCD0103 on growth of pancreatic carcinoma models following single agent treatment and in combination with gemcitabine. MGCD0103 inhibited tumor cell growth and acted synergistically with gemcitabine to enhance its cytotoxic effects. Gene expression analysis identified the cell cycle pathway as one of the most highly modulated gene groups. Our data suggest that MGCD0103 + gemcitabine might be an effective treatment for gemcitabine-refractory pancreatic cancer. © 2011 Japanese Cancer Association.

  11. 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.

  12. Effects of pyrrophenone, an inhibitor of group IVA phospholipase A2, on eicosanoid and PAF biosynthesis in human neutrophils

    Science.gov (United States)

    Flamand, N; Picard, S; Lemieux, L; Pouliot, M; Bourgoin, S G; Borgeat, P

    2006-01-01

    Background and Purpose: The biosynthesis of leukotrienes (LT) and platelet-activating factor (PAF) involves the release of their respective precursors, arachidonic acid (AA) and lyso-PAF by the group IVA PLA2 (cPLA2α). This paper aims at characterizing the inhibitory properties of the cPLA2α inhibitor pyrrophenone on eicosanoids and PAF in human neutrophils (PMN). Experimental Approach: Freshly isolated human PMN were activated with physiological and pharmacological agents (fMLP, PAF, exogenous AA, A23187 and thapsigargin) in presence and absence of the cPLA2α inhibitor pyrrophenone and biosynthesis of LT, PAF, and PGE2 was measured. Key Results: Pyrrophenone potently inhibited LT, PGE2 and PAF biosynthesis in PMN with IC50s in the range of 1–20 nM. These inhibitory effects of pyrrophenone were specific (the consequence of substrate deprivation), as shown by the reversal of inhibition by exogenous AA and lyso-PAF. Comparative assessment of pyrrophenone, methyl-arachidonoyl-fluoro-phosphonate (MAFP) and arachidonoyl-trifluoromethylketone (AACOCF3) demonstrated that pyrrophenone was more specific and 100-fold more potent than MAFP and AACOCF3 for the inhibition of LT biosynthesis in A23187-activated PMN. The inhibitory effect of pyrrophenone on LT biosynthesis was reversible as LT biosynthesis was recovered when pyrrophenone-treated PMN were washed with autologous plasma. No alteration of phospholipase D (PLD) activity in fMLP-activated PMN was observed with up to 10 μM pyrrophenone, suggesting that the cPLA2α inhibitor does not directly inhibit PLD. Conclusions and Implications: Pyrrophenone is a more potent and specific cPLA2α inhibitor than MAFP and AACOCF3 and represents an excellent pharmacological tool to investigate the biosynthesis and the biological roles of eicosanoids and PAF. PMID:16967052

  13. 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.

  14. [ALK inhibitor].

    Science.gov (United States)

    Mano, Hiroyuki

    2011-01-01

    While lung cancer is the leading cause of cancer deaths worldwide, the molecular mechanism underlying its carcinogenesis is mainly unknown. We have discovered a small, fusion-type tyrosine kinase EML4-ALK that is generated through a tiny inversion within the short arm of human chromosome 2. Transgenic mice expressing EML4-ALK in lung developed hundreds of lung cancer nodules soon after birth, but such nodules were readily eradicated upon treatment with an ALK inhibitor. Clinical trials for EML4-ALK-positive lung cancer with an ALK inhibitor is ongoing, with its interim results being highly promising.

  15. 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

  16. 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-gr...

  17. Placental transfer of the HIV integrase inhibitor dolutegravir in an ex vivo human cotyledon perfusion model

    NARCIS (Netherlands)

    Schalkwijk, S.J.; Greupink, R.; Colbers, A.P.; Wouterse, A.C.; Verweij, V.G.M.; Drongelen, J. van; Teulen, M.J.A.; Oetelaar, D. van den; Burger, D.M.; Russel, F.G.

    2016-01-01

    OBJECTIVES: Data on fetal exposure to antiretroviral agents during pregnancy are important to estimate their potential for prevention of mother-to-child transmission (PMTCT) and possible toxicity. For the recently developed HIV integrase inhibitor dolutegravir, clinical data on fetal disposition are

  18. 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.

  19. A first-in-human study of DS-1040, an inhibitor of the activated form of thrombin-activatable fibrinolysis inhibitor, in healthy subjects.

    Science.gov (United States)

    Zhou, J; Kochan, J; Yin, O; Warren, V; Zamora, C; Atiee, G; Pav, J; Orihashi, Y; Vashi, V; Dishy, V

    2017-05-01

    Essentials DS-1040 inhibits the activated form of thrombin-activatable fibrinolysis inhibitor (TAFIa). Infusion of DS-1040 was safe and well tolerated in healthy young and elderly subjects. DS-1040 substantially decreased TAFIa activity but had no impact on bleeding time. DS-1040 may provide an option of safer thrombolytic therapy. Background Current treatments for acute ischemic stroke and venous thromboembolism, such as recombinant tissue-type plasminogen activator and thrombectomy, are limited by a narrow time window and the risk of bleeding. DS-1040 is a novel low molecular weight compound that inhibits the activated form of thrombin-activatable fibrinolysis inhibitor (TAFIa), and was developed as a fibrinolysis enhancer for the treatment of thromboembolic diseases. Objectives This first-in-human, randomized, placebo-controlled, three-part, phase 1 study was conducted to evaluate the safety, pharmacokinetics and pharmacodynamics of DS-1040 in healthy subjects. Subjects/Methods Young (18-45 years) or elderly (65-75 years) subjects (N = 103) were randomized to receive single ascending doses of DS-1040 ranging from 0.1 mg to 40 mg, or placebo, administered either as a 0.5-h intravenous infusion or as a 24-h continuous infusion. Results All doses of DS-1040 were tolerated, and no serious adverse events (AEs) or discontinuations resulting from AEs occurred during the study. Bleeding time remained within the normal range for all doses tested in all subjects. Plasma exposure of DS-1040 increased proportionally with increase in dose. Elderly subjects had higher exposures to DS-1040 and prolonged elimination times, probably because of decreased renal clearance. DS-1040 caused a substantial dose-dependent and time-dependent decrease in TAFIa activity and in 50% clot lysis time. The levels of D-dimer, indicative of endogenous fibrinolysis, increased in some individuals following DS-1040 treatment. No effects of DS-1040 on coagulation parameters or platelet

  20. 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.

  1. 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.

  2. Identification of New Human Malaria Parasite Plasmodium Falciparum Dihydroorotate Dehydrogenase Inhibitors by Pharmacophore and Structure-Based Virtual Screening

    Science.gov (United States)

    Pavadai, Elumalai; El Mazouni, Farah; Wittlin, Sergio; de Kock, Carmen; Phillips, Margaret A.; Chibale, Kelly

    2016-01-01

    Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH), a key enzyme in the de novo pyrimidine biosynthesis pathway, which the Plasmodium falciparum relies on exclusively for survival, has emerged as a promising target for antimalarial drugs. In an effort to discover new and potent PfDHODH inhibitors, 3D-QSAR pharmacophore models were developed based on the structures of known PfDHODH inhibitors and the validated Hypo1 model was used as a 3D search query for virtual screening of the National Cancer Institute database. The virtual hit compounds were further filtered based on molecular docking and Molecular Mechanics/Generalized Born Surface Area binding energy calculations. The combination of the pharmacophore and structure-based virtual screening resulted in the identification of nine new compounds that showed >25% inhibition of PfDHODH at a concentration of 10 μM, three of which exhibited IC50 values in the range of 0.38–20 μM. The most active compound, NSC336047, displayed species-selectivity for PfDHODH over human DHODH and inhibited parasite growth with an IC50 of 26 μM. In addition to this, thirteen compounds inhibited parasite growth with IC50 values of ≤ 50 μM, four of which showed IC50 values in the range of 5–12 μM. These compounds could be further explored in the identification and development of more potent PfDHODH and parasite growth inhibitors. PMID:26915022

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

    Science.gov (United States)

    Amat-Ur-Rasool, Hafsa; Ahmed, Mehboob

    2015-01-01

    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.

  4. 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.

  5. 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.

  6. Restriction fragment length polymorphism caused by a deletion involving Alu sequences within the human. alpha. sub 2 -plasmin inhibitor gene

    Energy Technology Data Exchange (ETDEWEB)

    Miura, Osamu; Sugahara, Yuichi; Nakamura, Yuichi; Hirosawa, Shinsaku; Aoki, Nobuo (Tokyo Medical and Dental Univ. (Japan))

    1989-06-13

    A restriction fragment length polymorphism within the human {alpha}{sub 2}-plasmin inhibitor gene has been detected by Southern blot hybridization using an {alpha}{sub 2}-plasmin inhibitor cDNA probe. This restriction fragment length polymorphism can be attributed to the presence of two alleles, A and B, that are distributed in Hardy-Weinberg equilibrium with frequencies of 73.5% and 2.65%, respectively, in 66 unrelated Caucasian individuals or with frequencies of 51.0% and 49.0%, respectively, in 50 unrelated Japanese individuals. The minor allele, B, is due to a deletion of about 720 base pairs in intron 8 of the {alpha}{sub 2}-plasmin inhibitor gene. Sequence analysis of the deletion junction in allele B and the corresponding regions of allele A demonstrated the presence of oppositely oriented Alu sequences at the 5{prime} and 3{prime} deletion boundaries. These data suggest that this restriction fragment length polymorphism was caused by intrastrand recombination between Alu sequences.

  7. Pattern secretion of matrix Metalloproteinases and their biological tissue inhibitors by human glomerular mesangial cells in culture

    Directory of Open Access Journals (Sweden)

    "Hosseini R

    2001-08-01

    Full Text Available The glomerular mesangial cells (GMC play a central role in the synthesis and turnover of the glomerular mesangial matrix. The breakdown of the matrix likely depends on the balance between of a variety of proteinases including matrix metalloproteinases and their biological inhibitors secreted by the GMC, and any disturbance in the balance may result in appearance of various pathological states such as glomerulosclerosis. We therefore studied pattern secretion of matrix metalloproteinases (MMPs, MMP-1, MMP-2, MMP-3, MMP-9 and their biological tissue inhibitor of matrix metalloproteinases (TIMPs, TIMP-1 and TIMP-2 by cultured human GMC. We also measured MMP-1/TIMP-1 complex level in the cell culture supernatants. For this purpose, the GMC were incubated under serum-free conditions with medium (RPMI-1640 alone or in combination with TNF-α (30 ng/ml or phorbol myristate acetate (PMA (50 ng/ml for exactly 24, 48 and 72 hours. The above parameters were assayed by established ELISA techniques. Our results showed that the lowest and largest secretions were related to MMP-9 and MMP-2, respectively. The results indicated that the MMPs and TIMPs secretion were increased by TNF-α (MMP-1, MMP-2, TIMP-1 and TIMP-2 and PMA (MMP-2, TIMP-1 and TIMP-2, significantly (P<0.05. These results suggest that the GMC can synthesis and release various MMPs and their inhibitors (TIMPs that, in part, control turnover of extracellular matrix proteins.

  8. 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.

  9. Conversion of the HIV protease inhibitor nelfinavir to a bioactive metabolite by human liver CYP2C19.

    Science.gov (United States)

    Hirani, Vandana N; Raucy, Judy L; Lasker, Jerome M

    2004-12-01

    Antiretroviral therapy for human immunodeficiency virus (HIV) infection includes treatment with both reverse transcriptase inhibitors and protease inhibitors, which markedly suppress viral replication and circulating HIV RNA levels. Cytochrome P450 (P450) enzymes in human liver, chiefly CYP3A4, play a pivotal role in protease inhibitor biotransformation, converting these agents to largely inactive metabolites. However, the protease inhibitor nelfinavir (Viracept) is metabolized mainly to nelfinavir hydroxy-t-butylamide (M8), which exhibits potent antiviral activity, and to other minor products (termed M1 and M3) that are inactive. Since indirect evidence suggests that CYP2C19 underlies M8 formation, we examined the role of this inducible, polymorphic P450 enzyme in nelfinavir t-butylamide hydroxylation by human liver. Rates of microsomal M8 formation were 50.6 +/- 28.3 pmol of product formed/min/nmol P450 (n = 5 subjects), whereas kinetic analysis of the reaction revealed a KM of 21.6 microM and a Vmax of 24.6 pmol/min/nmol P450. In reconstituted systems, CYP2C19 catalyzed nelfinavir t-butylamide hydroxylation at a turnover rate of 2.2 min(-1), whereas CYP2C9, CYP2C8, and CYP3A4 were inactive toward nelfinavir. Polyclonal anti-CYP2C9 (cross-reactive with CYP2C19) and monoclonal anti-CYP2C19 completely inhibited microsomal M8 production, whereas monoclonal CYP2C9 and polyclonal CYP3A4 antibodies were without effect. Similarly, the CYP2C19 substrate omeprazole strongly inhibited (75%) hepatic nelfinavir t-butylamide hydroxylation at a concentration of only 12.5 microM. Our study shows that CYP2C19 underlies formation in human liver of M8, a bioactive nelfinavir metabolite. The inducibility of CYP2C19 by agents (e.g., rifampicin) often taken concurrently with nelfinavir, together with this P450's known polymorphic nature, may thus be important determinants of nelfinavir's antiviral potency.

  10. A Small Molecule Inhibitor of the BLM Helicase Modulates Chromosome Stability in Human Cells

    DEFF Research Database (Denmark)

    Nguyen, Giang Huong; Dexheimer, Thomas S; Rosenthal, Andrew S

    2013-01-01

    The Bloom's syndrome protein, BLM, is a member of the conserved RecQ helicase family. Although cell lines lacking BLM exist, these exhibit progressive genomic instability that makes distinguishing primary from secondary effects of BLM loss problematic. In order to be able to acutely disable BLM...... function in cells, we undertook a high throughput screen of a chemical compound library for small molecule inhibitors of BLM. We present ML216, a potent inhibitor of the DNA unwinding activity of BLM. ML216 shows cell-based activity and can induce sister chromatid exchanges, enhance the toxicity...... of aphidicolin, and exert antiproliferative activity in cells expressing BLM, but not those lacking BLM. These data indicate that ML216 shows strong selectivity for BLM in cultured cells. We discuss the potential utility of such a BLM-targeting compound as an anticancer agent....

  11. 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....... In the current study, we aimed to develop small molecule inhibitors of APE1 for cancer therapy....

  12. Protein kinase inhibitors CK59 and CID755673 alter primary human NK cell effector functions

    Directory of Open Access Journals (Sweden)

    Maxi eScheiter

    2013-03-01

    Full Text Available Natural killer (NK cells are part of the innate immune response and play a crucial role in the defense against tumors and virus-infected cells. Their effector functions include the specific killing of target cells, as well as the modulation of other immune cells by cytokine release. Kinases constitute a relevant part in signaling, are prime targets in drug research and the protein kinase inhibitor Dasatinib is already used for immune-modulatory theraphies. In this study, we have tested the effects of the kinase inhibitors CK59 and CID755673. These inhibitors are directed against CaMKII (CK59 and PKD family kinases (CID755673 that were previously suggested as novel components of NK activation pathways. Here, we use a multi-parameter, FACS-based assay to validate the influence of CK59 and CID755673 on the effector functions of primary NK cells. Dose dependent treatment with CK59 and CID755673 indeed results in a significant reduction of NK cell degranulation markers and cytokine release in freshly isolated PBMC populations from healthy blood donors. These results underline the importance of CaMKII for NK cell signaling and suggest PKD2 as a novel signaling component in NK cell activation. Notably, kinase inhibition studies on pure NK cell populations indicate significant donor variations.

  13. Functional and Transcriptional Characterization of Histone Deacetylase Inhibitor-Mediated Cardiac Adverse Effects in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

    Science.gov (United States)

    Kopljar, Ivan; Gallacher, David J; De Bondt, An; Cougnaud, Laure; Vlaminckx, Eddy; Van den Wyngaert, Ilse; Lu, Hua Rong

    2016-05-01

    Histone deacetylase (HDAC) inhibitors possess therapeutic potential to reverse aberrant epigenetic changes associated with cancers, neurological diseases, and immune disorders. Unfortunately, clinical studies with some HDAC inhibitors displayed delayed cardiac adverse effects, such as atrial fibrillation and ventricular tachycardia. However, the underlying molecular mechanism(s) of HDAC inhibitor-mediated cardiotoxicity remains poorly understood and is difficult to detect in the early stages of preclinical drug development because of a delayed onset of effects. In the present study, we show for the first time in human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) that HDAC inhibitors (dacinostat, panobinostat, vorinostat, entinostat, and tubastatin-a) induce delayed dose-related cardiac dysfunction at therapeutic concentrations associated with cardiac adverse effects in humans. HDAC inhibitor-mediated delayed effects on the beating properties of hiPS-CMs developed after 12 hours by decreasing the beat rate, shortening the field potential duration, and inducing arrhythmic behavior under form of sustained contractions and fibrillation-like patterns. Transcriptional changes that are common between the cardiotoxic HDAC inhibitors but different from noncardiotoxic treatments identified cardiac-specific genes and pathways related to structural and functional changes in cardiomyocytes. Combining the functional data with epigenetic changes in hiPS-CMs allowed us to identify molecular targets that might explain HDAC inhibitor-mediated cardiac adverse effects in humans. Therefore, hiPS-CMs represent a valuable translational model to assess HDAC inhibitor-mediated cardiotoxicity and support identification of better HDAC inhibitors with an improved benefit-risk profile. Histone deacetylase (HDAC) inhibitors are a promising class of drugs to treat certain cancers, autoimmune, and neurodegenerative diseases. However, treated patients can experience various

  14. 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

  15. Characterization of human liver enzymes involved in the biotransformation of boceprevir, a hepatitis C virus protease inhibitor.

    Science.gov (United States)

    Ghosal, Anima; Yuan, Yuan; Tong, Wei; Su, Ai-Duen; Gu, Chunyan; Chowdhury, Swapan K; Kishnani, Narendra S; Alton, Kevin B

    2011-03-01

    Boceprevir (SCH 503034), a protease inhibitor, is under clinical development for the treatment of human hepatitis C virus infections. In human liver microsomes, formation of oxidative metabolites after incubations with [(14)C]boceprevir was catalyzed by CYP3A4 and CYP3A5. In addition, the highest turnover was observed in recombinant CYP3A4 and CYP3A5. After a single radiolabeled dose to human, boceprevir was subjected to two distinct pathways, namely cytochrome P450-mediated oxidation and ketone reduction. Therefore, attempts were made to identify the enzymes responsible for the formation of carbonyl-reduced metabolites. Human liver S9 and cytosol converted ∼ 28 and ∼ 68% of boceprevir to M28, respectively, in the presence of an NADPH-generating system. Screening of boceprevir with recombinant human aldo-keto reductases (AKRs) revealed that AKR1C2 and AKR1C3 exhibited catalytic activity with respect to the formation of M+2 metabolites (M28 and M31). The formation of M28 was inhibited by 100 μM flufenamic acid (80.3%), 200 μM mefenamic acid (83.7%), and 100 μM phenolphthalein (86.1%), known inhibitors of AKRs, suggesting its formation through carbonyl reduction pathway. Formation of M28 was also inhibited by 100 μM diazepam (75.1%), 1 mM ibuprofen (70%), and 200 μM diflunisal (89.4%). These data demonstrated that CYP3A4 and CYP3A5 are primarily responsible for the formation of oxidative metabolites and the formation of M28 and M31, the keto-reduced metabolites, are most likely mediated by AKR1C2 and AKR1C3. Because the biotransformation and clearance of boceprevir involves two different enzymatic pathways, boceprevir is less likely to be a victim of significant drug-drug interaction with concomitant medication affecting either of these pathways.

  16. Phospholipase D inhibitors reduce human prostate cancer cell proliferation and colony formation.

    Science.gov (United States)

    Noble, Amanda R; Maitland, Norman J; Berney, Daniel M; Rumsby, Martin G

    2018-01-01

    Phospholipases D1 and D2 (PLD1/2) hydrolyse cell membrane glycerophospholipids to generate phosphatidic acid, a signalling lipid, which regulates cell growth and cancer progression through effects on mTOR and PKB/Akt. PLD expression and/or activity is raised in breast, colorectal, gastric, kidney and thyroid carcinomas but its role in prostate cancer (PCa), the major cancer of men in the western world, is unclear. PLD1 protein expression in cultured PNT2C2, PNT1A, P4E6, LNCaP, PC3, PC3M, VCaP, 22RV1 cell lines and patient-derived PCa cells was analysed by western blotting. PLD1 protein localisation in normal, benign prostatic hyperplasia (BPH), and castrate-resistant prostate cancer (CRPC) tissue sections and in a PCa tissue microarray (TMA) was examined by immunohistochemistry. PLD activity in PCa tissue was assayed using an Amplex Red method. The effect of PLD inhibitors on PCa cell viability was measured using MTS and colony forming assays. PLD1 protein expression was low in the luminal prostate cell lines (LNCaP, VCaP, 22RV1) compared with basal lines (PC3 and PC3M). PLD1 protein expression was elevated in BPH biopsy tissue relative to normal and PCa samples. In normal and BPH tissue, PLD1 was predominantly detected in basal cells as well in some stromal cells, rather than in luminal cells. In PCa tissue, luminal cells expressed PLD1. In a PCa TMA, the mean peroxidase intensity per DAB-stained Gleason 6 and 7 tissue section was significantly higher than in sections graded Gleason 9. In CRPC tissue, PLD1 was expressed prominently in the stromal compartment, in luminal cells in occasional glands and in an expanding population of cells that co-expressed chromogranin A and neurone-specific enolase. Levels of PLD activity in normal and PCa tissue samples were similar. A specific PLD1 inhibitor markedly reduced the survival of both prostate cell lines and patient-derived PCa cells compared with two dual PLD1/PLD2 inhibitors. Short-term exposure of PCa cells to the

  17. The danish protease inhibitor study: a randomized study comparing the virological efficacy of 3 protease inhibitor-containing regimens for the treatment of human immunodeficiency virus type 1 infection

    DEFF Research Database (Denmark)

    Katzenstein, TL; Kirk, O; Pedersen, C

    2000-01-01

    The Danish Protease Inhibitor (PI) Study has enrolled 318 human immunodeficiency virus (HIV)-infected, PI-naive patients for the purpose of comparing 3 PI-containing regimens for the treatment of HIV infection. The regimens include 2 nucleoside analogues in combination with indinavir (Idr...

  18. 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.

  19. Erectile properties of the Rho-kinase inhibitor SAR407899 in diabetic animals and human isolated corpora cavernosa

    Science.gov (United States)

    2012-01-01

    Background RhoA-Rho kinase complex contributes to keep the cavernosus smooth muscle contracted and its inhibition is considered a potential strategy for the therapy of erectile dysfunction (ED). Methods We compared the effects of SAR407899, the Rho-kinase inhibitor Y-27632 and the PDE5 inhibitor sildenafil for their ability to relax corpus cavernosum strips contracted with phenylephrine in healthy and diabetic animals. Strips were obtained from WKY, spontaneous hypertensive (SHR), control CD, and diabetic CD rats, humans, control and diabetic rabbits. Diabetes was induced by streptozotocin or alloxan injection. In vivo penile erection (length) induced by drugs was measured in conscious rabbits. Results SAR407899 dose-dependently relaxed the pre-contracted corpora cavernosa in all species, with similar potency and efficacy in healthy vs diabetic rats, WKY vs SHR rats, healthy vs diabetic rabbits (IC50 range from 0.05 to 0.29 μM, Emax range 89 to 97%). In the presence of the NO-synthase (NOS) inhibitor, L-NAME, the SAR407899 response did not decrease in any of the species or experimental conditions. The effect was confirmed in human strips where sildenafil was significantly less potent and effective, with IC50 respectively 0.13 and 0.51 μM; Emax 92 and 43%. Unlike SAR407899, the potency and efficacy of sildenafil and Y27632 were significantly reduced by diabetes and L-NAME. In vivo, SAR407899 dose-dependently induced rabbit penile erection, with greater potency and longer duration of action than sildenafil. Sildenafil, but not SAR407899, was less effective in alloxan-induced diabetes. Conclusion The induction of penile erection by SAR407899, unlike that by sildenafil, is largely independent of e-NO activity. This suggests its use in erectile dysfunction for diabetic and hypertensive patients where e-NO activity is impaired. PMID:22444253

  20. Conventional HPLC Method Used for Simultaneous Determination of the Seven HIV Protease Inhibitors and Nonnucleoside Reverse Transcription Inhibitor Efavirenz in Human Plasma

    National Research Council Canada - National Science Library

    Takahashi, Masaaki; Yoshida, Masao; Oki, Tsuyoshi; Okumura, Naoya; Suzuki, Tatsuo; Kaneda, Tsuguhiro

    2005-01-01

    We developed a simple HPLC method for the simultaneous quantitative determination of seven HIV protease inhibitors: amprenavir (APV), atazanavir (ATV), indinavir (IDV), lopinavir (LPV), nelfinavir (NFV), ritonavir (RTV), saquinavir (SQV...

  1. Efficacy and pharmacodynamic effects of bosutinib (SKI-606), a Src/Abl inhibitor, in freshly generated human pancreas cancer xenografts.

    Science.gov (United States)

    Messersmith, Wells A; Rajeshkumar, N V; Tan, Aik Choon; Wang, Xiao Fei; Diesl, Veronica; Choe, Sung E; Follettie, Max; Coughlin, Christina; Boschelli, Frank; Garcia-Garcia, Elena; Lopez-Rios, Fernando; Jimeno, Antonio; Hidalgo, Manuel

    2009-06-01

    Recently, Src tyrosine kinase has emerged as an attractive target for anticancer therapy, and Src is overexpressed in pancreatic cancer. The purpose of the study was to investigate the in vivo efficacy and pharmacodynamic effects of bosutinib (SKI-606), a Src/Abl inhibitor, using a panel of human pancreatic tumor xenografts. Surgically resected human pancreatic tumors were implanted into female nude mice and randomized to bosutinib versus control. Src and other pathways were analyzed by Western Blot, IHC, and Affymetrix U133 Plus 2.0 gene arrays. Of 15 patient tumors, 3 patient tumors were found to be sensitive to bosutinib, defined as tumor growth of bosutinib resulted in increased apoptosis. Phosphorylation of key signaling molecules downstream of Src, signal transducers and activators of transcription 3, and signal transducers and activators of transcription 3, were significantly inhibited by bosutinib. K-Top Scoring Pairs analysis of gene arrays gave a six-gene classifier that predicted resistance versus sensitivity in six validation cases. These results may aid the clinical development of bosutinib and other Src inhibitors in pancreas cancer.

  2. 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

  3. 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.

  4. Structure-activity relationships of amide-phosphonate derivatives as inhibitors of the human soluble epoxide hydrolase.

    Science.gov (United States)

    Kim, In-Hae; Park, Yong-Kyu; Nishiwaki, Hisashi; Hammock, Bruce D; Nishi, Kosuke

    2015-11-15

    Structure-activity relationships of amide-phosphonate derivatives as inhibitors of the human soluble epoxide hydrolase (sEH) were investigated. First, a series of alkyl or aryl groups were substituted on the carbon alpha to the phosphonate function in amide compounds to see whether substituted phosphonates can act as a secondary pharmacophore. A tert-butyl group (16) on the alpha carbon was found to yield most potent inhibition on the target enzyme. A 4-50-fold drop in inhibition was induced by other substituents such as aryls, substituted aryls, cycloalkyls, and alkyls. Then, the modification of the O-substituents on the phosphonate function revealed that diethyl groups (16 and 23) were preferable for inhibition to other longer alkyls or substituted alkyls. In amide compounds with the optimized diethylphosphonate moiety and an alkyl substitution such as adamantane (16), tetrahydronaphthalene (31), or adamantanemethane (36), highly potent inhibitions were gained. In addition, the resulting potent amide-phosphonate compounds had reasonable water solubility, suggesting that substituted phosphonates in amide inhibitors are effective for both inhibition potency on the human sEH and water solubility as a secondary pharmacophore. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. 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...

  6. 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.

  7. 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...... to 2.05 angstrom resolution to resolve apparent discrepancies between the complex structure and the previously published structure of the native enzyme. The new structure of the native enzyme is, within the experimental error, identical with the structure of the enzyme-inhibitor complex presented here...

  8. 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...... for the removal of u-PA after its complex formation with a specific inhibitor. The data suggest a novel mechanism by which receptor-mediated endocytosis is initiated by the binding of a secondary ligand. Full text...

  9. 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

  10. Kinetic and Thermodynamic Rationale for SAHA Being a Preferential Human HDAC8 Inhibitor as Compared to the Structurally Similar Ligand, TSA

    Science.gov (United States)

    Singh, Raushan K.; Lall, Naveena; Leedahl, Travis S.; McGillivray, Abigail; Mandal, Tanmay; Haldar, Manas; Mallik, Sanku; Cook, Gregory; Srivastava, D.K.

    2013-01-01

    Of the different hydroxamate-based histone deacetylase (HDAC) inhibitors, Suberoylanilide hydroxamic acid (SAHA) has been approved by the FDA for treatment of T-cell lymphoma. Interestingly, a structurally similar inhibitor, Trichostatin A (TSA), which has a higher in vitro inhibitory-potency against HDAC8, reportedly shows a poor efficacy in clinical settings. In order to gain the molecular insight into the above discriminatory feature, we performed transient kinetic and isothermal titration calorimetric studies for the interaction of SAHA and TSA to the recombinant form of human HDAC8. The transient kinetic data revealed that the binding of both the inhibitors to the enzyme showed the biphasic profiles, which represented an initial encounter of enzyme with the inhibitor followed by the isomerization of the transient enzyme-inhibitor complexes. The temperature-dependent transient kinetic studies with the above inhibitors revealed that the bimolecular process is primarily dominated by favorable enthalpic changes, as opposed to the isomerization step; which is solely contributed by entropic changes. The standard binding-enthalpy (ΔH0) of SAHA, deduced from the transient kinetic as well as the isothermal titration calorimetric experiments, was 2–3 kcal/mol higher as compared to TSA. The experimental data presented herein suggests that SAHA serves as a preferential (target-specific/selective) HDAC8 inhibitor as compared to TSA. Arguments are presented that the detailed kinetic and thermodynamic studies may guide in the rational design of HDAC inhibitors as therapeutic agents. PMID:24079912

  11. 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

  12. The Raf Kinase Inhibitor Sorafenib Inhibits JAK-STAT Signal Transduction in Human Immune Cells.

    Science.gov (United States)

    Martin del Campo, Sara E; Levine, Kala M; Mundy-Bosse, Bethany L; Grignol, Valerie P; Fairchild, Ene T; Campbell, Amanda R; Trikha, Prashant; Mace, Thomas A; Paul, Bonnie K; Jaime-Ramirez, Alena Cristina; Markowitz, Joseph; Kondadasula, Sri Vidya; Guenterberg, Kristan D; McClory, Susan; Karpa, Volodymyr I; Pan, Xueliang; Olencki, Thomas E; Monk, J Paul; Mortazavi, Amir; Tridandapani, Susheela; Lesinski, Gregory B; Byrd, John C; Caligiuri, Michael A; Shah, Manisha H; Carson, William E

    2015-09-01

    Sorafenib is an oral multikinase inhibitor that was originally developed as a Raf kinase inhibitor. We hypothesized that sorafenib would also have inhibitory effects on cytokine signaling pathways in immune cells. PBMCs from normal donors were treated with varying concentrations of sorafenib and stimulated with IFN-α or IL-2. Phosphorylation of STAT1 and STAT5 was measured by flow cytometry and confirmed by immunoblot analysis. Changes in IFN-α- and IL-2-stimulated gene expression were measured by quantitative PCR, and changes in cytokine production were evaluated by ELISA. Cryopreserved PBMCs were obtained from cancer patients before and after receiving 400 mg sorafenib twice daily. Patient PBMCs were thawed, stimulated with IL-2 or IFN-α, and evaluated for phosphorylation of STAT1 and STAT5. Pretreatment of PBMCs with 10 μM sorafenib decreased STAT1 and STAT5 phosphorylation after treatment with IFN-α or IL-2. This inhibitory effect was observed in PBMCs from healthy donors over a range of concentrations of sorafenib (5-20 μM), IL-2 (2-24 nM), and IFN-α (10(1)-10(6) U/ml). This effect was observed in immune cell subsets, including T cells, B cells, NK cells, regulatory T cells, and myeloid-derived suppressor cells. Pretreatment with sorafenib also inhibited PBMC expression of IFN-α- and IL-2-regulated genes and inhibited NK cell production of IFN-γ, RANTES, MIP1-α, and MIG in response to IFN-α stimulation. PBMCs from patients receiving sorafenib therapy showed decreased responsiveness to IL-2 and IFN-α treatment. Sorafenib is a Raf kinase inhibitor that could have off-target effects on cytokine-induced signal transduction in immune effector cells. Copyright © 2015 by The American Association of Immunologists, Inc.

  13. NF-κB inhibitor reverses temozolomide resistance in human glioma TR/U251 cells.

    Science.gov (United States)

    Wang, Xuan; Jia, Lili; Jin, Xiaohua; Liu, Qian; Cao, Wei; Gao, Xiangdong; Yang, Mingfeng; Sun, Baoliang

    2015-06-01

    Glioblastoma multiforme (GBM) demonstrates an unsatisfactory clinical prognosis due to the intrinsic or acquired resistance to temozolomide (TMZ) exhibited by the tumors. One possible cause of TMZ resistance in GBM is the overexpression of O6-methylguanine-DNA methyltransferase (MGMT), which can repair the TMZ-induced guanine damage in DNA. Additionally, excessive activated NF-κB is reported to be a component of the major inflammatory transcription pathway that is associated with TMZ resistance in GBM. However, the association between the NF-κB pathway and MGMT expression in GBM cells is unknown. Therefore, in the present study, the TMZ resistant (TR) U251 cell line (TR/U251) was successfully constructed to detect how the TR/U251 cell line and the parental U251 cell line each interact with TMZ in vitro. The TR/U251 cells were approximately five times more resistant to TMZ compared with the parental cells. Furthermore, it was found that the NF-κB inhibitor BAY 11-7082 suppressed the expression of MGMT in TR/U251 cells and enhanced TMZ-induced cytotoxicity and apoptosis, thereby indicating that the NF-κB pathway and MGMT interact to promote TMZ resistance. The inhibition of NF-κB may be a promising strategy to reverse drug resistance in TR glioma cells. The present results propose a potential mechanism for using the NF-κB inhibitor BAY 11-7082 as a potential therapy for the treatment of TR glioma. Although BAY 11-7082 is a well-known NF-κB inhibitor, the present study further investigated its underlying mechanisms through a series of new experiments.

  14. 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

  15. "Histopathologic and Immunohistochemical Characterization of Rash to Human Epidermal Growth Factor Receptor 1 (HER1) and HER1/2 Inhibitors in Cancer Patients

    OpenAIRE

    Nardone, Beatrice

    2011-01-01

    Introduction: Human epidermal receptor (HER) 1 inhibitors and HER 1/2 inhibitors have shown benefit against a wide range of solid tumors. However, their use is associated with rash in 40-90% of patients, which impacts quality of life and interrupts antineoplastic therapy. The pathologic characteristics of affected skin remain unclear, precluding development of rational therapies. The aim of this study is to evaluate differences in histologic and immunohistochemical (IHC) alterations in rash c...

  16. 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.

  17. 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.

  18. Inhibition by human recombinant tissue inhibitor of metalloproteinases of human amnion invasion and lung colonization by murine B16-F10 melanoma cells.

    Science.gov (United States)

    Schultz, R M; Silberman, S; Persky, B; Bajkowski, A S; Carmichael, D F

    1988-10-01

    The human tissue inhibitor of metalloproteinases (TIMP) is a glycoprotein with a molecular weight of 28,000. It appears to be ubiquitous in human mesoderm tissues and has previously been shown to be identical to the collagenase inhibitor isolated from human skin fibroblasts. TIMP inhibits type I- and IV-specific collagenases and other neutral metalloendoproteinases that may be responsible for the degradation of extracellular matrix in tumor cell metastasis. In this work we have utilized recombinant human TIMP (rTIMP) obtained by expression of its cDNA gene (Carmichael et al., Proc. Natl. Acad. Sci. USA, 83:2407, 1986). The rTIMP is shown to have similar inhibition properties as natural TIMP against human skin fibroblast collagenase. In an in vitro amnion invasion assay system, rTIMP inhibited the invasion of B16-F10 murine melanoma cells through the human amniotic membrane at an identical concentration to that reported previously for natural TIMP. The mechanism by which rTIMP inhibits amniotic membrane invasion was compared to the mechanism by which the fibronectin receptor binding peptide RGDS and the aminin receptor binding peptide YIGSR inhibit amnion invasion. RGDS and YIGSR inhibited strong binding of the tumor cells to the amniotic membrane. In contrast rTIMP did not inhibit the cell adhesion step in amnion invasion, but actually increased the number of tumor cells that were tightly bound to the amnion. Thus rTIMP appears to inhibit a later step in the amnion invasion process, following B16-F10 cell adhesion. C57BL/6 mice treated with i.p. injections of rTIMP every 12 h for 6.5 days showed a significant inhibition of metastatic lung colonization by B16-F10 murine melanoma cells. While the rTIMP inhibited the number of metastatic lung tumors formed, it had no significant effect on the size of the lung tumors. Furthermore, tumors grown s.c. in mice receiving 12-h i.p. injections of rTIMP for 6.5 days, as in the in vivo colonization assay, showed no difference

  19. Metabolite profiling and enzyme reaction phenotyping of luseogliflozin, a sodium-glucose cotransporter 2 inhibitor, in humans.

    Science.gov (United States)

    Miyata, Atsunori; Hasegawa, Masatoshi; Hachiuma, Kenji; Mori, Haruyuki; Horiuchi, Nobuko; Mizuno-Yasuhira, Akiko; Chino, Yukihiro; Jingu, Shigeji; Sakai, Soichi; Samukawa, Yoshishige; Nakai, Yasuhiro; Yamaguchi, Jun-Ichi

    2017-04-01

    1. To understand the clearance mechanism of luseogliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, we investigated its human metabolite profile and metabolic enzymes responsible for the primary metabolic pathways in human using reaction phenotyping. 2. Sixteen metabolites of luseogliflozin were found in human plasma and/or urine and their structural information indicated that the drug was metabolized via multiple metabolic pathways. The primary metabolic pathways involve (1) O-deethylation to form M2 and subsequent glucuronidation to form M12, (2) ω-hydroxylation at ethoxy group to form M3 followed by oxidation to form the corresponding carboxylic acid metabolite (M17) and (3) direct glucuronidation to form M8. 3. The reaction phenotyping studies indicated that the formation of M2 was mainly mediated by cytochrome P450 (CYP) 3A4/5, and subsequently M12 formation was catalyzed by UGT1A1, UGT1A8 and UGT1A9. The formation of M3 was mediated by CYP4A11, CYP4F2 and CYP4F3B, and the further oxidation of M3 to M17 was mediated by alcohol dehydrogenase and aldehyde dehydrogenase. The formation of M8 was catalyzed by UGT1A1. 4. These results demonstrate that luseogliflozin is metabolized through multiple pathways, including CYP-mediated oxidation and glucuronidation, in human.

  20. 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.

  1. Peroxisome proliferator-activated receptorα agonists differentially regulate inhibitor of DNA binding expression in rodents and human cells.

    Science.gov (United States)

    González, María Del Carmen; Corton, J Christopher; Acero, Nuria; Muñoz-Mingarro, Dolores; Quirós, Yolanda; Alvarez-Millán, Juan José; Herrera, Emilio; Bocos, Carlos

    2012-01-01

    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.

  2. Development of ML390: A Human DHODH Inhibitor That Induces Differentiation in Acute Myeloid Leukemia.

    Science.gov (United States)

    Lewis, Timothy A; Sykes, David B; Law, Jason M; Muñoz, Benito; Rustiguel, Joane K; Nonato, Maria Cristina; Scadden, David T; Schreiber, Stuart L

    2016-12-08

    Homeobox transcription factor A9 (HoxA9) is overexpressed in 70% of patients diagnosed with acute myeloid leukemia (AML), whereas only a small subset of AML patients respond to current differentiation therapies. A cell line overexpressing HoxA9 was derived from the bone marrow of a lysozyme-GFP mouse. In this fashion, GFP served as an endogenous reporter of differentiation, permitting a high-throughput phenotypic screen against the MLPCN library. Two chemical scaffolds were optimized for activity yielding compound ML390, and genetic resistance and sequencing efforts identified dihydroorotate dehydrogenase (DHODH) as the target enzyme. The DHODH inhibitor brequinar works against these leukemic cells as well. The X-ray crystal structure of ML390 bound to DHODH elucidates ML390s binding interactions.

  3. 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...... or malignant epithelial cells, in vascular cells or smooth muscle cells. Comparison of sections processed for TIMP-1 in situ hybridization with sections immunohistochemically stained with antibodies against TIMP-1 showed good correlation between TIMP-1 mRNA and immunoreactivity. Combining TIMP-1 in situ...... hybridization with immunohistochemical staining for alpha-smooth muscle actin or CD68 showed TIMP-1 mRNA in myofibroblasts but not in macrophages. TIMP-1 mRNA was detected in 2 of 7 adenomatous polyps in the adenoma area: in both cases associated with focal stromal inflammation at the epithelial...

  4. PAI-1 (Plasminogen Activator Inhibitor-1) Expression Renders Alternatively Activated Human Macrophages Proteolytically Quiescent.

    Science.gov (United States)

    Hohensinner, Philipp J; Baumgartner, Johanna; Kral-Pointner, Julia B; Uhrin, Pavel; Ebenbauer, Benjamin; Thaler, Barbara; Doberer, Konstantin; Stojkovic, Stefan; Demyanets, Svitlana; Fischer, Michael B; Huber, Kurt; Schabbauer, Gernot; Speidl, Walter S; Wojta, Johann

    2017-10-01

    Macrophages are versatile immune cells capable of polarizing into functional subsets depending on environmental stimulation. In atherosclerotic lesions, proinflammatory polarized macrophages are associated with symptomatic plaques, whereas Th2 (T-helper cell type 2) cytokine-polarized macrophages are inversely related with disease progression. To establish a functional cause for these observations, we analyzed extracellular matrix degradation phenotypes in polarized macrophages. We provide evidence that proinflammatory polarized macrophages rely on membrane-bound proteases including MMP-14 (matrix metalloproteinase-14) and the serine protease uPA (urokinase plasminogen activator) together with its receptor uPAR for extracellular matrix degradation. In contrast, Th2 cytokine alternatively primed macrophages do not show different proteolytic activity in comparison to unpolarized macrophages and lack increased localization of MMP-14 and uPA receptor to the cell membrane. Nonetheless, they express the highest amount of the serine protease uPA. However, uPA activity is blocked by similarly increased expression of its inhibitor PAI-1 (plasminogen activator inhibitor 1). When inhibiting PAI-1 or when analyzing macrophages deficient in PAI-1, Th2 cytokine-polarized macrophages display the same matrix degradation capability as proinflammatory-primed macrophages. Within atherosclerotic lesions, macrophages positive for the alternative activation marker CD206 express high levels of PAI-1. In addition, to test changed tissue remodeling capacities of alternatively activated macrophages, we used a bleomycin lung injury model in mice reconstituted with PAI-1(-/-) bone marrow. These results supported an enhanced remodeling phenotype displayed by increased fibrosis and elevated MMP activity in the lung after PAI-1 loss. We were able to demonstrate matrix degradation dependent on membrane-bound proteases in proinflammatory stimulated macrophages and a forced proteolytical quiescence

  5. The Checkpoint Kinase 1 Inhibitor Prexasertib Induces Regression of Preclinical Models of Human Neuroblastoma.

    Science.gov (United States)

    Lowery, Caitlin D; VanWye, Alle B; Dowless, Michele; Blosser, Wayne; Falcon, Beverly L; Stewart, Julie; Stephens, Jennifer; Beckmann, Richard P; Bence Lin, Aimee; Stancato, Louis F

    2017-08-01

    Purpose: Checkpoint kinase 1 (CHK1) is a key regulator of the DNA damage response and a mediator of replication stress through modulation of replication fork licensing and activation of S and G2-M cell-cycle checkpoints. We evaluated prexasertib (LY2606368), a small-molecule CHK1 inhibitor currently in clinical testing, in multiple preclinical models of pediatric cancer. Following an initial assessment of prexasertib activity, this study focused on the preclinical models of neuroblastoma.Experimental Design: We evaluated the antiproliferative activity of prexasertib in a panel of cancer cell lines; neuroblastoma cell lines were among the most sensitive. Subsequent Western blot and immunofluorescence analyses measured DNA damage and DNA repair protein activation. Prexasertib was investigated in several cell line-derived xenograft mouse models of neuroblastoma.Results: Within 24 hours, single-agent prexasertib promoted γH2AX-positive double-strand DNA breaks and phosphorylation of DNA damage sensors ATM and DNA-PKcs, leading to neuroblastoma cell death. Knockdown of CHK1 and/or CHK2 by siRNA verified that the double-strand DNA breaks and cell death elicited by prexasertib were due to specific CHK1 inhibition. Neuroblastoma xenografts rapidly regressed following prexasertib administration, independent of starting tumor volume. Decreased Ki67 and increased immunostaining of endothelial and pericyte markers were observed in xenografts after only 6 days of exposure to prexasertib, potentially indicating a swift reduction in tumor volume and/or a direct effect on tumor vasculature.Conclusions: Overall, these data demonstrate that prexasertib is a specific inhibitor of CHK1 in neuroblastoma and leads to DNA damage and cell death in preclinical models of this devastating pediatric malignancy. Clin Cancer Res; 23(15); 4354-63. ©2017 AACR. ©2017 American Association for Cancer Research.

  6. 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.

  7. 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.

  8. The cyclin-dependent kinase inhibitor flavopiridol sensitizes human hepatocellular carcinoma cells to TRAIL-induced apoptosis.

    Science.gov (United States)

    Miyashita, Kazumi; Shiraki, Katsuya; Fuke, Hiroyuki; Inoue, Tomoko; Yamanaka, Yutaka; Yamaguchi, Yumi; Yamamoto, Norihiko; Ito, Keiichi; Sugimoto, Kazushi; Nakano, Takeshi

    2006-08-01

    Flavopiridol was one of the first cyclin-dependent kinase inhibitors demonstrated to have an antitumor effect in several cancer types. Here, we investigated the effects of flavopiridol on TNF-related apoptosis-inducing ligand (TRAIL) in the human hepatocellular carcinoma (HCC) cell lines HLE and HepG2, and evaluated the role of flavopiridol in apoptosis. To better understand the mechanism of increased TRAIL sensitivity in HCC cells, we determined the effect of flavopiridol on cell surface expression of TRAIL and TRAIL receptors using flow cytometry analysis. The levels of survivin, FLIP, Bcl-xL and X-chromosome-linked IAP (XIAP) in treated and untreated cells was also determined. Flavopiridol decreased cell viability in a dose-dependent manner in the two HCC cell lines tested. The pan-caspase inhibitor z-VAD-FMK did not inhibit the effect. However, subtoxic levels of flavopiridol dramatically enhanced TRAIL-induced apoptosis in both cells. Flavopiridol up-regulated TRAIL, TRAIL-R1 and TRAIL-R2 in both cell lines. In addition, flavopiridol down-regulated expression of survivin in both cell lines, and expression of FLIP and Bcl-xL were down-regulated in HLE cells. In summary, flavopiridol augmented TRAIL sensitivity by up-regulation of TRAIL receptors and down-regulation of survivin, FLIP and Bcl-xL. Thus, combining flavopiridol with a TRAIL agonist may prove to be an effective new strategy for treatment of HCC.

  9. Effects of reverse transcriptase inhibitors on telomere length and telomerase activity in two immortalized human cell lines.

    Science.gov (United States)

    Strahl, C; Blackburn, E H

    1996-01-01

    The ribonucleoprotein telomerase, a specialized cellular reverse transcriptase, synthesizes one strand of the telomeric DNA of eukaryotes. We analyzed telomere maintenance in two immortalized human cell lines: the B-cell line JY616 and the T-cell line Jurkat E6-1, and determined whether known inhibitors of retroviral reverse transcriptases could perturb telomere lengths and growth rates of these cells in culture. Dideoxyguanosine (ddG) caused reproducible, progressive telomere shortening over several weeks of passaging, after which the telomeres stabilized and remained short. However, the prolonged passaging in ddG caused no observable effects on cell population doubling rates or morphology. Azidothymidine (AZT) caused progressive telomere shortening in some but not all T- and B-cell cultures. Telomerase activity was present in both cell lines and was inhibited in vitro by ddGTP and AZT triphosphate. Prolonged passaging in arabinofuranyl-guanosine, dideoxyinosine (ddI), dideoxyadenosine (ddA), didehydrothymidine (d4T), or phosphonoformic acid (foscarnet) did not cause reproducible telomere shortening or decreased cell growth rates or viabilities. Combining AZT, foscarnet, and/or arabinofuranyl-guanosine with ddG did not significantly augment the effects of ddG alone. Strikingly, with or without inhibitors, telomere lengths were often highly unstable in both cell lines and varied between parallel cell cultures. We propose that telomere lengths in these T- and B-cell lines are determined by both telomerase and telomerase-independent mechanisms.

  10. A Pan-BCL2 inhibitor renders bone-marrow-resident human leukemia stem cells sensitive to tyrosine kinase inhibition.

    Science.gov (United States)

    Goff, Daniel J; Court Recart, Angela; Sadarangani, Anil; Chun, Hye-Jung; Barrett, Christian L; Krajewska, Maryla; Leu, Heather; Low-Marchelli, Janine; Ma, Wenxue; Shih, Alice Y; Wei, Jun; Zhai, Dayong; Geron, Ifat; Pu, Minya; Bao, Lei; Chuang, Ryan; Balaian, Larisa; Gotlib, Jason; Minden, Mark; Martinelli, Giovanni; Rusert, Jessica; Dao, Kim-Hien; Shazand, Kamran; Wentworth, Peggy; Smith, Kristen M; Jamieson, Christina A M; Morris, Sheldon R; Messer, Karen; Goldstein, Lawrence S B; Hudson, Thomas J; Marra, Marco; Frazer, Kelly A; Pellecchia, Maurizio; Reed, John C; Jamieson, Catriona H M

    2013-03-07

    Leukemia stem cells (LSCs) play a pivotal role in the resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) and its progression to blast crisis (BC), in part, through the alternative splicing of self-renewal and survival genes. To elucidate splice-isoform regulators of human BC LSC maintenance, we performed whole-transcriptome RNA sequencing, splice-isoform-specific quantitative RT-PCR (qRT-PCR), nanoproteomics, stromal coculture, and BC LSC xenotransplantation analyses. Cumulatively, these studies show that the alternative splicing of multiple prosurvival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSCS that are quiescent in the marrow niche and that contribute to therapeutic resistance. Notably, sabutoclax, a pan-BCL2 inhibitor, renders marrow-niche-resident BC LSCs sensitive to TKIs at doses that spare normal progenitors. These findings underscore the importance of alternative BCL2 family splice-isoform expression in BC LSC maintenance and suggest that the combinatorial inhibition of prosurvival BCL2 family proteins and BCR-ABL may eliminate dormant LSCs and obviate resistance. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. 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.

  12. 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...

  13. Small Molecule Inhibitors of the LEDGF Site of Human Immunodeficiency Virus Integrase Identified by Fragment Screening and Structure Based Design

    Science.gov (United States)

    Peat, Thomas S.; Rhodes, David I.; Vandegraaff, Nick; Le, Giang; Smith, Jessica A.; Clark, Lisa J.; Jones, Eric D.; Coates, Jonathan A. V.; Thienthong, Neeranat; Newman, Janet; Dolezal, Olan; Mulder, Roger; Ryan, John H.; Savage, G. Paul; Francis, Craig L.; Deadman, John J.

    2012-01-01

    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. PMID:22808106

  14. 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.

  15. Identification of a human protein-derived HIV-1 fusion inhibitor targeting the gp41 fusion core structure.

    Directory of Open Access Journals (Sweden)

    Lijun Chao

    Full Text Available The HIV-1 envelope glycoprotein (Env gp41 plays a crucial role in the viral fusion process. The peptides derived from the C-terminal heptad repeat (CHR of gp41 are potent HIV fusion inhibitors. However, the activity of these anti-HIV-1 peptides in vivo may be attenuated by their induction of anti-gp41 antibodies. Thus, it is essential to identify antiviral peptides or proteins with low, or no, immunogenicity to humans. Here, we found that the C-terminal fragment (aa 462-521 of the human POB1 (the partner of RalBP1, designated C60, is an HIV-1 fusion inhibitor. It bound to N36, the peptide derived from the N-terminal heptad repeat (NHR of gp41, and to the six-helix bundle (6-HB formed by N36 and C34, a CHR-peptide, but it did not bind to C34. Unlike the CHR-peptides, C60 did not block gp41 6-HB formation. Rather, results suggest that C60 inhibits HIV-1 fusion by binding to the 6-HB, in particular, the residues in the gp41 NHR domain that are exposed on the surface of 6-HB. Since 6-HB plays a crucial role in the late stage of fusion between the viral envelope and endosomal membrane during the endocytic process of HIV-1, C60 may serve as a host restriction factor to suppress HIV-1 entry into CD4+ T lymphocytes. Taken together, it can be concluded from these results that C60 can be used as a lead for the development of anti-HIV-1 therapeutics or microbicides for the treatment and prevention of HIV-1 infection, as well as a molecular probe to study the fusogenic mechanism of HIV-1.

  16. O-GalNAcylation of RANTES Improves Its Properties as a Human Immunodeficiency Virus Type 1 Entry Inhibitor.

    Science.gov (United States)

    Guan, Xiaoyang; Chaffey, Patrick K; Chen, Huan; Feng, Wei; Wei, Xiuli; Yang, Liu-Meng; Ruan, Yuan; Wang, Xinfeng; Li, Yaohao; Barosh, Kimberly B; Tran, Amy H; Zhu, Jaimie; Liang, Wei; Zheng, Yong-Tang; Wang, Xu; Tan, Zhongping

    2018-01-09

    Many human proteins have the potential to be developed as therapeutic agents. However, side effects caused by direct administration of natural proteins have significantly slowed expansion of protein therapeutics into the clinic. Post-translational modifications (PTMs) can improve protein properties, but because of significant knowledge gaps, we are considerably limited in our ability to apply PTMs to generate better protein therapeutics. Here, we seek to fill the gaps by studying the PTMs of a small representative chemotactic cytokine, RANTES. RANTES can inhibit HIV-1 infection by competing with it for binding to receptor CCR5 and stimulating CCR5 endocytosis. Unfortunately, RANTES can induce strong signaling, leading to severe inflammatory side effects. We apply a chemical biology approach to explore the potential of post-translationally modified RANTES as safe inhibitors of HIV-1 infection. We synthesized and systematically tested a library of RANTES isoforms for their ability to inhibit inflammatory signaling and prevent HIV-1 infection of primary human cells. Through this research, we revealed that most of the glycosylated variants have decreased inflammation-associated properties and identified one particular glyco variant, a truncated RANTES containing a Galβ1-3GalNAc disaccharide α-linked to Ser4, which stands out as having the best overall properties: relatively high HIV-1 inhibition potency but also weak inflammatory properties. Moreover, our results provided a structural basis for the observed changes in the properties of RANTES. Taken together, this work highlights the potential importance of glycosylation as an alternative strategy for developing CCR5 inhibitors to treat HIV-1 infection and, more generally, for reducing or eliminating unwanted properties of therapeutic proteins.

  17. Simultaneous quantitation of HIV-protease inhibitors ritonavir, lopinavir and indinavir in human plasma by UPLC-ESI-MS-MS.

    Science.gov (United States)

    Das Mishra, Tulsi; Kurani, Hemal; Singhal, Puran; Shrivastav, Pranav S

    2012-08-01

    A selective, sensitive and high-throughput ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method has been developed and validated for the quantification of HIV-protease inhibitors ritonavir (RTV), lopinavir (LPV) and indinavir (IDV) in human plasma. Sample clean-up involved protein precipitation of both drugs and fluconazole used as internal standard from 100 µL human plasma. All the analytes were chromatographically separated on a Waters Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 µm particle size) analytical column using 0.1% formic acid and methanol (40:60, v/v) as the mobile phase. The parent → product ion transitions for ritonavir (m/z 721.40→ 296.10), lopinavir (m/z 629.40→ 447.40) and indinavir (m/z 614.4→ 421.0) IS (m/z 307.10 → 220.10) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion mode. The method was validated over the concentration range of 30-15,000 ng/mL for LPV and IDV and 3-1500 ng/mL for RTV. The method was successfully applied to a pilot bioequivalence study in 36 healthy human subjects after oral administration of lopinavir 200 mg and ritonavir 50 mg tablet formulation under fasting conditions.

  18. Loss of inhibitor of growth (ING-4) is implicated in the pathogenesis and progression of human astrocytomas.

    Science.gov (United States)

    Klironomos, George; Bravou, Vasiliki; Papachristou, Dionysios J; Gatzounis, George; Varakis, John; Parassi, Ekaterini; Repanti, Maria; Papadaki, Helen

    2010-03-01

    Inhibitor of growth 4 (ING-4) is a tumor suppressor gene that interacts with nuclear factor-kappaB (NF-kappaB) and represses its transcriptional activity. Several lines of evidence suggest that the tumor suppressor gene ING-4, the transcription factor NF-kappaB and its target genes matrix metalloproteases MMP-2, MMP-9 and urokinase plasminogen activator (u-PA) are critically involved in tumor invasion. The aim of the present study was to investigate immunohistochemically the expression pattern of ING-4, NF-kappaB and the NF-kappaB downstream targets MMP-2, MMP-9 and u-PA in human astrocytomas from 101 patients. We found that ING-4 expression was significantly decreased in astrocytomas, and ING-4 loss was associated with tumor grade progression. Expression of p65, a NF-kappaB subunit, was significantly higher in grade IV than in grade III and grade I/II tumors, and a statistical significant negative correlation between expression of ING-4 and expression of nuclear p65 was noticed. MMP-9, MMP-2 and u-PA were overexpressed in human astrocytomas. Of note, astrocytomas of advanced histologic grades (grade III, IV) displayed significantly higher expression levels of these proteins compared to tumors of lower grades (grade I, II). Collectively, our data suggest an essential role for ING-4 in human astrocytoma development and progression possibly through regulation of the NF-kappaB-dependent expression of genes involved in tumor invasion.

  19. 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.

  20. 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.

  1. Mitochondrial division inhibitor 1 (mdivi-1) enhances death receptor-mediated apoptosis in human ovarian cancer cells

    Science.gov (United States)

    Wang, Jingnan; Hansen, Karyn; Edwards, Robert; Van Houten, Bennett; Qian, Wei

    2014-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) based strategy is a promising targeted therapeutic approach for the treatment of a variety of cancers including ovarian cancer. However, the inherent or acquired resistance of tumor cells to TRAIL limits the potential application of TRAIL-mediated therapy. In this study, we identified that mitochondrial division inhibitor-1 (mdivi-1) is able to enhance the sensitivity of human ovarian cancer cells to death receptor ligands including TRAIL, FAS ligands, and TNF-α. Importantly, the combination of TRAIL and mdivi-1 has no apparent cytotoxic effect on non-transformed human cells, indicating a significant therapeutic window. We identified that caspase-8 and not the modulation of TRAIL receptors is required for the combination effect of TRAIL and mdivi-1. We further demonstrated that the enhanced efficacy of combination of mdivi-1 and death ligands is not dependent on the originally reported target of mdivi-1, Drp1, and is also not dependent on the two important pro-apoptotic Bcl-2 family proteins Bax and Bak. Thus, our study presents a novel strategy in enhancing the apoptotic effect of death receptor ligands and provides a new effective TRAIL-based combination approach for treating human ovarian cancer. PMID:25446129

  2. 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.

  3. Hypoxic regulation of plasminogen activator inhibitor-1 expression in human buccal mucosa fibroblasts stimulated with arecoline.

    Science.gov (United States)

    Tsai, Chung-Hung; Lee, Shiuan-Shinn; Chang, Yu-Chao

    2015-10-01

    Oral submucous fibrosis (OSF) is regarded as a pre-cancerous condition with fibrosis in oral subepithelial connective tissue. Hypoxia-inducible factor (HIF)-1α regulates a wide variety of profibrogenic genes, which are closely associated with tissue fibrosis. The aim of this study was to compare HIF-1α expression in normal buccal mucosa tissues and OSF specimens and further explore the potential mechanisms that may lead to the induction of HIF-1α expression. Twenty-five OSF specimens and six normal buccal mucosa were examined by immunohistochemistry. The expression of HIF-1α from fibroblasts cultured from OSF and normal buccal mucosa was measured by Western blot. Arecoline, a major areca nut alkaloid, was challenged to normal buccal mucosa fibroblasts (BMFs) to elucidate whether HIF-1α expression could affect by arecoline. In addition, the effects of arecoline on plasminogen activator inhibitor (PAI)-1 expression were evaluated in environmental hypoxia. HIF-1α expression was significantly higher in OSF specimens and expressed mainly by fibroblasts, epithelial cells, and inflammatory cells. Fibroblasts derived from OSF were found to exhibit higher HIF-1α protein expression than BMFs (P oral submucosa leading to fibrosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. 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.

  5. 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.

  6. RI-1: a chemical inhibitor of RAD51 that disrupts homologous recombination in human cells.

    Science.gov (United States)

    Budke, Brian; Logan, Hillary L; Kalin, Jay H; Zelivianskaia, Anna S; Cameron McGuire, William; Miller, Luke L; Stark, Jeremy M; Kozikowski, Alan P; Bishop, Douglas K; Connell, Philip P

    2012-08-01

    Homologous recombination serves multiple roles in DNA repair that are essential for maintaining genomic stability. We here describe RI-1, a small molecule that inhibits the central recombination protein RAD51. RI-1 specifically reduces gene conversion in human cells while stimulating single strand annealing. RI-1 binds covalently to the surface of RAD51 protein at cysteine 319 that likely destabilizes an interface used by RAD51 monomers to oligomerize into filaments on DNA. Correspondingly, the molecule inhibits the formation of subnuclear RAD51 foci in cells following DNA damage, while leaving replication protein A focus formation unaffected. Finally, it potentiates the lethal effects of a DNA cross-linking drug in human cells. Given that this inhibitory activity is seen in multiple human tumor cell lines, RI-1 holds promise as an oncologic drug. Furthermore, RI-1 represents a unique tool to dissect the network of reaction pathways that contribute to DNA repair in cells.

  7. Oncolytic Virotherapy Synergism with Signaling Inhibitors: Rapamycin Increases Myxoma Virus Tropism for Human Tumor Cells▿

    OpenAIRE

    Stanford, Marianne M.; Barrett, John W.; Nazarian, Steven H.; Werden, Steven; McFadden, Grant

    2006-01-01

    Myxoma virus is a rabbit-specific poxvirus pathogen that also exhibits a unique tropism for human tumor cells and is dramatically oncolytic for human cancer xenografts. Most tumor cell lines tested are permissive for myxoma infection in a fashion intimately tied to the activation state of Akt kinase. A host range factor of myxoma virus, M-T5, directly interacts with Akt and mediates myxoma virus tumor cell tropism. mTOR is a regulator of cell growth and metabolism downstream of Akt and is spe...

  8. Radiosensitizing potential of the selective cyclooygenase-2 (COX-2) inhibitor meloxicam on human glioma cells

    NARCIS (Netherlands)

    Bijnsdorp, Irene; Kuipers, Gitta; Lafleur, M.; Slotman, Ben; Sminia, Peter; Berg, van de Jaap; Rijn, van Johannes; Wedekind, Laurine

    2007-01-01

    The COX-2 protein is frequently overexpressed in human malignant gliomas. This expression has been associated with their aggressive growth characteristics and poor prognosis for patients. Targeting the COX-2 pathway might improve glioma therapy. In this study, the effects of the selective COX-2

  9. 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...

  10. 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.

  11. Recombinant human erythropoietin reduces plasminogen activator inhibitor and ameliorates pro-inflammatory responses following trauma

    Directory of Open Access Journals (Sweden)

    M Mojtahedzadeh

    2011-05-01

    Full Text Available "n  "n Background and the purpose of the study: Besides its hematopoietic effects, erythropoietin (EPO by mobilization of iron and modulation of some inflammatory cytokines has antioxidant and anti-inflammatory properties. The purpose of this study was to evaluate these effects of erythropoietin and its impact on organ function in traumatized patients. "n Methods: Twenty-six ICU-admitted traumatized patients within 24 hrs after trauma were randomly assigned to the EPO (received EPO, 300 units/Kg/day and Control (not received EPO groups. The inflammatory biomarkers including Tumor Necrosis Factor alpha (TNF-α, Interleukin 1 (IL-1, Plasminogen Activator Inhibitor 1 (PAI-1 and Nitrotyrosine were recorded at the admission, 3, 6 and 9 days thereafter. Acute Physiology and Chronic Health Evaluation (APACHE II and Sequential Organ Failure Assessment (SOFA scores were also recorded. "n Results: Among 12 patients (EPO group TNF-α level at the day of 9 (P=0.046, and within EPO group at the days of 3 (P=0.026 ameliorate, 6 (P=0.016, and 9 (P=0.052 were significantly lowered. Level of IL-1 and PAI-1 decreased significantly at days of 3, 6 and 9 post intervention. Also there were significant differences between two groups in the SOFA score during three measured time intervals (the first, third and seventh days. "n Conclusion: From the results of this study it seems that injection of erythrocyte stimulating agent is well tolerated and inhibits the inflammatory response and oxidative stress following trauma.

  12. 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.

  13. 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.

  14. Resveratrol as a Pan-HDAC Inhibitor Alters the Acetylation Status of Jistone Proteins in Human-Derived Hepatoblastoma Cells

    Science.gov (United States)

    Böcker, Alexander; Busch, Christian; Weiland, Timo; Noor, Seema; Leischner, Christian; Schleicher, Sabine; Mayer, Mascha; Weiss, Thomas S.; Bischoff, Stephan C.; Lauer, Ulrich M.; Bitzer, Michael

    2013-01-01

    The polyphenolic alcohol resveratrol has demonstrated promising activities for the prevention and treatment of cancer. Different modes of action have been described for resveratrol including the activation of sirtuins, which represent the class III histone deacetylases (HDACs). However, little is known about the activity of resveratrol on the classical HDACs of class I, II and IV, although these classes are involved in cancer development or progression and inhibitors of HDACs (HDACi) are currently under investigation as promising novel anticancer drugs. We could show by in silico docking studies that resveratrol has the chemical structure to inhibit the activity of different human HDAC enzymes. In vitro analyses of overall HDAC inhibition and a detailed HDAC profiling showed that resveratrol inhibited all eleven human HDACs of class I, II and IV in a dose-dependent manner. Transferring this molecular mechanism into cancer therapy strategies, resveratrol treatment was analyzed on solid tumor cell lines. Despite the fact that hepatocellular carcinoma (HCC) is known to be particularly resistant against conventional chemotherapeutics, treatment of HCC with established HDACi already has shown promising results. Testing of resveratrol on hepatoma cell lines HepG2, Hep3B and HuH7 revealed a dose-dependent antiproliferative effect on all cell lines. Interestingly, only for HepG2 cells a specific inhibition of HDACs and in turn a histone hyperacetylation caused by resveratrol was detected. Additional testing of human blood samples demonstrated a HDACi activity by resveratrol ex vivo. Concluding toxicity studies showed that primary human hepatocytes tolerated resveratrol, whereas in vivo chicken embryotoxicity assays demonstrated severe toxicity at high concentrations. Taken together, this novel pan-HDACi activity opens up a new perspective of resveratrol for cancer therapy alone or in combination with other chemotherapeutics. Moreover, resveratrol may serve as a lead

  15. The potential role of inhibitor of differentiation-3 in human adipose tissue remodeling and metabolic health

    DEFF Research Database (Denmark)

    Svendstrup, Mathilde; Vestergaard, Henrik

    2014-01-01

    Metabolic health in obesity is known to differ among individuals, and the distribution of visceral (VAT) and subcutaneous adipose tissue (SAT) plays an important role in this regard. Adipose tissue expansion is dependent on new blood vessel formation in order to prevent hypoxia and inflammation......-3 (ID3) gene in relation to adipose tissue and angiogenesis in humans in order to determine whether ID3 could be involved in the regulation of adipose tissue expansion and metabolic health in human obesity. We find evidence that ID3 is involved in regulatory mechanisms in adipose tissue...... and regulates angiogenesis in many tissues including adipose tissue. We discuss how this might influence obesity and metabolic health in obesity and further discuss some potential mechanisms by which ID3 might regulate visceral and subcutaneous adipose tissue expansion. The combined results from the reviewed...

  16. Direct comparisons of inhibitor sensitivities of reverse transcriptases from feline and human immunodeficiency viruses.

    OpenAIRE

    North, T W; Cronn, R C; Remington, K M; Tandberg, R T

    1990-01-01

    The sensitivities of reverse transcriptases (RTs) from feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (HIV) were directly compared. The two enzymes had similar sensitivities to three analogs of dTTP, namely, 3'-azido-3'-deoxythymidine 5'-triphosphate, 2',3'-dideoxythymidine 5'-triphosphate, and 2',3'-dideoxy-2',3'-didehydrothymidine 5'-triphosphate. Each of these analogs demonstrated competitive inhibition of both enzymes. Ki values for inhibition of FIV RT by the...

  17. Rho kinase inhibitor y-27632 prolongs the life span of adult human keratinocytes, enhances skin equivalent development, and facilitates lentiviral transduction.

    NARCIS (Netherlands)

    Bogaard, E.H.J. van den; Rodijk-Olthuis, D.; Jansen, P.A.M.; Vlijmen-Willems, I.M.J.J. van; Erp, P.E.J. van; Joosten, I.; Zeeuwen, P.L.J.M.; Schalkwijk, J.

    2012-01-01

    The use of tissue-engineered human skin equivalents (HSE) for fundamental research and industrial application requires the expansion of keratinocytes from a limited number of skin biopsies donated by adult healthy volunteers or patients. A pharmacological inhibitor of Rho-associated protein kinases,

  18. 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

  19. 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

  20. Zr-89-trastuzumab PET visualises HER2 downregulation by the HSP90 inhibitor NVP-AUY922 in a human tumour xenograft

    NARCIS (Netherlands)

    Munnink, Thijs H. Oude; de Korte, Maarten A.; Nagengast, Wouter B.; Timmer-Bosscha, Hetty; Schroder, Carolina P.; de Jong, Johan R.; van Dongen, Guus A. M. S.; Jensen, Michael Rugaard; Quadt, Cornelia; Lub-de Hooge, Marjolijn N.; de Vries, Elisabeth G. E.

    NVP-AUY922, a potent heat shock protein (HSP) 90 inhibitor, downregulates the expression of many oncogenic proteins, including the human epidermal growth factor receptor-2 (HER2). Because HER2 downregulation is a potential biomarker for early response to HSP90-targeted therapies, we used the

  1. Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2015-2016.

    Science.gov (United States)

    Gubareva, Larisa V; Besselaar, Terry G; Daniels, Rod S; Fry, Alicia; Gregory, Vicki; Huang, Weijuan; Hurt, Aeron C; Jorquera, Patricia A; Lackenby, Angie; Leang, Sook-Kwan; Lo, Janice; Pereyaslov, Dmitriy; Rebelo-de-Andrade, Helena; Siqueira, Marilda M; Takashita, Emi; Odagiri, Takato; Wang, Dayan; Zhang, Wenqing; Meijer, Adam

    2017-10-01

    Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) assessed antiviral susceptibility of 14,330 influenza A and B viruses collected by WHO-recognized National Influenza Centres (NICs) between May 2015 and May 2016. Neuraminidase (NA) inhibition assay was used to determine 50% inhibitory concentration (IC50) data for NA inhibitors (NAIs) oseltamivir, zanamivir, peramivir and laninamivir. Furthermore, NA sequences from 13,484 influenza viruses were retrieved from public sequence databases and screened for amino acid substitutions (AAS) associated with reduced inhibition (RI) or highly reduced inhibition (HRI) by NAIs. Of the viruses tested by WHO CCs 93% were from three WHO regions: Western Pacific, the Americas and Europe. Approximately 0.8% (n = 113) exhibited either RI or HRI by at least one of four NAIs. As in previous seasons, the most common NA AAS was H275Y in A(H1N1)pdm09 viruses, which confers HRI by oseltamivir and peramivir. Two A(H1N1)pdm09 viruses carried a rare NA AAS, S247R, shown in this study to confer RI/HRI by the four NAIs. The overall frequency of A(H1N1)pdm09 viruses containing NA AAS associated with RI/HRI was approximately 1.8% (125/6915), which is slightly higher than in the previous 2014-15 season (0.5%). Three B/Victoria-lineage viruses contained a new AAS, NA H134N, which conferred HRI by zanamivir and laninamivir, and borderline HRI by peramivir. A single B/Victoria-lineage virus harboured NA G104E, which was associated with HRI by all four NAIs. The overall frequency of RI/HRI phenotype among type B viruses was approximately 0.6% (43/7677), which is lower than that in the previous season. Overall, the vast majority (>99%) of the viruses tested by WHO CCs were susceptible to all four NAIs, showing normal inhibition (NI). Hence, NAIs remain the recommended antivirals for treatment of

  2. SKI-606 (bosutinib), a novel Src kinase inhibitor, suppresses migration and invasion of human breast cancer cells

    Science.gov (United States)

    Vultur, Adina; Buettner, Ralf; Kowolik, Claudia; Liang, Wei; Smith, David; Boschelli, Frank; Jove, Richard

    2009-01-01

    Src family kinase (SFK) activity is elevated in many human tumors, including breast cancer, and is often associated with aggressive disease. We examined the effects of SKI-606 (bosutinib), a selective SFK inhibitor, on human cancer cells derived from breast cancer patients in order to assess its potential for breast cancer treatment. Our results show that SKI-606 caused a decrease in cell motility and invasion of breast cancer cell lines with an IC50 of ~250 nM, which was also the IC50 for inhibition of c-Src kinase activity in intact tumor cells. These changes were accompanied by an increase in cell-to-cell adhesion and membrane localization of beta-catenin. By contrast, cell proliferation and survival were unaffected by SKI-606 at concentrations sufficient to block cell migration and invasion. Analysis of downstream effectors of Src revealed that SKI-606 inhibits the phosphorylation of focal adhesion kinase (FAK), proline-rich tyrosine kinase 2 (Pyk2) and Crk-associated substrate (p130Cas) with an IC50 similar to inhibition of c-Src kinase. Our findings indicate that SKI-606 inhibits signaling pathways involved in controlling tumor cell motility and invasion, suggesting that SKI-606 is a promising therapeutic for breast cancer. PMID:18483306

  3. 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.

  4. Radiosensitization to X-ray radiation by telomerase inhibitor MST-312 in human hepatoma HepG2 cells.

    Science.gov (United States)

    Wang, Yali; Sun, Chao; Mao, Aihong; Zhang, Xin; Zhou, Xin; Wang, Zhenhua; Zhang, Hong

    2015-02-15

    Previous studies in malignant cells have shown that irradiation-induced upregulation of telomerase activity, not only protected damaged telomeres, but also contributed to DNA damage repair by chromosomal healing and increased resistance to irradiation. The purpose of the present study was to investigate the radiosensitizing effect of telomerase inhibitor MST-312 and the corresponding mechanism in the human hepatoma cell line HepG2. Cell proliferation, telomerase activity, cell cycle distribution, DNA damage and repair, expression of p53, mitochondrial membrane potential, and cell apoptosis were measured with the MTT assay, real-time fluorescent quantitative PCR, flow cytometry, immunofluorescence, western blots, JC-1 staining, and Hoechst 33258 staining, respectively. MST-312 effectively inhibited telomerase activity and showed relative weak toxicity to HepG2 cells at 4 μM. Compared with irradiation alone, 4 μM MST-312 pretreatment, followed by X-ray treatment, significantly reduced clonogenic potential. Aggravated DNA damage and increased sub-G1 cell fractions were observed. Further investigation found that homologous recombination (HR) repair protein Rad51 foci nuclear formation was blocked, and expression of p53 was elevated. These led to the collapse of mitochondrial membrane potential, and enhanced the apoptotic rate. These data demonstrated that disturbances of telomerase function could enhance the radiosensitivity of HepG2 cells to X-ray irradiation by impairing HR repair processes. In addition, telomerase inhibitor MST-312 may be useful as an adjuvant treatment in combination with irradiation. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. RT-21Mre11-Rad50-Nbs1 COMPLEX INHIBITOR MIRIN ENHANCES RADIOSENSITIVITY IN HUMAN GLIOBLASTOMA CELLS

    Science.gov (United States)

    Mishima, Kazuhiko; Mishima-Kaneko, Masayo; Saya, Hideyuki; Ishimaru, Naozumi; Yamada, Kouichi; Fukada, Junichi; Nishikawa, Ryo; Kawata, Tetsuya

    2014-01-01

    PURPOSE: Radiation therapy plays a central part in the treatment of glioblastoma, however, it is not curative due to the high tumor radioresistance. Therefore, increasing the sensitivity of glioblastoma cells to radiation is a promising approach to improve survival in patients with glioblastoma. The Mre11, Rad 50 and Nbs1 proteins form a complex (MRN) that has a critical role in DNA damage detection and signaling. Because defects in MRN enhance radiosensitivity, it has been proposed that small molecule inhibitors targeted to these proteins might be used as radiosensitizers. Here, we investigated the effects of the MRN complex inhibitor, Mirin, on radiation response of human glioma cells. MATERIALS AND METHODS: Glioma cell lines (U251, LN229 and LN428) were irradiated with and without Mirin and then clonogenicity, apoptosis, and cell cycle change were examined. Western blot analysis was performed to determine the relative potency of Mirin to inhibit the radioresistance, through the signaling activity of AKT. We also examined the levels of H2AX phosphorylation (γH2AX), which is a marker of DNA double-strand breaks (DSBs) using Western blot. RESULTS: Glioblastoma cells pretreated with Mirin demonstrated an enhanced sensitivity to radiation. FACS analysis revealed that Mirin and radiation caused the glioma cells to accumulate in the G2/M-phase of the cell cycle and the combination of these two treatments further increased the G2/M fraction of the glioma cells. Mirin significantly enhanced radiation-induced apoptotic cell death. Also, Mirin blocked the basal and increase of radiation-induced AKT phosphorylation. We observed that the combination of Mirin and radiation increased persistence of γH2AX at 24 h suggesting the inhibition of DNA DSBs repair. CONCLUSION: These results indicate that Mirin can effectively enhance glioma cell radiosensitivity. It suggests that Mirin is a potent radiosensitizer for treating glioma cells.

  6. A small-molecule inhibitor of human DNA polymerase eta potentiates the effects of cisplatin in tumor cells.

    Science.gov (United States)

    Zafar, Maroof K; Maddukuri, Leena; Ketkar, Amit; Penthala, Narsimha; Reed, Megan R; Eddy, Sarah D; Crooks, Peter A; Eoff, Robert L

    2018-01-18

    Translesion DNA synthesis (TLS) performed by human DNA polymerase eta (hpol η) allows tolerance of damage from cis-diamminedichloroplatinum(II) (CDDP or cisplatin). We have developed hpol η inhibitors derived from N-aryl-substituted indole barbituric acid (IBA), indole thiobarbituric acid (ITBA), and indole quinuclidine scaffolds and identified 5-((5-chloro-1-(naphthalen-2-ylmethyl)-1H-indol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (PNR-7-02), an ITBA derivative that inhibited hpol η activity with an IC50 value of 8 μM and exhibited five- to ten-fold specificity for hpol η over replicative pols. We conclude from kinetic analyses, chemical footprinting assays, and molecular docking that PNR-7-02 binds to a site on the little finger domain and interferes with the proper orientation of template DNA to inhibit hpol η. A synergistic increase in CDDP toxicity was observed in hpol η-proficient cells co-treated with PNR-7-02 (combination index values = 0.4-0.6). Increased γH2AX formation accompanied treatment of hpol η-proficient cells with CDDP and PNR-7-02, indicative of increased DNA damage from CDDP through inhibition of hpol η TLS activity. Importantly, PNR-7-02 did not impact the effect of CDDP on cell viability or γH2AX in hpol η-deficient cells. In summary, we observed hpol η-dependent effects on DNA damage/replication stress and sensitivity to CDDP in cells treated with PNR-7-02. The ability to employ a small-molecule inhibitor of hpol η to improve the cytotoxic effect of CDDP may aid in the development of more effective chemotherapeutic strategies.

  7. 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

  8. 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

  9. Nef protein of human immunodeficiency virus type 1: evidence against its role as a transcriptional inhibitor.

    OpenAIRE

    Hammes, S. R.; Dixon, E P; Malim, M H; Cullen, B R; Greene, W C

    1989-01-01

    The type 1 human immunodeficiency virus (HIV-1) encodes a 27-kDa protein termed Nef (negative factor). Nef has been reported to down-regulate viral gene transcription directed by the HIV-1 long terminal repeat. To assess the possible role of Nef in the initiation or maintenance of viral latency, we prepared two different nef expression vectors (pNEF from the HXB-3 proviral clone; pNEF-2/3 from HXB-2 and HXB-3) and a control vector containing a frameshift mutation in the HXB-3 nef coding seque...

  10. Plant growth regulator daminozide is a selective inhibitor of human KDM2/7 histone demethylases.

    Science.gov (United States)

    Rose, Nathan R; Woon, Esther C Y; Tumber, Anthony; Walport, Louise J; Chowdhury, Rasheduzzaman; Li, Xuan Shirley; King, Oliver N F; Lejeune, Clarisse; Ng, Stanley S; Krojer, Tobias; Chan, Mun Chiang; Rydzik, Anna M; Hopkinson, Richard J; Che, Ka Hing; Daniel, Michelle; Strain-Damerell, Claire; Gileadi, Carina; Kochan, Grazyna; Leung, Ivanhoe K H; Dunford, James; Yeoh, Kar Kheng; Ratcliffe, Peter J; Burgess-Brown, Nicola; von Delft, Frank; Muller, Susanne; Marsden, Brian; Brennan, Paul E; McDonough, Michael A; Oppermann, Udo; Klose, Robert J; Schofield, Christopher J; Kawamura, Akane

    2012-07-26

    The JmjC oxygenases catalyze the N-demethylation of N(ε)-methyl lysine residues in histones and are current therapeutic targets. A set of human 2-oxoglutarate analogues were screened using a unified assay platform for JmjC demethylases and related oxygenases. Results led to the finding that daminozide (N-(dimethylamino)succinamic acid, 160 Da), a plant growth regulator, selectively inhibits the KDM2/7 JmjC subfamily. Kinetic and crystallographic studies reveal that daminozide chelates the active site metal via its hydrazide carbonyl and dimethylamino groups.

  11. Characterization of human S-adenosyl-homocysteine hydrolase in vitro and identification of its potential inhibitors.

    Science.gov (United States)

    Hao, Weiwei; Li, Yanhua; Shan, Qiuli; Han, Tian; Li, Wencheng; He, Sheng; Zhu, Kongkai; Li, Yumei; Tan, Xiaojun; Gu, Jinsong

    2017-12-01

    Human S-adenosyl-homocysteine hydrolase (SAHH, E.C.3.3.1.1) has been considered to be an attractive target for the design of medicines to treat human disease, because of its important role in regulating biological methylation reactions to catalyse the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine (Ado) and l-homocysteine (Hcy). In this study, SAHH protein was successfully cloned and purified with optimized, Pichia pastoris (P. pastoris) expression system. The biological activity results revealed that, among the tested compounds screened by ChemMapper and SciFinder Scholar, 4-(3-hydroxyprop-1-en-1-yl)-2-methoxyphenol (coniferyl alcohol, CAS: 458-35-5, ZINC: 12359045) exhibited the highest inhibition against rSAHH (IC50= 34 nM). Molecular docking studies showed that coniferyl alcohol was well docked into the active cavity of SAHH. And several H-bonds formed between them, which stabilized coniferyl alcohol in the active site of rSAHH with a proper conformation.

  12. 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

  13. 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.

  14. 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

  15. Docking-based Screening of Ficus religiosa Phytochemicals as Inhibitors of Human Histamine H2 Receptor.

    Science.gov (United States)

    Chaudhary, Amit; Yadav, Birendra Singh; Singh, Swati; Maurya, Pramod Kumar; Mishra, Alok; Srivastva, Shweta; Varadwaj, Pritish Kumar; Singh, Nand Kumar; Mani, Ashutosh

    2017-10-01

    Ficus religiosa L. is generally known as Peepal and belongs to family Moraceae. The tree is a source of many compounds having high medicinal value. In gastrointestinal tract, histamine H2 receptors have key role in histamine-stimulated gastric acid secretion. Their over stimulation causes its excessive production which is responsible for gastric ulcer. This study aims to screen the range of phytochemicals present in F. religiosa for binding with human histamine H2 and identify therapeutics for a gastric ulcer from the plant. In this work, a 3D-structure of human histamine H2 receptor was modeled by using homology modeling and the predicted model was validated using PROCHECK. Docking studies were also performed to assess binding affinities between modeled receptor and 34 compounds. Molecular dynamics simulations were done to identify most stable receptor-ligand complexes. Absorption, distribution, metabolism, excretion, and screening was done to evaluate pharmacokinetic properties of compounds. The results suggest that seven ligands, namely, germacrene, bergaptol, lanosterol, Ergost-5-en-3beta-ol, α-amyrin acetate, bergapten, and γ-cadinene showed better binding affinities. Among seven phytochemicals, lanosterol and α-amyrin acetate were found to have greater stability during simulation studies. These two compounds may be a suitable therapeutic agent against histamine H2 receptor. This study was performed to screen antiulcer compounds from F. religiosa. Molecular modeling, molecular docking and MD simulation studies were performed with selected phytochemicals from F. religiosa. The analysis suggests that Lanosterol and α-amyrin may be a suitable therapeutic agent against histamine H2 receptor. This study facilitates initiation of the herbal drug discovery process for the antiulcer activity. Abbreviations used: ADMET: Absorption, distribution, metabolism, excretion and toxicity, DOPE: Discrete Optimized Potential Energy, OPLS: Optimized potential for liquid

  16. N- and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits.

    Science.gov (United States)

    Koles, Kate; van Berkel, Patrick H C; Pieper, Frank R; Nuijens, Jan H; Mannesse, Maurice L M; Vliegenthart, Johannes F G; Kamerling, Johannis P

    2004-01-01

    Human C1 inhibitor (hC1INH) is a therapeutic N, O-glycoprotein with a growing number of clinical applications, but the current natural supplies are not likely to meet the clinical demands. Therefore, recombinant approaches are of interest, whereby specific attention has to be paid to the generated glycosylation patterns. Here, the N,O-glycoprotein was expressed in the mammary gland of transgenic rabbits and subjected to glycan analysis. After release of the N-glycans of recombinant-rabbit human C1 inhibitor (rhC1INH) by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the O-glycoprotein by centrifugal filtration, then fractionated by a combination of anion-exchange, normal-phase, and high-pH anion-exchange liquid chromatography. The O-glycans, released from the O-glycoprotein by alkaline borohydride treatment, were fractionated by anion-exchange high-performance liquid chromatography (HPLC). The structures of individual components were analysed by 500 MHz 1H NMR spectroscopy, in most cases combined with MALDI-TOF MS. In contrast to the structural data reported for native serum hC1INH, rhC1INH contained a broad array of different N-glycans, made up of oligomannose-, hybrid-, and complex-type structures. In the case of complex-type N-glycans (partially) (alpha2-6)-sialylated (N-acetylneuraminic acid only), mono- and diantennary chains were found; part of the diantennary structures were (alpha1-6)-core-fucosylated or (alpha1-3)-fucosylated in the lower or upper antenna (Lewis x). The manno-oligosaccharide pattern of part of the hybrid- and oligomannose-type structures indicates that besides the usual N-glycan processing route, also the alternative endo-mannosidase pathway is followed. The small core 1-type O-glycans showed the usual (alpha2-3)- and (alpha2-6)-sialylation pattern of O-glycoproteins of nonmucinous origin.

  17. A novel and selective inhibitor of PKC ζ potently inhibits human breast cancer metastasis in vitro and in mice.

    Science.gov (United States)

    Wu, Jing; Liu, Shuye; Fan, Zhijuan; Zhang, Lei; Tian, Yaqiong; Yang, Rui

    2016-06-01

    Cell motility and chemotaxis play pivotal roles in the process of tumor development and metastasis. Protein kinase C ζ (PKC ζ) mediates epidermal growth factor (EGF)-stimulated chemotactic signaling pathway through regulating cytoskeleton rearrangement and cell adhesion. The purpose of this study was to develop anti-PKC ζ therapeutics for breast cancer metastasis. In this study, a novel and high-efficient PKC ζ inhibitor named PKCZI195.17 was screened out through a substrate-specific strategy. MTT assay was used to determine the cell viability of human breast cancer MDA-MB-231, MDA-MB-435, and MCF-7 cells while under PKCZI195.17 treatment. Wound-healing, chemotaxis, and Matrigel invasion assays were performed to detect the effects of PKCZI195.17 on breast cancer cells migration and invasion. Adhesion, actin polymerization, and Western blotting were performed to detect the effects of PKCZI195.17 on cells adhesion and actin polymerization, and explore the downsteam signaling mechanisms involved in PKC ζ inhibition. MDA-MB-231 xenograft was used to measure the in vivo anti-metastasis efficacy of PKCZI195.17. The compound PKCZI195.17 selectively inhibited PKC ζ kinase activity since it failed to inhibit PKC α, PKC β, PKC δ, PKC η, AKT2, as well as FGFR2 activity. PKCZI195.17 significantly impaired spontaneous migration, chemotaxis, and invasion of human breast cancer MDA-MB-231, MDA-MB-435, and MCF-7 cells, while PKCZI195.17 did not obviously inhibited cells viability. PKCZI195.17 also inhibited cells adhesion and actin polymerization through attenuating the phosphorylations of integrin β1, LIMK, and cofilin, which might be the downstream effectors of PKC ζ-mediated chemotaxis in MDA-MB-231 cells. Furthermore, PKCZI195.17 suppressed the breast cancer metastasis and increased the survival time of breast tumor-bearing mice. In summary, PKCZI195.17 was a PKC ζ-specific inhibitor which dampened cancer cell migration and metastasis and may serve as a novel

  18. 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.

  19. 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.

  20. Protective effects of recombinant kunitz-domain 1 of human tissue factor pathway inhibitor-2 against 2-chloroethyl ethyl sulfide toxicity in vitro.

    Science.gov (United States)

    Choi, Moonsuk S; Parikh, Kalpana; Saxena, Ashima; Chilukuri, Nageswararao

    2007-12-03

    Sulfur mustard is a well-known blistering chemical warfare agent that has been investigated for its toxicological mechanisms and an efficacious antidote. Since sulfur mustard injury involves dermal:epidermal separation, proteolytic enzymes were suspected to be involved for this separation and eventual blister development. Therefore, protease inhibitors could be of therapeutic utility against sulfur mustard injury. In this study, the effects of Kunitz-domain 1 of human tissue factor pathway inhibitor-2 were evaluated against the toxic effects of 2-chloroethyl ethyl sulfide, a surrogate agent of sulfur mustard. Tissue factor pathway inhibitor-2 is a 32-kDa serine protease inhibitor produced by a variety of cell types including human epidermal keratinocytes, fibroblasts, and endothelial cells. It consists of 3 Kunitz-domains and the first Kunitz-domain contains the putative P(1) residue (arginine at position 24) responsible for protease inhibitory activity. Recombinant wild-type and R24Q mutant Kunitz-domain 1s were expressed in Escherichia coli and purified. The purified proteins were refolded, and their effects were tested in an in vitro human epidermal keratinocyte cell wounding assay. Wild-type but not R24Q Kunitz-domain 1 inhibited the amidolytic activity of trypsin and plasmin. Wild-type Kunitz-domain 1 was stable for 4 weeks at 42 degrees C and for more than 8 weeks at room temperature. Wild-type Kunitz-domain 1 significantly improved wound healing of unexposed and 2-chloroethyl ethyl sulfide-exposed cells without influencing cell proliferation. Although R24Q Kunitz-domain 1 lacked trypsin and plasmin inhibitory activity, it promoted wound closure of untreated and 2-chloroethyl ethyl sulfide-treated cells but to a much lesser degree. These data suggest that wild-type Kunitz-domain 1 of human tissue factor pathway inhibitor-2 can be developed as a medical countermeasure against sulfur mustard cutaneous injury.

  1. Enhanced anti-hyperproliferative activity of human thymidylate synthase inhibitor peptide by solid lipid nanoparticle delivery.

    Science.gov (United States)

    Sacchetti, Francesca; Marraccini, Chiara; D'Arca, Domenico; Pelà, Michela; Pinetti, Diego; Maretti, Eleonora; Hanuskova, Miriam; Iannuccelli, Valentina; Costi, Maria Paola; Leo, Eliana

    2015-12-01

    Recently, octapeptide LSCQLYQR (LRp), reducing growth of cis-platinum (cDDP) resistant ovarian carcinoma cells by inhibiting the monomer-monomer interface of the human enzyme thymidylate synthase, has been identified. As the peptide is not able to cross the cell membrane it requires an appropriate delivery system. In this work the application of SLNs, biocompatible and efficient tools for the intracellular drug transport, applied especially for lipophilic drugs, was exploited for the delivery of the hydrophilic peptide LRp. SLNs formulated in the absence/presence of small amount of squalene showed dimensions below 150 nm, negative zeta potential and good stability to the freeze-drying process. Even though the particles formulated with squalene exhibited a less ordered crystal lattice and a lower surface hydrophobicity, a rapid drug release from these nanocarriers occurred as a result of the relevant expulsion of the drug from the lipid core during lipid crystallization. On the contrary, SLNs formulated in the absence of squalene were able to incorporate more stably the peptide showing considerable cytotoxic effect on cDDP resistant C13* ovarian carcinoma cell line at concentration 50 times lower than that used previously with a marketed delivery system. From the cell cycle analysis by the propidium iodide test in SLNs-peptide treated cancer cells an increase of apoptosis percentage was observed, indicating that SLNs were able to carry efficiently the peptide until its enzymatic target. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Selective growth inhibitor toward human intestinal bacteria derived from Pulsatilla cernua root.

    Science.gov (United States)

    Lee, H S; Beon, M S; Kim, M K

    2001-10-01

    Among 21 medicinal plants, the growth-inhibiting activity of Pulsatilla cernua root-derived materials toward human intestinal bacteria was examined by using an impregnated paper disk method. The biologically active components of P. cernua roots were characterized as 4-hydroxy-3-methoxycinnamic acid and 3,4-dihydroxycinnamic acid by spectroscopic analysis. The activity was compared with that of six commercially available cinnamic acid derivatives trans-cinnamaldehyde, trans-cinnamic acid, cinnamyl alcohol, 2-methoxycinnamic acid, 3-methoxycinnamic acid, and 4-methoxycinnamic acid. The growth responses varied with each bacterial strain tested. Two isolated compounds revealed a potent inhibition against Clostridium perfringens, and moderate to weak activity against Escherichia coli was exhibited by 4-hydroxy-3-methoxycinnamic acid. Weak or no inhibitory activity was obtained against the bifidobacteria or Lactobacillus acidophilus. The inhibitory effect was much more pronounced in C. perfringens and E. coli as compared to B. adolescentis, B. bifidum, B. fragilis, B. longum, or L. acidophilus. Cinnamaldehyde exhibited a strong growth-inhibiting activity, but no inhibition was observed from treatments with trans-cinnamic acid, cinnamyl alcohol, 2-methoxycinnamic acid, 3-methoxycinnamic acid, and 4-methoxycinnamic acid. These results may be an indication of at least one of the pharmacological actions of P. cernua root.

  3. Synthesis and structure-activity relationship of Huprine derivatives as human acetylcholinesterase inhibitors.

    Science.gov (United States)

    Ronco, Cyril; Sorin, Geoffroy; Nachon, Florian; Foucault, Richard; Jean, Ludovic; Romieu, Anthony; Renard, Pierre-Yves

    2009-07-01

    New series of Huprine (12-amino-6,7,10,11-tetrahydro-7,11-methanocycloocta[b]quinolines) derivatives have been synthesized and their inhibiting activities toward recombinant human acetylcholinesterase (rh-AChE) are reported. We have synthesized two series of Huprine analogues; in the first one, the benzene ring of the quinoline moiety has been replaced by different heterocycles or electron-withdrawing or electron-donating substituted phenyl group. The second one has been designed in order to evaluate the influence of modification at position 12 where different short linkers have been introduced on the Huprine X, Y skeletons. All these molecules have been prepared from ethyl- or methyl-bicyclo[3.3.1]non-6-en-3-one via Friedländer reaction involving selected o-aminocyano aromatic compounds. The synthesis of two heterodimers based on these Huprines has been also reported. Activities from moderate to same range than the most active Huprines X and Y taken as references have been obtained, the most potent analogue being about three times less active than parent Huprines X and Y. Topologic data have been inferred from molecular dockings and variations of activity between the different linkers suggest future structural modifications for activity improvement.

  4. 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).

  5. Design, synthesis, X-ray crystallographic analysis, and biological evaluation of thiazole derivatives as potent and selective inhibitors of human dihydroorotate dehydrogenase.

    Science.gov (United States)

    Zhu, Junsheng; Han, Le; Diao, Yanyan; Ren, Xiaoli; Xu, Minghao; Xu, Liuxin; Li, Shiliang; Li, Qiang; Dong, Dong; Huang, Jin; Liu, Xiaofeng; Zhao, Zhenjiang; Wang, Rui; Zhu, Lili; Xu, Yufang; Qian, Xuhong; Li, Honglin

    2015-02-12

    Human dihydroorotate dehydrogenase (HsDHODH) is a flavin-dependent mitochondrial enzyme that has been certified as a potential therapeutic target for the treatment of rheumatoid arthritis and other autoimmune diseases. On the basis of lead compound 4, which was previously identified as potential HsDHODH inhibitor, a novel series of thiazole derivatives were designed and synthesized. The X-ray complex structures of the promising analogues 12 and 33 confirmed that these inhibitors bind at the putative ubiquinone binding tunnel and guided us to explore more potent inhibitors, such as compounds 44, 46, and 47 which showed double digit nanomolar activities of 26, 18, and 29 nM, respectively. Moreover, 44 presented considerable anti-inflammation effect in vivo and significantly alleviated foot swelling in a dose-dependent manner, which disclosed that thiazole-scaffold analogues can be developed into the drug candidates for the treatment of rheumatoid arthritis by suppressing the bioactivity of HsDHODH.

  6. 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,

  7. Development of a human dihydroorotate dehydrogenase (hDHODH pharma-similarity index approach with scaffold-hopping strategy for the design of novel potential inhibitors.

    Directory of Open Access Journals (Sweden)

    Kuei-Chung Shih

    Full Text Available Human dihydroorotate dehydrogenase (hDHODH is a class-2 dihydroorotate dehydrogenase. Because it is extensively used by proliferating cells, its inhibition in autoimmune and inflammatory diseases, cancers, and multiple sclerosis is of substantial clinical importance. In this study, we had two aims. The first was to develop an hDHODH pharma-similarity index approach (PhSIA using integrated molecular dynamics calculations, pharmacophore hypothesis, and comparative molecular similarity index analysis (CoMSIA contour information techniques. The approach, for the discovery and design of novel inhibitors, was based on 25 diverse known hDHODH inhibitors. Three statistical methods were used to verify the performance of hDHODH PhSIA. Fischer's cross-validation test provided a 98% confidence level and the goodness of hit (GH test score was 0.61. The q(2, r(2, and predictive r(2 values were 0.55, 0.97, and 0.92, respectively, for a partial least squares validation method. In our approach, each diverse inhibitor structure could easily be aligned with contour information, and common substructures were unnecessary. For our second aim, we used the proposed approach to design 13 novel hDHODH inhibitors using a scaffold-hopping strategy. Chemical features of the approach were divided into two groups, and the Vitas-M Laboratory fragment was used to create de novo inhibitors. This approach provides a useful tool for the discovery and design of potential inhibitors of hDHODH, and does not require docking analysis; thus, our method can assist medicinal chemists in their efforts to identify novel inhibitors.

  8. Development of a human dihydroorotate dehydrogenase (hDHODH) pharma-similarity index approach with scaffold-hopping strategy for the design of novel potential inhibitors.

    Science.gov (United States)

    Shih, Kuei-Chung; Lee, Chi-Ching; Tsai, Chi-Neu; Lin, Yu-Shan; Tang, Chuan-Yi

    2014-01-01

    Human dihydroorotate dehydrogenase (hDHODH) is a class-2 dihydroorotate dehydrogenase. Because it is extensively used by proliferating cells, its inhibition in autoimmune and inflammatory diseases, cancers, and multiple sclerosis is of substantial clinical importance. In this study, we had two aims. The first was to develop an hDHODH pharma-similarity index approach (PhSIA) using integrated molecular dynamics calculations, pharmacophore hypothesis, and comparative molecular similarity index analysis (CoMSIA) contour information techniques. The approach, for the discovery and design of novel inhibitors, was based on 25 diverse known hDHODH inhibitors. Three statistical methods were used to verify the performance of hDHODH PhSIA. Fischer's cross-validation test provided a 98% confidence level and the goodness of hit (GH) test score was 0.61. The q(2), r(2), and predictive r(2) values were 0.55, 0.97, and 0.92, respectively, for a partial least squares validation method. In our approach, each diverse inhibitor structure could easily be aligned with contour information, and common substructures were unnecessary. For our second aim, we used the proposed approach to design 13 novel hDHODH inhibitors using a scaffold-hopping strategy. Chemical features of the approach were divided into two groups, and the Vitas-M Laboratory fragment was used to create de novo inhibitors. This approach provides a useful tool for the discovery and design of potential inhibitors of hDHODH, and does not require docking analysis; thus, our method can assist medicinal chemists in their efforts to identify novel inhibitors.

  9. 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.

  10. [Therapeutic effect of flavopiridol, a small molecular cyclin-dependent kinase inhibitor, in human ovarian carcinoma].

    Science.gov (United States)

    Song, Yue; Shen, Keng; Tang, Ping-ping

    2007-11-01

    To investigate the antitumor effect of flavopiridol in ovarian cancer. After the treatment with flavopiridol of AO cells, cell apoptotic rate and cell cycle distribution were detected by flow cytometer and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL). Real time PCR was used to detect the expression of cyclin D and active caspase-3 in AO cells. Subcutaneous tumor models and abdominally spread tumor models of human ovarian carcinoma using AO cells in BALB/c nude mice were established. The mouse survival rates were measured for abdominally spread tumor models and the volume of tumor nodules was determined for subcutaneous tumor models following the treatments of flavopiridol. TUNEL was used to detect cell apoptosis, and immunohistochemistry was used to measure microvessel density (MVD) in tumor tissues. AO cells showed apoptotic rates of 4.1%, 10.7% and 7.6% following the treatments with flavopiridol at 150, 300 and 500 nmol/L respectively, accompanied by an increase in G(1) progression and a decrease in S phase progression. The level of active caspase-3 increased (2.55 vs 2.49) and the level of cyclin D expression decreased significantly (0.25 vs 0.69, P flavopiridol. Flavopiridol prolonged mouse survival [mean survival time of (141 +/- 14) days] and suppressed tumor growth significantly (tumor growth suppression rate of 40%), when compared with treatment using phosphate-buffered saline [(106 +/- 11) days, P flavopiridol. MVD of tumor tissue was 12 +/- 5 following flavopiridol treatment, significantly higher than that of 35 +/- 10 treated with phosphate-buffered saline (P Flavopiridol results in significant suppression of ovarian carcinoma cell growth and prolongs survival of mice.

  11. 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.

  12. Jak Inhibitors Modulate Production of Replication Competent Zika Virus in Human Hofbauer, Trophoblasts, and Neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    Christina Gavegnano

    2017-05-01

    Full Text Available Zika Virus (ZIKV is a Flavivirus that has been implicated in brain deformations, birth defects, and microcephaly of unborn fetuses and associated with Guillain-Barre syndrome.  Mechanisms responsible for transmission of ZIKV across the placenta to the fetus are incompletely understood.  Herein, we define key events modulating infection in clinically relevant cells, including primary placental macrophages (human hofbauer cells; HC, trophoblasts, and neuroblastoma cells. Consistent with previous findings, HC and trophoblasts are permissive to ZIKV infection. Decrease of interferon signaling by Jak 1/2 inhibition (via ruxolitinib significantly increased ZIKV replicationin HC, trophoblasts, and neuroblasts. Enhanced ZIKV production in ruxolitinib treated HC was associated with increased expression of HLA-DR and DC-SIGN. Nucleoside analogs blocked ruxolitinib-mediated production of extracellular virus. Although low-level ZIKV infection occurred in untreated HC and trophoblasts, the produced virus was incapable of infecting naïve Vero cells.  These deficient virions from untreated HC present “thin-coats” suggesting immature virion structure. Blocking Jak 1/2 signaling (with ruxolitinib restored replication competence as virions produced under these conditions confer CPE in naïve Vero cells.  These data demonstrate that Jak-STAT signaling directly impacts the ability of primary placental cells to produce replication competent virus and is a key gatekeeper in production of mature virions in clinically relevant cells including HC and trophoblasts. Design of targeted agents to prevent ZIKV replication in the placenta should consider Jak 1/2 signaling and the impact of its block on ZIKV infection and subsequent transmission to the fetus.

  13. Identification of human telomerase inhibitors having the core of N-acyl-4,5-dihydropyrazole with anticancer effects.

    Science.gov (United States)

    Xiao, Xuan; Ni, Yong; Jia, Ying-Ming; Zheng, Min; Xu, Han-Fei; Xu, Jun; Liao, Chenzhong

    2016-03-15

    Eight human telomerase inhibitors (5a-5h) having the core of N-acyl-4,5-dihydropyrazole with anticancer effects were identified in this study. Biological results revealed that a few compounds had potent anticancer activities against three common tumor cell lines (SGC-7901, HepG2 and MGC-803). Among them, compound 5c, with a molecular weight of only 272.2 Da, had antiproliferative activities against SGC-7901 and MGC-803 with EC50 values of 2.06 ± 0.17 and 2.89 ± 0.62 μM, respectively, better than 5-Fluorouracil. Compound 5c inhibited the enzyme of telomerase with an IC50 value of 1.86 ± 0.51 μM, surpassing the control compound, ethidium bromide. Modeling study showed that this compound can reside in the binding pocket of the telomerase/TNA:DNA hairpin complex. When the moiety of N-acyl was changed to N-sulfonyl, the gotten compounds (8a-8i) had deteriorative activities against both these three cancer cell lines and the enzyme of telomerase. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. L-glutamine Schiff base copper complex as a proteasome inhibitor and an apoptosis inducer in human cancer cells.

    Science.gov (United States)

    Xiao, Yan; Bi, Caifeng; Fan, Yuhua; Cui, Cindy; Zhang, Xia; Dou, Q Ping

    2008-11-01

    Interest in the use of metallic compounds for cancer treatment has been increasing since the discovery of cisplatin. Clinical studies suggest the use of proteasome inhibitors as potential novel anticancer agents. L-glutamine is the most abundant free amino acid in the body, and has been shown to play a regulatory role in several cellular processes, including metabolism, degradation, redox potential and cellular integrity. Although glutamine is reported to play a role in the regulation of apoptosis, the effect of glutamine copper complex on tumor cells and the involved molecular mechanism have not been investigated. Here, for the first time, we report that a newly synthesized L-glutamine-containing copper complex has proteasome-inhibitory activity in human breast cancer and leukemia cells. The inhibition of the tumor proteasomal activity results in the accumulation of ubiquitinated proteins and ubiquitinated form of IkappaB-alpha, a natural proteasome substrate, followed by induction of apoptosis. Furthermore, this glutamine Schiff base copper complex selectively inhibits the proteasomal activity and induces cell death in cultured breast cancer cells, but not normal, immortalized breast cells. Our data suggest that glutamine Schiff base copper complexes have a potential use for to be used in cancer treatment and prevention.

  15. 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.

  16. A first-in-human study of AMG 208, an oral MET inhibitor, in adult patients with advanced solid tumors.

    Science.gov (United States)

    Hong, David S; Rosen, Peter; Lockhart, A Craig; Fu, Siqing; Janku, Filip; Kurzrock, Razelle; Khan, Rabia; Amore, Benny; Caudillo, Isaac; Deng, Hongjie; Hwang, Yuying C; Loberg, Robert; Ngarmchamnanrith, Gataree; Beaupre, Darrin M; Lee, Peter

    2015-07-30

    This first-in-human study evaluated AMG 208, a small-molecule MET inhibitor, in patients with advanced solid tumors. Three to nine patients were enrolled into one of seven AMG 208 dose cohorts (25, 50, 100, 150, 200, 300, and 400 mg). Patients received AMG 208 orally on days 1 and days 4-28 once daily. The primary objectives were to evaluate the safety, tolerability, pharmacokinetics, and maximum tolerated dose (MTD) of AMG 208. Fifty-four patients were enrolled. Six dose-limiting toxicities were observed: grade 3 increased aspartate aminotransferase (200 mg), grade 3 thrombocytopenia (200 mg), grade 4 acute myocardial infarction (300 mg), grade 3 prolonged QT (300 mg), and two cases of grade 3 hypertension (400 mg). The MTD was not reached. The most frequent grade ≥3 treatment-related adverse event was anemia (n = 3) followed by hypertension, prolonged QT, and thrombocytopenia (two patients each). AMG 208 exposure increased linearly with dose; mean plasma half-life estimates were 21.4-68.7 hours. One complete response (prostate cancer) and three partial responses (two in prostate cancer, one in kidney cancer) were observed. In this study, AMG 208 had manageable toxicities and showed evidence of antitumor activity, particularly in prostate cancer.

  17. 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.

  18. 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.

  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. Retinoic acids and trichostatin A (TSA), a histone deacetylase inhibitor, induce human pyruvate dehydrogenase kinase 4 (PDK4) gene expression.

    Science.gov (United States)

    Kwon, Hye-Sook; Huang, Boli; Ho Jeoung, Nam; Wu, Pengfei; Steussy, Calvin N; Harris, Robert A

    2006-01-01

    Induction of pyruvate dehydrogenase kinase 4 (PDK4) conserves glucose and substrates for gluconeogenesis and thereby helps regulate blood glucose levels during starvation. We report here that retinoic acids (RA) as well as Trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), regulate PDK4 gene expression. Two retinoic acid response elements (RAREs) to which retinoid X receptor alpha (RXRalpha) and retinoic acid receptor alpha (RARalpha) bind and activate transcription are present in the human PDK4 (hPDK4) proximal promoter. Sp1 and CCAAT box binding factor (CBF) bind to the region between two RAREs. Mutation of either the Sp1 or the CBF site significantly decreases basal expression, transactivation by RXRalpha/RARalpha/RA, and the ability of TSA to stimulate hPDK4 gene transcription. By the chromatin immunoprecipitation assay, RA and TSA increase acetylation of histones bound to the proximal promoter as well as occupancy of CBP and Sp1. Interaction of p300/CBP with E1A completely prevented hPDK4 gene activation by RXRalpha/RARalpha/RA and TSA. The p300/CBP may enhance acetylation of histones bound to the hPDK4 promoter and cooperate with Sp1 and CBF to stimulate transcription of the hPDK4 gene in response to RA and TSA.

  1. Angiotensin-converting enzyme inhibitors attenuate propofol-induced pro-oxidative and antifibrinolytic effect in human endothelial cells.

    Science.gov (United States)

    Wojewodzka-Zelezniakowicz, Marzena; Gromotowicz-Poplawska, Anna; Kisiel, Wioleta; Konarzewska, Emilia; Szemraj, Janusz; Ladny, Jerzy Robert; Chabielska, Ewa

    2017-01-01

    The aim of this study was to investigate the effects of plasma and tissue angiotensin-converting enzyme inhibitors (ACE-Is) against propofol-induced endothelial dysfunction and to elucidate the involved mechanisms in vitro. We examined the effects of propofol (50 μM), quinaprilat and enalaprilat (10-5 M) on fibrinolysis (t-PA, PAI-1, TAFI antigen levels), oxidative stress parameters (H2O2 and MDA antigen levels and SOD and NADPH oxidase mRNA levels) and nitric oxide bioavailability (NO2/NO3 concentration and NOS expression at the level of mRNA) in human umbilical vein endothelial cells (HUVECs). We found that both ACE-Is promoted similar endothelial fibrinolytic properties and decreased oxidative stress in vitro. Propofol alone increased the release of antifibrinolytic and pro-oxidative factors from the endothelium and increased mRNA iNOS expression. We also found that the incubation of HUVECs in the presence of propofol following ACE-Is pre-incubation caused weakness of the antifibrinolytic and pro-oxidative potential of propofol and this effect was similar after both ACE-Is. This observation suggests that the studied ACE-Is exerted protective effects against endothelial cell dysfunction caused by propofol, independently of hemodynamics.

  2. 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.

  3. A direct thrombin inhibitor suppresses protein C activation and factor Va degradation in human plasma: Possible mechanisms of paradoxical enhancement of thrombin generation.

    Science.gov (United States)

    Kamisato, Chikako; Furugohri, Taketoshi; Morishima, Yoshiyuki

    2016-05-01

    We have demonstrated that antithrombin (AT)-independent thrombin inhibitors paradoxically increase thrombin generation (TG) in human plasma in a thrombomodulin (TM)- and protein C (PC)-dependent manner. We determined the effects of AT-independent thrombin inhibitors on the negative-feedback system, activation of PC and production and degradation of factor Va (FVa), as possible mechanisms underlying the paradoxical enhancement of TG. TG in human plasma containing 10nM TM was assayed by means of the calibrated automated thrombography. As an index of PC activation, plasma concentration of activated PC-PC inhibitor complex (aPC-PCI) was measured. The amounts of FVa heavy chain and its degradation product (FVa(307-506)) were examined by western blotting. AT-independent thrombin inhibitors, melagatran and dabigatran (both at 25-600nM) and 3-30μg/ml active site-blocked thrombin (IIai), increased peak levels of TG. Melagatran, dabigatran and IIai significantly decreased plasma concentration of aPC-PCI complex at 25nM or more, 75nM or more, and 10 and 30μg/ml, respectively. Melagatran (300nM) significantly increased FVa and decreased FVa(307-506). In contrast, a direct factor Xa inhibitor edoxaban preferentially inhibited thrombin generation (≥25nM), and higher concentrations were required to inhibit PC activation (≥150nM) and FVa degradation (300nM). The present study suggests that the inhibitions of protein C activation and subsequent degradation of FVa and increase in FVa by antithrombin-independent thrombin inhibitors may contribute to the paradoxical TG enhancement, and edoxaban may inhibit PC activation and FVa degradation as a result of TG suppression. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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    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.

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

    Science.gov (United States)

    Olesen, Sanne H.; Ingles, Donna J.; Zhu, Jin-Yi; Martin, Mathew P.; Betzi, Stephane; Georg, Gunda I.; Tash, Joseph S.; Schönbrunn, Ernst

    2015-01-01

    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. PMID:25608045

  6. 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...

  7. Stability of the human Hsp90-p50Cdc37 chaperone complex against nucleotides and Hsp90 inhibitors, and the influence of phosphorylation by casein kinase 2.

    Science.gov (United States)

    Olesen, Sanne H; Ingles, Donna J; Zhu, Jin-Yi; Martin, Mathew P; Betzi, Stephane; Georg, Gunda I; Tash, Joseph S; Schönbrunn, Ernst

    2015-01-19

    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.

  8. 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.

  9. 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.

  10. 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.

  11. The orally active renin inhibitor A-74273. In vivo and in vitro morpholine ring metabolism in rats, dogs, and humans.

    Science.gov (United States)

    Denissen, J F; Grabowski, B A; Johnson, M K; Boyd, S A; Uchic, J T; Stein, H; Cepa, S; Hill, P

    1994-01-01

    The metabolism and disposition of [14C]A-74273--a potent, orally active renin inhibitor--were investigated in beagle dogs and Sprague-Dawley rats. Two male and two female dogs received a single 10 mg/kg oral or 1 mg/kg intravenous dose in a cross-over experiment and urine and feces were collected for 5 days. After both intravenous and oral dosing, > 92% of the dose was recovered in the feces and < 3% was recovered in the urine. The predominance of hepatobiliary elimination in the clearance of A-74273 was verified in a bile-exteriorized dog, where 79.8% of a 1 mg/kg intravenous dose was excreted in the bile within 6 hr after administration. Similarly, administration of a 1 mg/kg intravenous dose to a bile-exteriorized rat resulted in biliary excretion of 60.5% of the dose within 6 hr. Radio-HPLC analysis of bile and feces from both species indicated extensive metabolism of A-74273 to three major morpholine ring-opened metabolites; the ethanolamine A-78242, the amine A-78030, and the carboxylic acid A-81307. These three metabolites each contributed 12.0-20.2% of the biliary radioactivity after intravenous dosing, while unchanged A-74273 contributed 5-17%. Incubation of [14C]A-74273 with rat, dog, and human liver microsomes afforded nearly equal amounts of the three in vivo metabolites for all three species, suggesting that the in vitro system was representative of A-74273 in vivo metabolism and that humans should also convert A-74273 to the morpholine ring-opened metabolites in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. 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

  13. 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.

  14. Expression of serine/threonine protein-kinases and related factors in normal monkey and human retinas: the mechanistic understanding of a CDK2 inhibitor induced retinal toxicity.

    Science.gov (United States)

    Saturno, Grazia; Pesenti, Manuela; Cavazzoli, Cristiano; Rossi, Anna; Giusti, Anna M; Gierke, Berthold; Pawlak, Michael; Venturi, Miro

    2007-12-01

    Protein-kinase inhibitors are among the most advanced compounds in development using the new drug discovery paradigm of developing small-molecule drugs against specific molecular targets in cancer. After treatment with a cyclin dependent kinase CDK2 inhibitor in monkey, histopathological analysis of the eye showed specific cellular damage in the photoreceptor layer. Since this CDK2 inhibitor showed activity also on other CDKs, in order to investigate the mechanism of toxicity of this compound, we isolated cones and rods from the retina of normal monkey and humans by Laser Capture Microdissection. Using Real-Time PCR we first measured the expression of cyclin dependent protein-kinases (CDK)1, 2, 4, 5, Glycogen synthase kinase 3beta (GSK3beta) and microtubule associated protein TAU. We additionally verified the presence of these proteins in monkey eye sections by immuno-histochemistry and immunofluorescence analysis and afterwards quantified GSK3beta, phospho-GSK3beta and TAU by Reverse Phase Protein Microarrays. With this work we demonstrate how complementary gene expression and protein-based technologies constitute a powerful tool for the understanding of the molecular mechanism of a CDK2 inhibitor induced toxicity. Moreover, this investigative approach is helpful to better understand and characterize the mechanism of species-specific toxicities and further support a rational, molecular mechanism-based safety assessment in humans.

  15. Improvement of Cell Permeability of Human Neuronal Nitric Oxide Synthase Inhibitors Using Potent and Selective 2-Aminopyridine-Based Scaffolds with a Fluorobenzene Linker.

    Science.gov (United States)

    Do, Ha T; Wang, Heng-Yen; Li, Huiying; Chreifi, Georges; Poulos, Thomas L; Silverman, Richard B

    2017-11-22

    Inhibition of neuronal nitric oxide synthase (nNOS) is a promising therapeutic approach to treat neurodegenerative diseases. Recently, we have achieved considerable progress in improving the potency and isoform selectivity of human nNOS inhibitors bearing a 2-aminopyridine scaffold. However, these inhibitors still suffered from too low cell membrane permeability to enter into CNS drug development. We report herein our studies to improve permeability of nNOS inhibitors as measured by both PAMPA-BBB and Caco-2 assays. The most permeable compound (12) in this study still preserves excellent potency with human nNOS (Ki = 30 nM) and very high selectivity over other NOS isoforms, especially human eNOS (hnNOS/heNOS = 2799, the highest hnNOS/heNOS ratio we have obtained to date). X-ray crystallographic analysis reveals that 12 adopts a similar binding mode in both rat and human nNOS, in which the 2-aminopyridine and the fluorobenzene linker form crucial hydrogen bonds with glutamate and tyrosine residues, respectively.

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

    Energy Technology Data Exchange (ETDEWEB)

    Santiago, Francisco [Division of Hematology-Oncology, Department of Medicine, Weill Cornell Medical College, New York, NY (United States); Oguma, Junya; Brown, Anthony M.C. [Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, NY (United States); Laurence, Jeffrey, E-mail: jlaurenc@med.cornell.edu [Division of Hematology-Oncology, Department of Medicine, Weill Cornell Medical College, New York, NY (United States)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer First demonstration of direct role for noncanonical Wnt in osteoclast differentiation. Black-Right-Pointing-Pointer Demonstration of Ryk as a Wnt5a/b receptor in inhibition of canonical Wnt signaling. Black-Right-Pointing-Pointer Modulation of noncanonical Wnt signaling by a clinically important drug, ritonavir. Black-Right-Pointing-Pointer Establishes a mechanism for an important clinical problem: HIV-associated bone loss. -- Abstract: Wnt proteins that signal via the canonical Wnt/{beta}-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 {beta}-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, {beta}-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

  17. [Adriamycin and mitomycin dose-dependently downregulate X-kinked inhibitor of apoptosis protein in human bladder cancer cells].

    Science.gov (United States)

    Yin, Zhen-fei; Li, Ming; Lai, Yong-qing; Yuan, Yi-ming; Liu, Gang; Chen, Liang; Gan, Lin; Na, Yan-qun

    2006-07-04

    To investigate the efficacy of inducing apoptosis in human bladder cancer cells by adriamycin and mitomycin and relevant mechanism. Human bladder cancer cells of the lines RT4, MGH-U1, FJ, and T24 were cultured. Adriamycin of the concentrations of 0.1, 1, and 10 microg/ml, and mitomycin of the concentrations of 0.01, 0.1, and 1 microg/ml were added into the culture fluid respectively. CCK-8 colorimetric assay was used to detect the survival rates of the cells so as to select the cell line sensitive and tolerable to the drugs. Flow cytometry was used to detect the cell apoptosis. Western blotting was used to detect the levels of X-kinked inhibitor of apoptosis protein (XIAP) and the cleavage of caspase-3 precursor. It was found that RT4 cells were the most sensitive and the T24 cells were the most tolerable to adriamycin and mitomycin. Treated with adriamycin of the concentrations of 0.1, 1, and 10 microg/ml for 24 hours, the apoptotic rates of the RT4 cells were 15.3% +/- 4.3%, 29.3% +/- 6.4%, and 45.0% +/- 5.5% respectively; and the apoptotic rates of the T24 cells were 7.3% +/- 3.1%, 12.5% +/- 4.3%, and 18.2% +/- 6.3% respectively, all significantly lower than those of the RT4 cells (P RT4 cells were 12.7% +/- 2.9%, 31.3% +/- 4.4%, and 48.9% +/- 5.8% respectively, and the apoptotic rates of the RT4 cells were 7.2% +/- 3.3%, 15.5% +/- 5.2%, and 21.2% +/- 4.4% respectively, all significantly lower than those of the RT4 cells (all P cell lines before the adriamycin and mitomycin treatment. After the treatment of adriamycin and mitomycin, the expression of XIAP was down-regulated dose-dependently, however, being weaker in the T24 cells than in the RT4 cells; and caspase-3 precursor cleavage was enhanced, however, being weaker in the T24 cells too. Adriamycin and mitomycin dose-dependently kill the human bladder cancer cells. Such cytotoxic effect may be realized through inducing the cell apoptosis which is associated with the down-regulation of XIAP and cleavage of

  18. Exploring the interaction between human focal adhesion kinase and inhibitors: a molecular dynamic simulation and free energy calculations.

    Science.gov (United States)

    Zhan, Jiu-Yu; Zhang, Ji-Long; Wang, Yan; Li, Ye; Zhang, Hong-Xing; Zheng, Qing-Chuan

    2016-11-01

    Focal adhesion kinase is an important target for the treatment of many kinds of cancers. Inhibitors of FAK are proposed to be the anticancer agents for multiple tumors. The interaction characteristic between FAK and its inhibitors is crucial to develop new inhibitors. In the present article, we used Molecular Dynamic (MD) simulation method to explore the characteristic of interaction between FAK and three inhibitors (PHM16, TAE226, and ligand3). The MD simulation results together with MM-GB/SA calculations show that the combinations are enthalpy-driven process. Cys502 and Asp564 are both essential residues due to the hydrogen bond interactions with inhibitors, which was in good agreement with experimental data. Glu500 can form a non-classical hydrogen bond with each inhibitor. Arg426 can form electrostatic interactions with PHM16 and ligand3, while weaker with TAE226. The electronic static potential was employed, and we found that the ortho-position methoxy of TAE226 has a weaker negative charge than the meta-position one in PHM16 or ligand3. Ile428, Val436, Ala452, Val484, Leu501, Glu505, Glu506, Leu553, Gly563 Leu567, Ser568 are all crucial residues in hydrophobic interactions. The key residues in this work will be available for further inhibitor design of FAK and also give assistance to further research of cancer.

  19. Protective Effects of Recombinant Kunitz-Domain 1 of Human Tissue Factor Pathway Inhibitor-2 Against 2-Chloroethyl Ethyl Sulfide Toxicity In Vitro

    OpenAIRE

    Moonsuk S. Choi; Kalpana Parikh; Ashima Saxena; Nageswararao Chilukuri

    2008-01-01

    Objective: Sulfur mustard is a well-known blistering chemical warfare agent that has been investigated for its toxicological mechanisms and an efficacious antidote. Since sulfur mustard injury involves dermal:epidermal separation, proteolytic enzymes were suspected to be involved for this separation and eventual blister development. Therefore, protease inhibitors could be of therapeutic utility against sulfur mustard injury. In this study, the effects of Kunitz-domain 1 of human tissue factor...

  20. Protective Effects of Recombinant Kunitz-Domain 1 of Human Tissue Factor Pathway Inhibitor-2 Against 2-Chloroethyl Ethyl Sulfide Toxicity In Vitro

    OpenAIRE

    Choi, Moonsuk S.; Parikh, Kalpana; Saxena, Ashima; Chilukuri, Nageswararao

    2007-01-01

    Objective: Sulfur mustard is a well-known blistering chemical warfare agent that has been investigated for its toxicological mechanisms and an efficacious antidote. Since sulfur mustard injury involves dermal:epidermal separation, proteolytic enzymes were suspected to be involved for this separation and eventual blister development. Therefore, protease inhibitors could be of therapeutic utility against sulfur mustard injury. In this study, the effects of Kunitz-domain 1 of human tissue factor...

  1. Efficient substrate screening and inhibitor testing of human CYP4Z1 using permeabilized recombinant fission yeast.

    Science.gov (United States)

    Yan, Qi; Machalz, David; Zöllner, Andy; Sorensen, Erik J; Wolber, Gerhard; Bureik, Matthias

    2017-12-15

    We have established a protocol for the preparation of permeabilized fission yeast cells (enzyme bags) that recombinantly express human cytochrome P450 enzymes (CYPs). A direct comparison of CYP3A4 activity gave an eightfold higher space-time yield for enzyme bag-catalyzed biotransformation as compared to whole-cell biotransformation, even though the total number of cells employed was lower by a factor of 150. Biotransformation of the luminogenic substrate Luciferin-H using CYP2C9-containing enzyme bags proceeded efficiently and stably for 24h. CYP4Z1 is of interest because it is strongly overexpressed both in breast cancer cells and in breast cancer metastases; however, current knowledge about its catalytic properties is very limited. Screening of CYP4Z1-containing enzyme bags with 15 luminogenic substrates enabled us to identify two new hydroxylations and eleven ether cleavage reactions that are catalyzed by CYP4Z1. By far the best substrate found in this study was Luciferin benzyl ether (Luciferin-BE). On the basis of the recently published crystal structure of CYP4B1 we created a new homology model of CYP4Z1 and performed molecular docking experiments, which indicate that all active substrates show a highly similar binding geometry compared to the endogenous substrates. The model predicts that Ser113, Ser222, Asn381, and Ser383 are key hydrogen bonding residues. We also identified five new inhibitors of CYP4Z1: miconazole, econazole, aminobenzotriazole, tolazoline, and 1-benzylimidazole respectively, with the last compound being the most potent giving an IC50 value of 180nM in our test system. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. 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.

  3. A retrograde trafficking inhibitor of ricin and Shiga-like toxins inhibits infection of cells by human and monkey polyomaviruses.

    Science.gov (United States)

    Nelson, Christian D S; Carney, Dan W; Derdowski, Aaron; Lipovsky, Alex; Gee, Gretchen V; O'Hara, Bethany; Williard, Paul; DiMaio, Daniel; Sello, Jason K; Atwood, Walter J

    2013-11-12

    Polyomaviruses are ubiquitous pathogens that cause severe disease in immunocompromised individuals. JC polyomavirus (JCPyV) is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), whereas BK polyomavirus (BKPyV) causes polyomavirus-induced nephropathy and hemorrhagic cystitis. Vaccines or antiviral therapies targeting these viruses do not exist, and treatments focus on reducing the underlying causes of immunosuppression. We demonstrate that retro-2(cycl), an inhibitor of ricin and Shiga-like toxins (SLTs), inhibits infection by JCPyV, BKPyV, and simian virus 40. Retro-2(cycl) inhibits retrograde transport of polyomaviruses to the endoplasmic reticulum, a step necessary for productive infection. Retro-2(cycl) likely inhibits polyomaviruses in a way similar to its ricin and SLT inhibition, suggesting an overlap in the cellular host factors used by bacterial toxins and polyomaviruses. This work establishes retro-2(cycl) as a potential antiviral therapy that broadly inhibits polyomaviruses and possibly other pathogens that use retrograde trafficking. The human polyomaviruses JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) cause rare but severe diseases in individuals with reduced immune function. During immunosuppression, JCPyV disseminates from the kidney to the central nervous system and destroys oligodendrocytes, resulting in the fatal disease progressive multifocal leukoencephalopathy. Kidney transplant recipients are at increased risk of BKPyV-induced nephropathy, which results in kidney necrosis and loss of the transplanted organ. There are currently no effective therapies for JCPyV and BKPyV. We show that a small molecule named retro-2(cycl) protects cells from infection with JCPyV and BKPyV by inhibiting intracellular viral transport. Retro-2(cycl) treatment reduces viral spreading in already established infections and may therefore be able to control infection in affected patients. Further optimization of

  4. Small-molecule inhibitor which reactivates p53 in human T-cell leukemia virus type 1-transformed cells.

    Science.gov (United States)

    Jung, Kyung-Jin; Dasgupta, Arindam; Huang, Keven; Jeong, Soo-Jin; Pise-Masison, Cynthia; Gurova, Katerina V; Brady, John N

    2008-09-01

    Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of the aggressive and fatal disease adult T-cell leukemia. Previous studies have demonstrated that the HTLV-1-encoded Tax protein inhibits the function of tumor suppressor p53 through a Tax-induced NF-kappaB pathway. Given these attributes, we were interested in the activity of small-molecule inhibitor 9-aminoacridine (9AA), an anticancer drug that targets two important stress response pathways, NF-kappaB and p53. In the present study, we have examined the effects of 9AA on HTLV-1-transformed cells. Treatment of HTLV-1-transformed cells with 9AA resulted in a dramatic decrease in cell viability. Consistent with these results, we observed an increase in the percentage of cells in sub-G(1) and an increase in the number of cells positive by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay following treatment of HTLV-1-transformed cells with 9AA. In each assay, HTLV-1-transformed cells C8166, Hut102, and MT2 were more sensitive to treatment with 9AA than control CEM and peripheral blood mononuclear cells. Analyzing p53 function, we demonstrate that treatment of HTLV-1-transformed cells with 9AA resulted in an increase in p53 protein and activation of p53 transcription activity. Of significance, 9AA-induced cell death could be blocked by introduction of a p53 small interfering RNA, linking p53 activity and cell death. These results suggest that Tax-repressed p53 function in HTLV-1-transformed cells is "druggable" and can be restored by treatment with 9AA. The fact that 9AA induces p53 and inhibits NF-kappaB suggests a promising strategy for the treatment of HTLV-1-transformed cells.

  5. 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.

  6. The effect of antenatal depression and selective serotonin reuptake inhibitor treatment on nerve growth factor signaling in human placenta

    NARCIS (Netherlands)

    Kaihola, Helena; Olivier, Jocelien; Poromaa, Inger Sundström; Åkerud, Helena

    2015-01-01

    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

  7. Identification of novel human dipeptidyl peptidase-IV inhibitors of natural origin (Part II): in silico prediction in antidiabetic extracts.

    Science.gov (United States)

    Guasch, Laura; Sala, Esther; Ojeda, María José; Valls, Cristina; Bladé, Cinta; Mulero, Miquel; Blay, Mayte; Ardévol, Anna; Garcia-Vallvé, Santiago; Pujadas, Gerard

    2012-01-01

    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. 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. 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.

  8. 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.

  9. Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase

    Energy Technology Data Exchange (ETDEWEB)

    Filgueira de Azevedo, W. Jr.; Mueller-Dieckmann, H.J.; Schulze-Gahmen, U. [Lawrence Berkeley National Lab., CA (United States)] [and others

    1996-04-02

    The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for studying inhibitory molecules that can modify the degree of cell proliferation. The discovery of specific inhibitors of CDKs such as polyhydroxylated flavones has opened the way to investigation and design of antimitotic compounds. A novel flavone, (-)-cis-5,7-dihydroxyphenyl-8-[4-(3-hydroxy-1-methyl)piperidinyl]-4H-1-benzopyran-4-one hydrochloride hemihydrate (L868276), is a potent inhibitor of CDKs. A chlorinated form, flavopiridol, is currently in phase I clinical trials as a drug against breast tumors. We determined the crystal structure of a complex between CDK2 and L868276 at 2.33-{Angstrom} resolution and refined to an R{sub factor} of 20.3%. The aromatic portion of the inhibitor binds to the adenine-binding pocket of CDK2, and the position of the phenyl group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP complex structure. The analysis of the position of this phenyl ring not only explains the great differences of kinase inhibition among the flavonoid inhibitors but also explains the specificity of L868276 to inhibit CDK2 and CDC2. 36 refs., 4 figs., 2 tabs.

  10. Disruption of the EGFR E884-R958 ion pair conserved in the human kinome differentially alters signaling and inhibitor sensitivity.

    Science.gov (United States)

    Tang, Z; Jiang, S; Du, R; Petri, E T; El-Telbany, A; Chan, P S O; Kijima, T; Dietrich, S; Matsui, K; Kobayashi, M; Sasada, S; Okamoto, N; Suzuki, H; Kawahara, K; Iwasaki, T; Nakagawa, K; Kawase, I; Christensen, J G; Hirashima, T; Halmos, B; Salgia, R; Boggon, T J; Kern, J A; Ma, P C

    2009-01-29

    Targeted therapy against epidermal growth factor receptor (EGFR) represents a major therapeutic advance in lung cancer treatment. Somatic mutations of the EGFR gene, most commonly L858R (exon 21) and short in-frame exon 19 deletions, have been found to confer enhanced sensitivity toward the inhibitors gefitinib and erlotinib. We have recently identified an EGFR mutation E884K, in combination with L858R, in a patient with advanced lung cancer who progressed on erlotinib maintenance therapy, and subsequently had leptomeningeal metastases that responded to gefitinib. The somatic E884K substitution appears to be relatively infrequent and resulted in a mutant lysine residue that disrupts an ion pair with residue R958 in the EGFR kinase domain C-lobe, an interaction that is highly conserved within the human kinome as demonstrated by our sequence analysis and structure analysis. Our studies here, using COS-7 transfection model system, show that E884K works in concert with L858R in-cis, in a dominant manner, to change downstream signaling, differentially induce Mitogen-activated protein kinase (extracellular signaling-regulated kinase 1/2) signaling and associated cell proliferation and differentially alter sensitivity of EGFR phosphorylation inhibition by ERBB family inhibitors in an inhibitor-specific manner. Mutations of the conserved ion pair E884-R958 may result in conformational changes that alter kinase substrate recognition. The analogous E1271K-MET mutation conferred differential sensitivity toward preclinical MET inhibitors SU11274 (unchanged) and PHA665752 (more sensitive). Systematic bioinformatics analysis of the mutation catalog in the human kinome revealed the presence of cancer-associated mutations involving the conserved E884 homologous residue, and adjacent residues at the ion pair, in known proto-oncogenes (KIT, RET, MET and FAK) and tumor-suppressor gene (LKB1). Targeted therapy using small-molecule inhibitors should take into account potential

  11. 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.

  12. Kinetic factors control efficiencies of cell entry, efficacies of entry inhibitors, and mechanisms of adaptation of human immunodeficiency virus.

    Science.gov (United States)

    Platt, Emily J; Durnin, James P; Kabat, David

    2005-04-01

    Replication of human immunodeficiency virus type 1 (HIV-1) in diverse conditions limiting for viral entry into cells frequently leads to adaptive mutations in the V3 loop of the gp120 envelope glycoprotein. This has suggested that the V3 loop limits the efficiencies of HIV-1 infections, possibly by directly affecting gp120-coreceptor affinities. In contrast, V3 loop mutations that enable HIV-1(JR-CSF) to use the low-affinity mutant coreceptor CCR5(Y14N) are shown here to have negligible effects on the virus-coreceptor affinity but to dramatically accelerate the irreversible conformational conversion of the envelope gp41 subunits from a three-stranded coil into a six-helix bundle. This slow step is blocked irreversibly by the inhibitor T-20. To further evaluate the role of entry rates in controlling infection efficiencies and viral adaptations, we developed methods to quantitatively measure viral entry kinetics. The virions were adsorbed by spinoculation at 4 degrees C onto HeLa-CD4/CCR5 cell clones that either had limiting or saturating concentrations of CCR5. After warming to 37 degrees C, the completion of entry was monitored over time by the resistance of infections to the competitive CCR5 inhibitor TAK-779. Our results suggest that the efficiency of entry of cell-attached infectious HIV-1 is principally controlled by three kinetic processes. The first is a lag phase that is caused in part by the concentration-dependent reversible association of virus with CD4 and CCR5 to form an equilibrium assemblage of complexes. Second, this assembly step lowers but does not eliminate a large activation energy barrier for a rate-limiting, CCR5-dependent conformational change in gp41 that is sensitive to blockage by T-20. The rate of infection therefore depends on the fraction of infectious virions that are sufficiently saturated with CCR5 to undergo this conformational change and on the magnitude of the activation energy barrier. Although only a small fraction of fully

  13. Binding and Inactivation Mechanism of a Humanized Fatty Acid Amide Hydrolase by [alpha]-Ketoheterocycle Inhibitors Revealed from Cocrystal Structures

    Energy Technology Data Exchange (ETDEWEB)

    Mileni, Mauro; Garfunkle, Joie; DeMartino, Jessica K.; Cravatt, Benjamin F.; Boger, Dale L.; Stevens, Raymond C.; (Scripps)

    2010-08-17

    The cocrystal X-ray structures of two isomeric {alpha}-ketooxazole inhibitors (1 (OL-135) and 2) bound to fatty acid amide hydrolase (FAAH), a key enzymatic regulator of endocannabinoid signaling, are disclosed. The active site catalytic Ser241 is covalently bound to the inhibitors electrophilic carbonyl groups, providing the first structures of FAAH bound to an inhibitor as a deprotonated hemiketal mimicking the enzymatic tetrahedral intermediate. The work also offers a detailed view of the oxyanion hole and an exceptional 'in-action' depiction of the unusual Ser-Ser-Lys catalytic triad. These structures capture the first picture of inhibitors that span the active site into the cytosolic port providing new insights that help to explain FAAH's interaction with substrate leaving groups and their role in modulating inhibitor potency and selectivity. The role for the activating central heterocycle is clearly defined and distinguished from that observed in prior applications with serine proteases, reconciling the large electronic effect of attached substituents found unique to this class of inhibitors with FAAH. Additional striking active site flexibility is seen upon binding of the inhibitors, providing insights into the existence of a now well-defined membrane access channel with the disappearance of a spatially independent portion of the acyl chain-binding pocket. Finally, comparison of the structures of OL-135 (1) and its isomer 2 indicates that they bind identically to FAAH, albeit with reversed orientations of the central activating heterocycle, revealing that the terminal 2-pyridyl substituent and the acyl chain phenyl group provide key anchoring interactions and confirming the distinguishing role of the activating oxazole.

  14. Uterus-relaxing effect of β2-agonists in combination with phosphodiesterase inhibitors: studies on pregnant rat in vivo and on pregnant human myometrium in vitro.

    Science.gov (United States)

    Verli, Judit; Klukovits, Anna; Kormányos, Zsolt; Hajagos-Tóth, Judit; Ducza, Eszter; Seres, Adrienn B; Falkay, George; Gáspár, Róbert

    2013-01-01

    Our aims were to examine the effects of a simultaneous stimulation of β(2) -adrenergic receptors and inhibition of uterine phosphodiesterases (PDE), in the pregnant rat uterus in vivo and on human uterine tissue in vitro. We also set out to measure cAMP levels and detect the expressions of the isoenzymes PDE4B and PDE4D in human uterine tissue samples. Preterm birth was induced in Sprague-Dawley rats with bacterial lipopolysaccharide. The uterine effects of terbutaline alone or in combination with rolipram were tested in vivo. Human myometrial strips from cesarean sections at full-term pregnancy and at preterm labor were stimulated with oxytocin, and the inhibitory effects of theophylline, rolipram and terbutaline were studied. The myometrial accumulation of cAMP in the presence of rolipram and terbutaline was determined by enzyme immunoassay. The expressions of PDE4B and PDE4D proteins were detected by Western blotting. The selective PDE4 inhibitor rolipram was more effective than the non-selective PDE inhibitor theophylline in inhibiting the oxytocin-induced contractions in the human uterus. The uterus-relaxing effects of low doses of terbutaline were markedly potentiated by rolipram, both in rats and in human tissues. The changes in uterine cAMP levels correlated with these results. At preterm labor, PDE4B was the predominant form of PDE4 expressed; at full term, PDE4D was expressed more strongly. A combination of selective PDE4 inhibitors and β(2) -agonists should be considered for the treatment of preterm contractions. © 2012 The Authors. Journal of Obstetrics and Gynaecology Research © 2012 Japan Society of Obstetrics and Gynecology.

  15. High performance liquid chromatography using UV detection for the simultaneous quantification of the new non-nucleoside reverse transcriptase inhibitor etravirine (TMC-125), and 4 protease inhibitors in human plasma.

    Science.gov (United States)

    Hirano, Atsushi; Takahashi, Masaaki; Kinoshita, Eri; Shibata, Masaaki; Nomura, Toshiharu; Yokomaku, Yoshiyuki; Hamaguchi, Motohiro; Sugiura, Wataru

    2010-01-01

    Etravirine (TMC-125, ETV) is a second-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) that demonstrates potent activity against NNRTI-resistant strains of human immunodeficiency virus type-1 (HIV-1). Thus, ETV has been used in combination with ritonavir-boosted protease inhibitor (PI) and integrase inhibitor for therapy-experienced HIV-1-infected patients. On the other hand, as ETV is a substrate and inducer of cytochrome P450 3A4 (CYP3A4), ETV may induce metabolism of PI and alter the concentrations of co-administered PIs. In order to ensure optimal drug efficacy and prevention of resistance, it is essential to monitor plasma concentrations of ETV and PIs. Here we describe the application of HPLC with UV detection for the simulataneous assay of ETV and 4 PIs, darunavir (DRV), atazanavir (ATV), ritonavir (RTV) and lopinavir (LPV). In this study, the calibration curve of each drug was linear with the average accuracy ranging from 93.6 to 110.9%. Both intra- and interday coefficients of variation for each drug were less than 11.6%. The mean recovery of all drugs ranged from 88.0 to 97.5%. The limit of quantification was 0.04, 0.04, 0.04, 0.05 and 0.07 microg/ml for ETV, DRV, ATV, RTV and LPV, respectively. These results demonstrate that our HPLC-UV method can be used for routine determination of plasma concentrations of ETV and 4 PIs in clinical settings.

  16. Gene expression levels of matrix metalloproteinases in human atherosclerotic plaques and evaluation of radiolabeled inhibitors as imaging agents for plaque vulnerability.

    Science.gov (United States)

    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

    2014-08-01

    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. 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. 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. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Synergistic antineoplastic action of 5-aza-2'deoxycytidine (decitabine in combination with different inhibitors of enhancer of zeste homolog 2 (EZH2 on human lung carcinoma cells

    Directory of Open Access Journals (Sweden)

    Nascimento ASF

    2016-10-01

    Full Text Available Patients with metastatic lung cancer have a very poor prognosis indicating an urgent need to develop more effective chemotherapy. Aberrant promoter DNA methylation can result in the epigenetic silencing of tumor suppressor genes (TSGs in lung cancer. 5-Aza-2’deoxycytidine (5-Aza-CdR, decitabine, an inhibitor of DNA methylation, is able to reactivate silent TSGs. Trimethylation of histone H3 on lysine 27 (H3K27me3 by enhancer of zeste homolog 2 (EZH2 histone methyltransferase can also silence TSGs in lung cancer. 3-Deazaneplanocin-A (DZNep, an inhibitor of EZH2, up-regulates the expression of genes silenced by H3K27me3. In this study we compared the in vitro antineoplastic activity of different inhibitors of EZH2; DZNep, U-4149 and Gsk-126, alone and in combination with 5-Aza-CdR, on the human A549 lung adenocarcinoma cells. U-4149, an analogue of DZNep, was more potent than either DZNep or Gsk-126. The reduction in colony formation was dose- and time-dependent for each EZH2 inhibitors. Combination treatment of 5-Aza-CdR with the EZH2 inhibitors showed a synergistic antineoplastic activity. 5-Aza-CdR and U-4149 was the most potent combination. The in vitro antineoplastic activity of these agents was evaluated by inhibition of growth, colony formation, induction of senescence and apoptosis. All the drug combinations induced signs of senescence and apoptosis. Analysis by gene expression by qRT-PCR showed that the combinations increased the expression of several TSGs to a greater extent that either agent alone. In conclusion, epigenetic therapy that specifically targets DNA and histone methylation has interesting potential for the treatment of lung cancer and merits further investigation.

  18. Critical factors governing the difference in antizyme-binding affinities between human ornithine decarboxylase and antizyme inhibitor.

    Directory of Open Access Journals (Sweden)

    Yen-Chin Liu

    Full Text Available Both ornithine decarboxylase (ODC and its regulatory protein, antizyme inhibitor (AZI, can bind with antizyme (AZ, but the latter has a higher AZ-binding affinity. The results of this study clearly identify the critical amino acid residues governing the difference in AZ-binding affinities between human ODC and AZI. Inhibition experiments using a series of ODC mutants suggested that residues 125 and 140 may be the key residues responsible for the differential AZ-binding affinities. The ODC_N125K/M140K double mutant demonstrated a significant inhibition by AZ, and the IC(50 value of this mutant was 0.08 µM, three-fold smaller than that of ODC_WT. Furthermore, the activity of the AZ-inhibited ODC_N125K/M140K enzyme was hardly rescued by AZI. The dissociation constant (K(d of the [ODC_N125K/M140K]-AZ heterodimer was approximately 0.02 µM, which is smaller than that of WT_ODC by approximately 10-fold and is very close to the K(d value of AZI_WT, suggesting that ODC_N125K/M140K has an AZ-binding affinity higher than that of ODC_WT and similar to that of AZI. The efficiency of the AZI_K125N/K140M double mutant in the rescue of AZ-inhibited ODC enzyme activity was less than that of AZI_WT. The K(d value of [AZI_K125N/K140M]-AZ was 0.18 µM, nine-fold larger than that of AZI_WT and close to the K(d value of ODC_WT, suggesting that AZI_K125N/K140M has an AZ-binding affinity lower than that of AZI_WT and similar to that of ODC. These data support the hypothesis that the differences in residues 125 and 140 in ODC and AZI are responsible for the differential AZ-binding affinities.

  19. 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...

  20. Evaluation of Cytotoxicity Effects of Chalcone Epoxide Analogues as a Selective COX-II Inhibitor in the Human Liver Carcinoma Cell Line

    Directory of Open Access Journals (Sweden)

    Pouran Makhdoumi

    2017-09-01

    Full Text Available Objectives: Study of the mechanisms involved in cancer progression suggests that cyclooxygenase enzymes play an important role in the induction of inflammation, tumor formation, and metastasis of cancer cells. Thus, cyclooxygenase enzymes could be considered for cancer chemotherapy. Among these enzymes, cyclooxygenase 2 (COX-2 is associated with liver carcinogenesis. Various COX-2 inhibitors cause growth inhibition of human hepatocellular carcinoma cells, but many of them act in the COX-2 independent mechanism. Thus, the introduction of selective COX-2 inhibitors is necessary to achieve a clear result. The present study was aimed to determine the growth-inhibitory effects of new analogues of chalcone epoxide as selective COX-2 inhibitors on the human hepatocellular carcinoma (HepG2 cell line. Methods: Estimation of both cell growth and the amount of prostaglandin E2 (PGE2 production were used to study the effect of selective COX-2 inhibitors on the hepatocellular carcinoma cell. Cell growth determination has done by MTT assay in 24 h, 48 h and 72 h, and PGE2 production has estimated by using ELYSA kit in 48 h and 72 h. Results: The results showed growth inhibition of the HepG2 cell line in a concentration and time-dependent manner, as well as a reduction in the formation of PGE2 as a product of COX-2 activity. Among the compounds those analogues with methoxy and hydrogen group showed more inhibitory effect than others. Conclusion: The current in-vitro study indicates that the observed significant growth-inhibitory effect of chalcone-epoxide analogues on the HepG2 cell line may involve COX-dependent mechanisms and the PGE2 pathway parallel to the effect of celecoxib. It can be said that these analogues might be efficient compounds in chemotherapy of COX-2 dependent carcinoma specially preventing and treatment of hepatocellular carcinomas.

  1. 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.

  2. Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells

    Science.gov (United States)

    Li, Luyuan; Paz, Ana C.; Wilky, Breelyn A.; Johnson, Britt; Galoian, Karina; Rosenberg, Andrew; Hu, Guozhi; Tinoco, Gabriel; Bodamer, Olaf; Trent, Jonathan C.

    2015-01-01

    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. PMID:26368816

  3. Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells.

    Science.gov (United States)

    Li, Luyuan; Paz, Ana C; Wilky, Breelyn A; Johnson, Britt; Galoian, Karina; Rosenberg, Andrew; Hu, Guozhi; Tinoco, Gabriel; Bodamer, Olaf; Trent, Jonathan C

    2015-01-01

    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.

  4. 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...

  5. Effect of L-NAME, an inhibitor of nitric oxide synthesis, on cardiopulmonary function in human septic shock

    NARCIS (Netherlands)

    J.A.M. Avontuur; R.P. Tutein Nolthenius; S.L.C.E. Buijk (Steven); K.J.K. Kanhai; H.A. Bruining (Hajo)

    1998-01-01

    textabstractSTUDY OBJECTIVES: We tested the effects of continuous infusion of N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, on cardiovascular performance and pulmonary gas exchange in patients with hyperdynamic septic shock.

  6. Different induction of two plasminogen activator inhibitor 1 mRNA species by phorbol ester in human hepatoma cells

    NARCIS (Netherlands)

    Bosma, P.J.; Kooistra, T.

    1991-01-01

    In man, the plasminogen activator inhibitor 1 (PAI-1) gene codes for two mRNA species, one of 3.2 kilobases (kb) and the other of 2.4 kb. We report that the protein kinase C activating phorbol ester, phorbol 12-myristate-13-acetate (PMA), causes a different induction of the two PAI-1 mRNA species in

  7. 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

    The sirtuin enzymes are important regulatory deacylases in a variety of biochemical contexts and may therefore be potential therapeutic targets through either activation or inhibition by small molecules. Here, we describe the discovery of the most potent inhibitor of sirtuin 5 (SIRT5) reported to...

  8. 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.

  9. Synthesis, Biological Evaluation and Molecular Modeling of Substituted Indeno[1,2-b]indoles as Inhibitors of Human Protein Kinase CK2

    Directory of Open Access Journals (Sweden)

    Faten Alchab

    2015-06-01

    Full Text Available Due to their system of annulated 6-5-5-6-membered rings, indenoindoles have sparked great interest for the design of ATP-competitive inhibitors of human CK2. In the present study, we prepared twenty-one indeno[1,2-b]indole derivatives, all of which were tested in vitro on human CK2. The indenoindolones 5a and 5b inhibited human CK2 with an IC50 of 0.17 and 0.61 µM, respectively. The indeno[1,2-b]indoloquinone 7a also showed inhibitory activity on CK2 at a submicromolar range (IC50 = 0.43 µM. Additionally, a large number of indenoindole derivatives was evaluated for their cytotoxic activities against the cell lines 3T3, WI-38, HEK293T and MEF.

  10. Functional expression of human dihydroorotate dehydrogenase (DHODH) in pyr4 mutants of ustilago maydis allows target validation of DHODH inhibitors in vivo.

    Science.gov (United States)

    Zameitat, Elke; Freymark, Gerald; Dietz, Cornelia D; Löffler, Monika; Bölker, Michael

    2007-05-01

    Dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) is a central enzyme of pyrimidine biosynthesis and catalyzes the oxidation of dihydroorotate to orotate. DHODH is an important target for antiparasitic and cytostatic drugs since rapid cell proliferation often depends on the de novo synthesis of pyrimidine nucleotides. We have cloned the pyr4 gene encoding mitochondrial DHODH from the basidiomycetous plant pathogen Ustilago maydis. We were able to show that pyr4 contains a functional mitochondrial targeting signal. The deletion of pyr4 resulted in uracil auxotrophy, enhanced sensitivity to UV irradiation, and a loss of pathogenicity on corn plants. The biochemical characterization of purified U. maydis DHODH overproduced in Escherichia coli revealed that the U. maydis enzyme uses quinone electron acceptor Q6 and is resistant to several commonly used DHODH inhibitors. Here we show that the expression of the human DHODH gene fused to the U. maydis mitochondrial targeting signal is able to complement the auxotrophic phenotype of pyr4 mutants. While U. maydis wild-type cells were resistant to the DHODH inhibitor brequinar, strains expressing the human DHODH gene became sensitive to this cytostatic drug. Such engineered U. maydis strains can be used in sensitive in vivo assays for the development of novel drugs specifically targeted at either human or fungal DHODH.

  11. Functional Expression of Human Dihydroorotate Dehydrogenase (DHODH) in pyr4 Mutants of Ustilago maydis Allows Target Validation of DHODH Inhibitors In Vivo▿

    Science.gov (United States)

    Zameitat, Elke; Freymark, Gerald; Dietz, Cornelia D.; Löffler, Monika; Bölker, Michael

    2007-01-01

    Dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) is a central enzyme of pyrimidine biosynthesis and catalyzes the oxidation of dihydroorotate to orotate. DHODH is an important target for antiparasitic and cytostatic drugs since rapid cell proliferation often depends on the de novo synthesis of pyrimidine nucleotides. We have cloned the pyr4 gene encoding mitochondrial DHODH from the basidiomycetous plant pathogen Ustilago maydis. We were able to show that pyr4 contains a functional mitochondrial targeting signal. The deletion of pyr4 resulted in uracil auxotrophy, enhanced sensitivity to UV irradiation, and a loss of pathogenicity on corn plants. The biochemical characterization of purified U. maydis DHODH overproduced in Escherichia coli revealed that the U. maydis enzyme uses quinone electron acceptor Q6 and is resistant to several commonly used DHODH inhibitors. Here we show that the expression of the human DHODH gene fused to the U. maydis mitochondrial targeting signal is able to complement the auxotrophic phenotype of pyr4 mutants. While U. maydis wild-type cells were resistant to the DHODH inhibitor brequinar, strains expressing the human DHODH gene became sensitive to this cytostatic drug. Such engineered U. maydis strains can be used in sensitive in vivo assays for the development of novel drugs specifically targeted at either human or fungal DHODH. PMID:17369345

  12. Growth inhibitory effects of the dual ErbB1/ErbB2 tyrosine kinase inhibitor PKI-166 on human prostate cancer xenografts.

    Science.gov (United States)

    Mellinghoff, Ingo K; Tran, Chris; Sawyers, Charles L

    2002-09-15

    Experiments with human prostate cancer cell lines have shown that forced overexpression of the ErbB2-receptor tyrosine kinase (RTK) promotes androgen-independent growth and increases androgen receptor-transcriptional activity in a ligand-independent fashion. To investigate the relationship between ErbB-RTK signaling and androgen in genetically unmanipulated human prostate cancer, we performed biochemical and biological studies with the dual ErbB1/ErbB2 RTK inhibitor PKI-166 using human prostate cancer xenograft models with isogenic sublines reflecting the transition from androgen-dependent to androgen-independent growth. In the presence of low androgen concentrations, PKI-166 showed profound growth-inhibitory effects on tumor growth, which could be partially reversed by androgen add-back. At physiological androgen concentrations, androgen withdrawal greatly enhanced the ability of PKI-166 to retard tumor growth. The level of extracellular signal-regulated kinase activation correlated with the response to PKI-166 treatment, whereas the expression levels of ErbB1 and ErbB2 did not. These results suggest that ErbB1/ErbB2 RTKs play an important role in the biology of androgen-independent prostate cancer and provide a rationale for clinical evaluation of inhibitors targeted to this pathway.

  13. Thrombospondin-1 (TSP-1) up-regulates tissue inhibitor of metalloproteinase-1 (TIMP-1) production in human tumor cells: exploring the functional significance in tumor cell invasion.

    Science.gov (United States)

    John, Anitha S; Hu, Xioulong; Rothman, Vicki L; Tuszynski, George P

    2009-12-01

    Thrombospondin-1 (TSP-1), a matrix-bound adhesive glycoprotein, has been shown to modulate tumor progression. We previously demonstrated that TSP-1 up-regulates matrix metalloproteinases MMP-2 and MMP-9. Our studies suggested that the balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) is a key determinant in tumor cell invasion. We now report that TSP-1 up-regulates TIMP-1 expression in both human breast and prostate cancer cell lines. The effect of TSP-1 on TIMP-1 expression was examined in human breast adenocarcinoma cell lines (MDA-MB-231) and human prostate cancer cell lines (PC3-NI and PC3-ML) treated with exogenous TSP-1. TIMP-1 expression was also examined in TSP-1 stably transfected breast cancer cell line (MDA-MB-435). Northern and western blot analysis revealed TIMP-1 mRNA and TIMP-1 protein expression increased with increasing concentrations of TSP-1. This effect was inhibited by antibodies against the type I repeat domain of TSP-1 further suggesting that TSP-1 mediates TIMP-1 secretion. Inhibition of TSP-1 induced TIMP-1 levels increased tumor cell invasion. We conclude that TSP-1 is involved in influencing the critical balance between MMPs and their inhibitors, maintaining the controlled degradation of the extracellular matrix needed to support metastasis and our results may provide an explanation for the divergent activities reported for TSP-1 in tumor progression.

  14. Ninety-Nine Is Not Enough: Molecular Characterization of Inhibitor-Resistant Human Immunodeficiency Virus Type 1 Protease Mutants with Insertions in the Flap Region

    Energy Technology Data Exchange (ETDEWEB)

    Koiek, Milan; Saskova, Klara Grantz; Rezaova, Pavlina; Brynda, Jii; van Maarseveen, Noortje M.; De Jong, Dorien; Boucher, Charles A.; Kagan, Ron M.; Nijhuis, Monique; Konvalinka, Jan (Quest); (Charles U); (Utrecht)

    2008-07-21

    While the selection of amino acid insertions in human immunodeficiency virus (HIV) reverse transcriptase (RT) is a known mechanism of resistance against RT inhibitors, very few reports on the selection of insertions in the protease (PR) coding region have been published. It is still unclear whether these insertions impact protease inhibitor (PI) resistance and/or viral replication capacity. We show that the prevalence of insertions, especially between amino acids 30 to 41 of HIV type 1 (HIV-1) PR, has increased in recent years. We identified amino acid insertions at positions 33 and 35 of the PR of HIV-1-infected patients who had undergone prolonged treatment with PIs, and we characterized the contribution of these insertions to viral resistance. We prepared the corresponding mutated, recombinant PR variants with or without insertions at positions 33 and 35 and characterized them in terms of enzyme kinetics and crystal structures. We also engineered the corresponding recombinant viruses and analyzed the PR susceptibility and replication capacity by recombinant virus assay. Both in vitro methods confirmed that the amino acid insertions at positions 33 and 35 contribute to the viral resistance to most of the tested PIs. The structural analysis revealed local structural rearrangements in the flap region and in the substrate binding pockets. The enlargement of the PR substrate binding site together with impaired flap dynamics could account for the weaker inhibitor binding by the insertion mutants. Amino acid insertions in the vicinity of the binding cleft therefore represent a novel mechanism of HIV resistance development.

  15. 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.

  16. 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.

  17. 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

  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

    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...... 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...

  19. The ACAT inhibitor VULM1457 significantly reduced production and secretion of adrenomedullin (AM) and down-regulated AM receptors on human hepatoblastic cells.

    Science.gov (United States)

    Drímal, J; Fáberová, V; Schmidtová, L; Bednáriková, M; Drímal, J; Drímal, D

    2005-12-01

    Acyl-CoA:cholesterol acyltransferase (ACAT) is an important enzyme in the pathways of cholesterol esterification. It has been shown that new ACAT inhibitor 1-(2,6-diisopropyl-phenyl)-3-[4-(4'-nitrophenylthio)phenyl] urea (VULM1457) significantly reduced atherogenic activity in animal experimental atherosclerosis. Proliferative hormone adrenomedullin (AM) has been shown to be released in response to hypoxia, however, its role in cellular protection has remained elusive. The effect of increased local production of AM in cells and resultant down-regulation of AM receptors has not been investigated yet. We hypothesized that increased expression of AM in hypoxic cells was the result of excessive AM production with resultant AM receptor down-regulation, surface-membrane protein degradation and that the new specific ACAT inhibitor would reduce AM induction in hypoxia and thus proliferation of cells. In order to investigate specific cellular AM signaling and protection induced by VULM1457, we characterized specific surface-membrane [125I]AM receptors expressed on cells, evaluated AM secretion (RIA assays), AM mRNA expression in cultured cells (RT-PCR analysis) and proliferation (incorporation of [3H]thymidine) in control, hypoxic and metabolically stressed human hepatoblastoma cell lines exposed to gradually increasing concentrations of VULM1457. The new ACAT inhibitor VULM1457 in concentration 0.03 and 0.1 micromol/l significantly down-regulated specific AM receptors on HepG2 cells, reduced AM secretion of HepG2 cells exposed to hypoxia. These results suggest that VULM1457, as new member of ACAT family of inhibitors could negatively regulate cell proliferation induced by AM, which may correlate with down-regulation of membrane-bound AM receptors on HepG2 cells, and moreover, with the induction and expression of AM in hypoxia.

  20. In vitro modulation of MMP-2 and MMP-9 in pediatric human sarcoma cell lines by cytokines, inducers and inhibitors

    OpenAIRE

    ROOMI, M.W.; KALINOVSKY, T.; RATH, M.; NIEDZWIECKI, A.

    2013-01-01

    The highly aggressive pediatric sarcomas are characterized by high levels of matrix metalloproteinase (MMP)-2 and MMP-9, which play crucial roles in tumor invasion and metastasis by degradation of the extracellular membrane leading to cancer cell spread to distal organs. We examined the effects of cytokines, mitogens, inducers and inhibitors on MMP-2 and -9 expression in osteosarcoma (U2OS) and rhabdomyosarcoma (RD). The selected compounds included natural cytokines and growth factors, as wel...

  1. 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...

  2. Acridine Derivatives as Inhibitors of the IRE1α-XBP1 Pathway Cytotoxic to Human Multiple Myeloma

    Science.gov (United States)

    Jiang, Dadi; Tam, Arvin; Alagappan, Muthuraman; Hay, Michael P.; Gupta, Aparna; Kozak, Margaret; Solow-Cordero, David E.; Lum, Pek Y; Denko, Nicholas; Giaccia, Amato J.; Le, Quynh-Thu; Niwa, Maho; Koong, Albert C.

    2016-01-01

    Using a luciferase reporter-based high throughput chemical library screen and topological data analysis (TDA), we identified N-acridine-9-yl-N′,N′-dimethylpropane-1,3-diamine (DAPA) as a inhibitor of the IRE1α-XBP1 pathway of the unfolded protein response (UPR). We designed a collection of analogues based on the structure of DAPA to explore structure-activity relationships (SAR) and identified N9-(3-(dimethylamino)propyl)-N3,N3,N6,N6-tetramethylacridine-3,6,9-triamine (3,6-DMAD), with 3,6-dimethylamino substitution on the chromophore, as a potent inhibitor. 3,6-DMAD inhibited both IRE1α oligomerization and in vitro endoribonuclease (RNase) activity, while the other analogues only blocked IRE1α oligomerization. Consistent with the inhibition of IRE1α-mediated XBP1 splicing, which is critical for multiple myeloma (MM) cell survival, these analogues were cytotoxic to MM cell lines. Furthermore, 3,6-DMAD inhibited XBP1 splicing and the growth of MM tumor xenografts. Our study not only confirmed the utilization of topological data analysis in drug discovery but also identified a class of compounds with a unique mechanism of action as potent IRE1α-XBP1 inhibitors in the treatment of MM. PMID:27307600

  3. Locostatin, a disrupter of Raf kinase inhibitor protein, inhibits extracellular matrix production, proliferation, and migration in human uterine leiomyoma and myometrial cells.

    Science.gov (United States)

    Janjusevic, Milijana; Greco, Stefania; Islam, Md Soriful; Castellucci, Clara; Ciavattini, Andrea; Toti, Paolo; Petraglia, Felice; Ciarmela, Pasquapina

    2016-11-01

    To investigate the presence of Raf kinase inhibitor protein (RKIP) in human myometrium and leiomyoma as well as to determine the effect of locostatin (RKIP inhibitor) on extracellular matrix (ECM) production, proliferation, and migration in human myometrial and leiomyoma cells. Laboratory study. Human myometrium and leiomyoma. Thirty premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Myometrial and leiomyoma tissues were used to investigate the localization and the expression level of RKIP through immunohistochemistry and Western blotting. Myometrial and leiomyoma cells were treated with locostatin (10 μM) to measure ECM expression by real-time polymerase chain reaction, GSK3β expression by Western blotting, cell migration by wound-healing assay, and cell proliferation by MTT assay and immunocytochemistry. The expression of RKIP in human myometrial and leiomyoma tissue; ECM components and GSK3β expression, migration, and proliferation in myometrial and leiomyoma cells. RKIP is expressed in human myometrial and leiomyoma tissue. Locostatin treatment resulted in the activation of the mitogen-activated protein kinase (MAPK) signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, RKIP inhibition by locostatin reduces ECM components. Moreover, the inhibition of RKIP by locostatin impaired cell proliferation and migration in both leiomyoma and myometrial cells. Finally, locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells. Our results indicate that RKIP may be involved in leiomyoma pathophysiology. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  4. Use of recombinant Entamoeba histolytica cysteine proteinase 1 to identify a potent inhibitor of amebic invasion in a human colonic model.

    Science.gov (United States)

    Meléndez-López, Samuel G; Herdman, Scott; Hirata, Ken; Choi, Min-Ho; Choe, Youngchool; Craik, Charles; Caffrey, Conor R; Hansell, Elisabeth; Chávez-Munguía, Bibiana; Chen, Yen Ting; Roush, William R; McKerrow, James; Eckmann, Lars; Guo, Jianhua; Stanley, Samuel L; Reed, Sharon L

    2007-07-01

    Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.

  5. MiR-143 and miR-135 inhibitors treatment induces skeletal myogenic differentiation of human adult dental pulp stem cells.

    Science.gov (United States)

    Li, Dongxia; Deng, Tianzheng; Li, Hongshi; Li, Ying

    2015-11-01

    Dental pulp stem cells (DPSCs) possess pluripotent properties that allow them to differentiate into multiple cell lineages, which can be potentially used in tissue regeneration. The aim of this in vitro study is to explore the effect of miRNAs on the myogenic differentiation of human adult DPSCs and seek for some potential biological factors for stable and feasible application in DPSC myogenic differentiation. Human adult DPSCs were isolated from normal impacted third molars were treated with 5-Aza-2'-deoxycytidine to induce to myogenic differentiation in vitro. During this process the levels of myomiRNAs and myogenic marker genes were detected by real-time qPCR and Western blotting. Then antisense oligonucleotides of miR-143 and miR-135 were transfected into DPSCs to explore their effects on myogenic differentiation. Gene expression detection and MyHC immunofluorescence microscopy analysis were applied to characterize the myogenic differentiation of DPSCs. Expression of miR-135 and miR-143 was markedly decreased in myoblast DPSCs induced by 5-Aza. Part of the DPSCs treated with miR-135 or miR-143 inhibitors showed apparent myocytic properties and eventually fused to form myotubes. Co-transfection of miR-135 and miR-143 inhibitors impelled half of DPSCs to form myotubes. MiR-135 and miR-143 inhibitors could induce myogenic differentiation of DPSCs. Our findings indicated that miRNAs could exert a decisive function in induction of myogenic differentiation of DPSCs. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Combined treatment of curcumin and small molecule inhibitors suppresses proliferation of A549 and H1299 human non-small-cell lung cancer cells.

    Science.gov (United States)

    Lin, Hui-Ping; Kuo, Li-Kuo; Chuu, Chih-Pin

    2012-01-01

    Curcumin (diferuloylmethane) is a phenolic compound present in turmeric and is ingested daily in many parts of the world. Curcumin has been reported to cause inhibition on proliferation and induction of apoptosis in many human cancer cell lines, including non-small cell lung cancer cells (NSCLC). However, the clinical application of curcumin is restricted by its low bioavailability. In this report, it was observed that combined treatment of a low dosage of curcumin (5-10 µM) with a low concentration (0.1-2.5 µM) of small molecule inhibitors, including AG1478, AG1024, PD173074, LY294002 and caffeic acid phenethyl ester (CAPE) increased the growth inhibition in two human NSCLC cell lines: A549 and H1299 cells. The observation suggested that combined treatment of a low dosage of curcumin with inhibitors against epidermal growth factor receptor (EGFR), insulin-like growth factor 1 (IGF-1R), fibroblast growth factors receptor (FGFR), phosphatidylinositol 3-kinases (PI3K) or NF-κB signaling pathway may be a potential adjuvant therapy beneficial to NSCLC patients. Copyright © 2011 John Wiley & Sons, Ltd.

  7. 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.

  8. Design and synthesis of 2-substituted-5-(4-trifluoromethylphenyl-sulphonamido)benzoxazole derivatives as human GST P1-1 inhibitors.

    Science.gov (United States)

    Ertan-Bolelli, Tugba; Bolelli, Kayhan; Musdal, Yaman; Yildiz, Ilkay; Aki-Yalcin, Esin; Mannervik, Bengt; Yalcin, Ismail

    2017-05-14

    The glutathione transferases (GSTs) are a family of widely distributed Phase II detoxification enzymes. GST P1-1 is frequently overexpressed in rat and human tumours. It is suggested that overexpression of hGST P1-1 by human tumor cells may play a role in resistance to cancer chemotherapy. Hence, hGST P1-1 can be a promising target for cancer treatment. In this study, new hGST P1-1 inhibitors, 2-(4-substitutedphenyl/benzyl)-5-(4-trifluoromethylphenylsulphonamido) benzoxazole derivatives (Va-Vk) have been designed and synthesized. Surprisingly, in vitro hGST P1-1 enzyme inhibition studies demonstrated that all of the tested compounds except Vj had better activity than the reference drug EA and it is also correlated with the docking results. Additionally we compared the interactions with hGST P1-1 enzyme of newly synthesized compound Vh (bearing CF3 group) and previously synthesized compound 5f (bearing NO2 group). According to the docking results, compound Vh bound to the hGST P1-1 enzyme with a higher affinity compared to 5f. Therefore, we can consider that these data make a sense and can explain its higher activity. The compounds that obtained from this research could be used as scaffolds in design of new potent hGST P1-1 inhibitors useful in the treatment of the resistance of cancer chemotherapy.

  9. 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.

  10. In vitro modulation of MMP-2 and MMP-9 in pediatric human sarcoma cell lines by cytokines, inducers and inhibitors.

    Science.gov (United States)

    Roomi, M W; Kalinovsky, T; Rath, M; Niedzwiecki, A

    2014-01-01

    The highly aggressive pediatric sarcomas are characterized by high levels of matrix metalloproteinase (MMP)-2 and MMP-9, which play crucial roles in tumor invasion and metastasis by degradation of the extracellular membrane leading to cancer cell spread to distal organs. We examined the effects of cytokines, mitogens, inducers and inhibitors on MMP-2 and -9 expression in osteosarcoma (U2OS) and rhabdomyosarcoma (RD). The selected compounds included natural cytokines and growth factors, as well as chemical compounds applied in therapy of sarcoma and natural compounds that have demonstrated anticancer therapeutic potential. These cell lines were cultured in their respective media to near confluence and the cells were washed with PBS and incubated in serum-free medium with various concentrations of several cytokines, mitogens and inhibitors. After 24 h the media were removed and analyzed for MMP-2 and -9 by gelatinase zymography and quantitated by densitometry. Osteosarcoma and rhabdomyosarcoma showed bands corresponding to MMP-2 and -9 with dose-dependent enhancement of MMP-9 with phorbol 12-myristate 13-acetate (PMA) treatment. Tumor necrosis factor-α, interleukin-1β and LPS enhanced osteosarcoma U2OS MMP-9 secretion but had no effect on MMP-2 secretion. Tumor necrosis factor-α stimulated rhabdomyosarcoma MMP-2 expression, but had no effect on MMP-9 secretion. Doxycycline, epigallocatechin gallate, nutrient mixture (NM), actinomycin-D, cyclohexamide, retinoic acid and dexamethasone inhibited MMP-2 and -9 in U2OS osteosarcoma cells. PMA-treated RD cells showed dose-response inhibition of MMP-9 by doxycycline and epigallocatechin gallate and both MMPs by NM. Dexamethasone and actinomycin-D showed inhibition of MMP-2 secretion of RD cells. Our results show that cytokines, mitogens and inducers show variable upregulation of U2OS osteosarcoma and RD rhabdomyosarcoma MMP-2 and -9 secretion, and inhibitors demonstrate downregulation under stimulatory conditions

  11. Kinetic and mutational analysis of human immunodeficiency virus type 1 reverse transcriptase inhibition by inophyllums, a novel class of non-nucleoside inhibitors.

    Science.gov (United States)

    Taylor, P B; Culp, J S; Debouck, C; Johnson, R K; Patil, A D; Woolf, D J; Brooks, I; Hertzberg, R P

    1994-03-04

    Inophyllums are novel non-nucleoside inhibitors of human immunodeficiency virus (HIV) type 1 reverse transcriptase identified through an enzyme screening program and isolated from the plant Calophyllum inophyllum. The kinetics of reverse transcriptase inhibition by inophyllum B were characterized using recombinant purified enzyme, a heteropolymeric RNA template, and a scintillation proximity assay. Preincubation of inhibitor with the enzyme-template-primer complex for 11 min was required for maximal inhibition of reverse transcriptase to occur, suggesting that inophyllum B had a slow on-rate and that template-primer must bind to reverse transcriptase prior to inhibitor binding. Inhibition of reverse transcriptase by inophyllums was shown to be reversible. When thymidine triphosphate was the variable substrate, inophyllum B inhibited reverse transcriptase noncompetitively with a Ki of 42 nM. Enzyme inhibition with respect to template-primer was uncompetitive with a Ki of 26 nM. Reverse transcriptase enzymes containing point mutations in which tyrosine 181 was changed to either cysteine or isoleucine exhibited marginal resistance to inophyllums but were resistant to (+)-(5S)-4,5,6,7-tetrahydro-9-chloro-5-methyl-6- (3-methyl-2-butenyl)-imidazo[4,5,1-j,k][1,4]benzodiazepin-2-(1H)-t hione (TIBO R82913). A mutant enzyme in which tyrosine 188 was changed to leucine was cross-resistant to both inophyllum B and TIBO R82913, as was HIV type 2 reverse transcriptase. These studies suggest that inophyllum B and TIBO R82913 bind to distinct but overlapping sites. Inhibition of avian myeloblastosis virus reverse transcriptase and Moloney murine leukemia virus reverse transcriptase by inophyllum B was detectible, suggesting that these inhibitors may be more promiscuous than other previously described non-nucleoside inhibitors. Inophyllums were active against HIV type 1 in cell culture with IC50 values of approximately 1.5 microM. These studies imply that the inophyllums have a

  12. 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

  13. 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.

  14. 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.

  15. Valproic acid inhibits the release of soluble CD40L induced by non-nucleoside reverse transcriptase inhibitors in human immunodeficiency virus infected individuals.

    Directory of Open Access Journals (Sweden)

    Donna C Davidson

    Full Text Available Despite the use of highly active antiretroviral therapies (HAART, a majority of Human Immunodeficiency Virus Type 1 (HIV infected individuals continually develop HIV - Associated Neurocognitive Disorders (HAND, indicating that host inflammatory mediators, in addition to viral proteins, may be contributing to these disorders. Consistent with this notion, we have previously shown that levels of the inflammatory mediator soluble CD40 ligand (sCD40L are elevated in the plasma and cerebrospinal fluid (CSF of HIV infected, cognitively impaired individuals, and that excess sCD40L can contribute to blood brain barrier (BBB permeability in vivo, thereby signifying the importance of this inflammatory mediator in the pathogenesis of HAND. Here we demonstrate that the non-nucleoside reverse transcriptase inhibitor (NNRTI efavirenz (EFV induces the release of circulating sCD40L in both HIV infected individuals and in an in vitro suspension of washed human platelets, which are the main source of circulating sCD40L. Additionally, EFV was found to activate glycogen synthase kinase 3 beta (GSK3β in platelets, and we now show that valproic acid (VPA, a known GSK3β inhibitor, was able to attenuate the release of sCD40L in HIV infected individuals receiving EFV, and in isolated human platelets. Collectively these results have important implications in determining the pro-inflammatory role that some antiretroviral regimens may have. The use of antiretrovirals remains the best strategy to prevent HIV-associated illnesses, including HAND, however these drugs have clear limitations to this end, and thus, these results underscore the need to develop adjunctive therapies for HAND that can also minimize the undesired negative effects of the antiretrovirals.

  16. 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.

  17. 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.

  18. Potent inhibitors of human LAT1 (SLC7A5) transporter based on dithiazole and dithiazine compounds for development of anticancer drugs.

    Science.gov (United States)

    Napolitano, Lara; Scalise, Mariafrancesca; Koyioni, Maria; Koutentis, Panayiotis; Catto, Marco; Eberini, Ivano; Parravicini, Chiara; Palazzolo, Luca; Pisani, Leonardo; Galluccio, Michele; Console, Lara; Carotti, Angelo; Indiveri, Cesare

    2017-11-01

    The LAT1 transporter is acknowledged as a pharmacological target of tumours since it is strongly overexpressed in many human cancers. The purpose of this work was to find novel compounds exhibiting potent and prolonged inhibition of the transporter. To this aim, compounds based on dithiazole and dithiazine scaffold have been screened in the proteoliposome experimental model. Inhibition was tested on the antiport catalysed by hLAT1 as transport of extraliposomal [3H]histidine in exchange with intraliposomal histidine. Out of 59 compounds tested, 8 compounds, showing an inhibition higher than 90% at 100µM concentration, were subjected to dose-response analysis. Two of them exhibited IC50 lower than 1µM. Inhibition kinetics, performed on the two best inhibitors, indicated a mixed type of inhibition with respect to the substrate. Furthermore, inhibition of the transporter was still present after removal of the compounds from the reaction mixture, but was reversed on addition of dithioerythritol, a S-S reducing agent, indicating the formation of disulfide(s) between the compounds and the protein. Molecular docking of the two best inhibitors on the hLAT1 homology structural model, highlighted interaction with the substrate binding site and formation of a covalent bond with the residue C407. Indeed, the inhibition was impaired in the hLAT1 mutant C407A confirming the involvement of that Cys residue. Treatment of SiHa cells expressing hLAT1 at relatively high level, with the two most potent inhibitors led to cell death which was not observed after treatment with a compound exhibiting very poor inhibitory effect. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. 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.

  20. 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.

  1. 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

    2015-08-01

    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 Ca(2+) 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. 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. GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca(2+) currents after Ca(2+) 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. Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis

  2. Characterization of HJ-PI01 as a novel Pim-2 inhibitor that induces apoptosis and autophagic cell death in triple-negative human breast cancer.

    Science.gov (United States)

    Zhao, Yu-Qian; Yin, Yi-Qiong; Liu, Jie; Wang, Gui-Hua; Huang, Jian; Zhu, Ling-Juan; Wang, Jin-Hui

    2016-09-01

    Pim-2 is a short-lived serine/threonine kinase, which plays a key role in metastasis of breast cancer through persistent activation of STAT3. Although the crystal structure of Pim-2 has been reported, but thus far no specific Pim-2-targeted compounds have been reported. In this study, we identified a novel Pim-2 inhibitor, HJ-PI01, by in silico analysis and experimental validation. The protein-protein interaction (PPI) network, chemical synthesis, molecular docking, and molecular dynamics (MD) simulations were used to design and discover the new Pim-2 inhibitor HJ-PI01. The anti-tumor effects of HJ-PI01 were evaluated in human breast MDA-MB-231, MDA-MB-468, MDA-MB-436, MCF-7 cells in vitro and in MDA-MB-231 xenograft mice, which were treated with HJ-PI01 (40 mg·kg(-1)·d(-1), ig) with or without lienal polypeptide (50 mg·kg(-1)·d(-1), ip) for 10 d. The apoptosis/autophage-inducing mechanisms of HJ-PI01 were elucidated using Western blots, immunoblots, flow cytometry, transmission electron microscopy and fluorescence microscopy. Based on the PrePPI network, the potential partners interacting with Pim-2 in regulating apoptosis (160 protein pairs) and autophagy (47 protein pairs) were identified. Based on the structural characteristics of Pim-2, a total of 15 compounds (HJ-PI01 to HJ-P015) were synthesized, which showed moderate or remarkable anti-proliferative potency in the human breast cancer cell lines tested. The most effective compound HJ-PI01 exerted a robust inhibition on MDA-MB-231 cells compared with chlorpromazine and the pan-Pim inhibitor PI003. Molecular dynamics (MD) simulation revealed that HJ-PI01 had a good binding score with Pim-2. Moreover, HJ-PI01 (300 nmol/L) induced death receptor-dependent and mitochondrial apoptosis as well as autophagic death in MDA-MB-231 cells. In MDA-MB-231 xenograft mice, administration of HJ-PI01 remarkably inhibited the tumor growth and induced tumor cell apoptosis in vivo. Co-administration of HJ-PI01 with lienal

  3. Role of the lysozyme inhibitor Ivy in growth or survival of Escherichia coli and Pseudomonas aeruginosa bacteria in hen egg white and in human saliva and breast milk.

    Science.gov (United States)

    Deckers, Daphne; Vanlint, Dietrich; Callewaert, Lien; Aertsen, Abram; Michiels, Chris W

    2008-07-01

    Ivy is a lysozyme inhibitor that protects Escherichia coli against lysozyme-mediated cell wall hydrolysis when the outer membrane is permeabilized by mutation or by chemical or physical stress. In the current work, we have investigated whether Ivy is necessary for the survival or growth of E. coli MG1655 and Pseudomonas aeruginosa PAO1 in hen egg white and in human saliva and breast milk, which are naturally rich in lysozyme and in membrane-permeabilizing components. Wild-type E. coli was able to grow in saliva and breast milk but showed partial inactivation in egg white. The knockout of Ivy did not affect growth in breast milk but slightly increased sensitivity to egg white and caused hypersensitivity to saliva, resulting in the complete inactivation of 10(4) CFU ml(-1) of bacteria within less than 5 hours. The depletion of lysozyme from saliva completely restored the ability of the ivy mutant to grow like the parental strain. P. aeruginosa, in contrast, showed growth in all three substrates, which was not affected by the knockout of Ivy production. These results indicate that lysozyme inhibitors like Ivy promote bacterial survival or growth in particular lysozyme-rich secretions and suggest that they may promote the bacterial colonization of specific niches in the animal host.

  4. 3D pharmacophore-based virtual screening, docking and density functional theory approach towards the discovery of novel human epidermal growth factor receptor-2 (HER2) inhibitors.

    Science.gov (United States)

    Gogoi, Dhrubajyoti; Baruah, Vishwa Jyoti; Chaliha, Amrita Kashyap; Kakoti, Bibhuti Bhushan; Sarma, Diganta; Buragohain, Alak Kumar

    2016-12-21

    Human epidermal growth factor receptor 2 (HER2) is one of the four members of the epidermal growth factor receptor (EGFR) family and is expressed to facilitate cellular proliferation across various tissue types. Therapies targeting HER2, which is a transmembrane glycoprotein with tyrosine kinase activity, offer promising prospects especially in breast and gastric/gastroesophageal cancer patients. Persistence of both primary and acquired resistance to various routine drugs/antibodies is a disappointing outcome in the treatment of many HER2 positive cancer patients and is a challenge that requires formulation of new and improved strategies to overcome the same. Identification of novel HER2 inhibitors with improved therapeutics index was performed with a highly correlating (r=0.975) ligand-based pharmacophore model (Hypo1) in this study. Hypo1 was generated from a training set of 22 compounds with HER2 inhibitory activity and this well-validated hypothesis was subsequently used as a 3D query to screen compounds in a total of four databases of which two were natural product databases. Further, these compounds were analyzed for compliance with Veber's drug-likeness rule and optimum ADMET parameters. The selected compounds were then subjected to molecular docking and Density Functional Theory (DFT) analysis to discern their molecular interactions at the active site of HER2. The findings thus presented would be an important starting point towards the development of novel HER2 inhibitors using well-validated computational techniques. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Rational Design of Benzylidenehydrazinyl-Substituted Thiazole Derivatives as Potent Inhibitors of Human Dihydroorotate Dehydrogenase with in Vivo Anti-arthritic Activity

    Science.gov (United States)

    Li, Shiliang; Luan, Guoqin; Ren, Xiaoli; Song, Wenlin; Xu, Liuxin; Xu, Minghao; Zhu, Junsheng; Dong, Dong; Diao, Yanyan; Liu, Xiaofeng; Zhu, Lili; Wang, Rui; Zhao, Zhenjiang; Xu, Yufang; Li, Honglin

    2015-01-01

    Human dihydroorotate dehydrogenase (hDHODH) is an attractive therapeutic target for the treatment of rheumatoid arthritis, transplant rejection and other autoimmune diseases. Based on the X-ray structure of hDHODH in complex with lead compound 7, a series of benzylidenehydrazinyl-substituted thiazole derivatives as potent inhibitors of hDHODH were designed and synthesized, of which 19 and 30 were the most potent with IC50 values in the double-digit nanomolar range. Moreover, compound 19 displayed significant anti-arthritic effects and favorable pharmacokinetic profiles in vivo. Further X-ray structure and SAR analyses revealed that the potencies of the designed inhibitors were partly attributable to additional water-mediated hydrogen bond networks formed by an unexpected buried water between hDHODH and the 2-(2-methylenehydrazinyl)thiazole scaffold. This work not only elucidates promising scaffolds targeting hDHODH for the treatment of rheumatoid arthritis, but also demonstrates that the water-mediated hydrogen bond interaction is an important factor in molecular design and optimization. PMID:26443076

  6. Review of issues concerning the use of reproductive inhibitors, with particular emphasis on resolving human-wildlife conflicts in North America.

    Science.gov (United States)

    Fagerstone, Kathleen A; Miller, Lowell A; Killian, Gary; Yoder, Christi A

    2010-03-01

    This manuscript provides an overview of past wildlife contraception efforts and discusses the current state of research. Two fertility control agents, an avian reproductive inhibitor containing the active ingredient nicarbazin and an immunocontraceptive vaccine, have received regulatory approval with the Environmental Protection Agency and are commercially available in the USA. OvoControl G Contraceptive Bait for Canada Geese and Ovo Control for pigeons are delivered as oral baits. An injectable immunocontraceptive vaccine (GonaCon Immunocontraceptive Vaccine) was registered with the Environmental Protection Agency for use in female white-tailed deer in September 2009. An injectable product (GonaCon Immunocontraceptive Vaccine) is registered for use in female white-tailed deer. Both products are labeled for use in urban/suburban areas where these species are overabundant. Several other compounds are currently being tested for use in wildlife in the USA, Europe, Australia and New Zealand that could have promise in the future. The development and use of reproductive inhibitors for resolving human-wildlife conflicts will depend on a number of factors, including meeting the requirements of regulatory agencies for use in the environment and on the biological and economical feasibility of their use. Use will also be dependent on health and safety issues and on public acceptance of the techniques. © 2010 ISZS, Blackwell Publishing and IOZ/CAS.

  7. 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.

  8. Sanguinarine causes cell cycle blockade and apoptosis of human prostate carcinoma cells via modulation of cyclin kinase inhibitor-cyclin-cyclin-dependent kinase machinery.

    Science.gov (United States)

    Adhami, Vaqar Mustafa; Aziz, Moammir Hasan; Reagan-Shaw, Shannon R; Nihal, Minakshi; Mukhtar, Hasan; Ahmad, Nihal

    2004-08-01

    Prostate cancer is the second leading cause of cancer-related deaths in males in the United States. This warrants the development of novel mechanism-based strategies for the prevention and/or treatment of prostate cancer. Several studies have shown that plant-derived alkaloids possess remarkable anticancer effects. Sanguinarine, an alkaloid derived from the bloodroot plant Sanguinaria canadensis, has been shown to possess antimicrobial, anti-inflammatory, and antioxidant properties. Previously, we have shown that sanguinarine possesses strong antiproliferative and proapoptotic properties against human epidermoid carcinoma A431 cells and immortalized human HaCaT keratinocytes. Here, employing androgen-responsive human prostate carcinoma LNCaP cells and androgen-unresponsive human prostate carcinoma DU145 cells, we studied the antiproliferative properties of sanguinarine against prostate cancer. Sanguinarine (0.1-2 micromol/L) treatment of LNCaP and DU145 cells for 24 hours resulted in dose-dependent (1) inhibition of cell growth [as evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay], (2) arrest of cells in G0-G1 phase of the cell cycle (as assessed by DNA cell cycle analysis), and (3) induction of apoptosis (as evaluated by DNA ladder formation and flow cytometry). To define the mechanism of antiproliferative effects of sanguinarine against prostate cancer, we studied the effect of sanguinarine on critical molecular events known to regulate the cell cycle and the apoptotic machinery. Immunoblot analysis showed that sanguinarine treatment of both LNCaP and DU145 cells resulted in significant (1) induction of cyclin kinase inhibitors p21/WAF1 and p27/KIP1; (2) down-regulation of cyclin E, D1, and D2; and (3) down-regulation of cyclin-dependent kinase 2, 4, and 6. A highlight of this study was the fact that sanguinarine induced growth inhibitory and antiproliferative effects in human prostate carcinoma cells irrespective of their androgen

  9. Synthesis of 17beta-N-substituted 19-Nor-10-azasteroids as inhibitors of human 5alpha-reductases I and II.

    Science.gov (United States)

    Scarpi, Dina; Occhiato, Ernesto G; Danza, Giovanna; Serio, Mario; Guarna, Antonio

    2002-11-01

    The synthesis of 17beta-[N-(phenyl)methyl/phenyl-amido] substituted 10-azasteroids has been accomplished by either the TiCl4- or TMSOTf-catalysed reaction of carbamates 11 and 12 with Danishefsky's diene. The reaction provided 5alpha-H isomers 3a-5a and 5beta-H isomers 3b-5b depending on the reaction conditions. Both epimers of each compound were tested against human 5alpha-reductase types I and II. Unexpectedly, 5beta-H compounds were found more active than their 5alpha-H counterparts, the best inhibitors being 3b (IC50=279 and 2000 nM toward isoenzyme I and II, respectively) and 5b (IC50=913 and 247 nM toward isoenzymes I and II, respectively).

  10. Discovery of an Orally Bioavailable Benzimidazole Diacylglycerol Acyltransferase 1 (DGAT1) Inhibitor That Suppresses Body Weight Gain in Diet-Induced Obese Dogs and Postprandial Triglycerides in Humans.

    Science.gov (United States)

    Nakajima, Katsumasa; Chatelain, Ricardo; Clairmont, Kevin B; Commerford, Renee; Coppola, Gary M; Daniels, Thomas; Forster, Cornelia J; Gilmore, Thomas A; Gong, Yongjin; Jain, Monish; Kanter, Aaron; Kwak, Youngshin; Li, Jingzhou; Meyers, Charles D; Neubert, Alan D; Szklennik, Paul; Tedesco, Vivienne; Thompson, James; Truong, David; Yang, Qing; Hubbard, Brian K; Serrano-Wu, Michael H

    2017-06-08

    Modification of a gut restricted class of benzimidazole DGAT1 inhibitor 1 led to 9 with good oral bioavailability. The key structural changes to 1 include bioisosteric replacement of the amide with oxadiazole and α,α-dimethylation of the carboxylic acid, improving DGAT1 potency and gut permeability. Since DGAT1 is expressed in the small intestine, both 1 and 9 can suppress postprandial triglycerides during acute oral lipid challenges in rats and dogs. Interestingly, only 9 was found to be effective in suppressing body weight gain relative to control in a diet-induced obese dog model, suggesting the importance of systemic inhibition of DGAT1 for body weight control. 9 has advanced to clinical investigation and successfully suppressed postprandial triglycerides during an acute meal challenge in humans.

  11. Gas chromatographic-mass spectrometric analysis of hydroxylamine for monitoring the metabolic hydrolysis of metalloprotease inhibitors in rat and human liver microsomes.

    Science.gov (United States)

    Peng, S X; Strojnowski, M J; Hu, J K; Smith, B J; Eichhold, T H; Wehmeyer, K R; Pikul, S; Almstead, N G

    1999-03-05

    A gas chromatographic-mass spectrometric (GC-MS) method was developed for the analysis of hydroxylamine (HA) in supernatants obtained from liver microsomes. HA monitoring was used to determine the metabolic hydrolysis of two hydroxamic acid-based matrix metalloprotease inhibitors in rat and human liver microsomes. The hydrolysis of the hydroxamic acids to their corresponding carboxylic acids releases HA as a common metabolic product. HA was derivatized to acetone oxime by addition of acetone to the liver microsomal supernatant, followed by direct injection of the supernatant into the GC-MS, with detection of the oxime by selected-ion-monitoring. The method is simple, reproducible, and sensitive for the determination of the hydrolysis of hydroxamic acid compounds, where hydrolysis is the major metabolic pathway. The methodology can be used for rank ordering and selecting hydroxamic acid analogs based on their susceptibility to hydrolysis.

  12. 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.

  13. 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.

  14. In vitro screen of a small molecule inhibitor drug library identifies multiple compounds that synergize with oncolytic myxoma virus against human brain tumor-initiating cells.

    Science.gov (United States)

    McKenzie, Brienne A; Zemp, Franz J; Pisklakova, Alexandra; Narendran, Aru; McFadden, Grant; Lun, Xueqing; Kenchappa, Rajappa S; Kurz, Ebba U; Forsyth, Peter A

    2015-08-01

    Brain tumor-initiating cells (BTICs) are stem-like cells hypothesized to form a disease reservoir that mediates tumor recurrence in high-grade gliomas. Oncolytic virotherapy uses replication-competent viruses to target and kill malignant cells and has been evaluated in clinic for glioma therapy with limited results. Myxoma virus (MyxV) is a safe and highly effective oncolytic virus (OV) in conventional glioma models but, as seen with other OVs, is only modestly effective for patient-derived BTICs. The objective of this study was to determine whether MyxV treatment against human BTICs could be improved by combining chemotherapeutics and virotherapy. A 73-compound library of drug candidates in clinical use or preclinical development was screened to identify compounds that sensitize human BTICs to MyxV treatment in vitro, and synergy was evaluated mathematically in lead compounds using Chou-Talalay analyses. The effects of combination therapy on viral gene expression and viral replication were also assessed. Eleven compounds that enhance MyxV efficacy were identified, and 6 were shown to synergize with the virus using Chou-Talalay analyses. Four of the synergistic compounds were shown to significantly increase viral gene expression, indicating a potential mechanism for synergy. Three highly synergistic compounds (axitinib, a VEGFR inhibitor; rofecoxib, a cyclooxygenase-2 inhibitor; and pemetrexed, a folate anti-metabolite) belong to classes of compounds that have not been previously shown to synergize with oncolytic viruses in vitro. This study has identified multiple novel drug candidates that synergistically improve MyxV efficacy in a preclinical BTIC glioma model. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. 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.

  16. 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

  17. Novel small molecule XPO1/CRM1 inhibitors induce nuclear accumulation of TP53, phosphorylated MAPK and apoptosis in human melanoma cells.

    Directory of Open Access Journals (Sweden)

    Jennifer Yang

    Full Text Available XPO1/CRM1 is a key nuclear exporter protein that mediates translocation of numerous cellular regulatory proteins. We investigated whether XPO1 is a potential therapeutic target in melanoma using novel selective inhibitors of nuclear export (SINE. In vitro effects of SINE on cell growth and apoptosis were measured by MTS assay and flow cytometry [Annexin V/propidium iodide (PI], respectively in human metastatic melanoma cell lines. Immunoblot analysis was used to measure nuclear localization of key cellular proteins. The in vivo activity of oral SINE was evaluated in NOD/SCID mice bearing A375 or CHL-1 human melanoma xenografts. SINE compounds induced cytostatic and pro-apoptotic effects in both BRAF wild type and mutant (V600E cell lines at nanomolar concentrations. The cytostatic and pro-apoptotic effects of XPO1 inhibition were associated with nuclear accumulation of TP53, and CDKN1A induction in the A375 cell line with wild type TP53, while pMAPK accumulated in the nucleus regardless of TP53 status. The orally bioavailable KPT-276 and KPT-330 compounds significantly inhibited growth of A375 (p<0.0001 and CHL-1 (p = 0.0087 human melanoma cell lines in vivo at well tolerated doses. Inhibition of XPO1 using SINE represents a potential therapeutic approach for melanoma across cells with diverse molecular phenotypes by promoting growth inhibition and apoptosis.

  18. Screening of mammalian DNA polymerase and topoisomerase inhibitors from Garcinia mangostana L. and analysis of human cancer cell proliferation and apoptosis.

    Science.gov (United States)

    Onodera, Takefumi; Takenaka, Yukiko; Kozaki, Sachiko; Tanahashi, Takao; Mizushina, Yoshiyuki

    2016-03-01

    We purified and identified eight xanthones from mangosteen (Garcinia mangostana L.) and investigated whether these compounds inhibited the activities of mammalian DNA polymerases (Pols) and human DNA topoisomerases (Topos). β-Mangostin was the strongest inhibitor of both mammalian Pols and human Topos among the isolated xanthones, with 50% inhibitory concentration (IC50) values of 6.4-39.6 and 8.5-10 µM, respectively. Thermal transition analysis indicated that β-mangostin did not directly bind to double-stranded DNA, suggesting that this compound directly bound the enzyme protein rather than the DNA substrate. β-Mangostin showed the strongest suppression of human cervical cancer HeLa cell proliferation among the eight compounds tested, with a 50% lethal dose (LD50) of 27.2 µM. This compound halted cell cycle in S phase at 12-h treatment and induced apoptosis. These results suggest that decreased proliferation by β-mangostin may be a result of the inhibition of cellular Pols rather than Topos, and β-mangostin might be an anticancer chemotherapeutic agent.

  19. 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.

  20. 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

  1. A Rho-associated coiled-coil containing kinases (ROCK) inhibitor, Y-27632, enhances adhesion, viability and differentiation of human term placenta-derived trophoblasts in vitro.

    Science.gov (United States)

    Motomura, Kenichiro; Okada, Naoko; Morita, Hideaki; Hara, Mariko; Tamari, Masato; Orimo, Keisuke; Matsuda, Go; Imadome, Ken-Ichi; Matsuda, Akio; Nagamatsu, Takeshi; Fujieda, Mikiya; Sago, Haruhiko; Saito, Hirohisa; Matsumoto, Kenji

    2017-01-01

    Although human term placenta-derived primary cytotrophoblasts (pCTBs) represent a good human syncytiotrophoblast (STB) model, in vitro culture of pCTBs is not always easily accomplished. Y-27632, a specific inhibitor of Rho-associated coiled-coil containing kinases (ROCK), reportedly prevented apoptosis and improved cell-to-substrate adhesion and culture stability of dissociated cultured human embryonic stem cells and human corneal endothelial cells. The Rho kinase pathway regulates various kinds of cell behavior, some of which are involved in pCTB adhesion and differentiation. In this study, we examined Y-27632's potential for enhancing pCTB adhesion, viability and differentiation. pCTBs were isolated from term, uncomplicated placentas by trypsin-DNase I-Dispase II treatment and purified by HLA class I-positive cell depletion. Purified pCTBs were cultured on uncoated plates in the presence of epidermal growth factor (10 ng/ml) and various concentrations of Y-27632. pCTB adhesion to the plates was evaluated by phase-contrast imaging, viability was measured by WST-8 assay, and differentiation was evaluated by immunofluorescence staining, expression of fusogenic genes and hCG-β production. Ras-related C3 botulinum toxin substrate 1 (Rac1; one of the effector proteins of the Rho family) and protein kinase A (PKA) involvement was evaluated by using their specific inhibitors, NSC-23766 and H-89. We found that Y-27632 treatment significantly enhanced pCTB adhesion to plates, viability, cell-to-cell fusion and hCG-β production, but showed no effects on pCTB proliferation or apoptosis. Furthermore, NSC-23766 and H-89 each blocked the effects of Y-27632, suggesting that Y-27632 significantly enhanced pCTB differentiation via Rac1 and PKA activation. Our findings suggest that Rac1 and PKA may be interactively involved in CTB differentiation, and addition of Y-27632 to cultures may be an effective method for creating a stable culture model for studying CTB and STB biology

  2. 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...

  3. 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

  4. Exploring the active center of human acetylcholinesterase with stereomers of an organophosphorus inhibitor with two chiral centers

    NARCIS (Netherlands)

    Ordentlich, A.; Barak, D.; Kronman, C.; Benschop, H.P.; Jong, L.P.A. de; Ariel, N.; Barak, R.; Segall, Y.; Velan, B.; Shafferman, A.

    1999-01-01

    The stereoselectivity of the phosphonylation reaction and the effects of adduct configuration on the aging process were examined for human acetylcholinesterase (HuAChE) and its selected active center mutants, using the four stereomers of 1,2,2-trimethylpropyl methylphosphonofluoridate (soman). The

  5. 2-Pyridyl P1′-Substituted Symmetry-Based Human Immunodeficiency Virus Protease Inhibitors (A-792611 and A-790742) with Potential for Convenient Dosing and Reduced Side Effects

    Energy Technology Data Exchange (ETDEWEB)

    DeGoey, David A.; Grampovnik, David J.; Flentge, Charles A.; Flosi, William J.; Chen, Hui-ju; Yeung, Clinton M.; Randolph, John T.; Klein, Larry L.; Dekhtyar, Tatyana; Colletti, Lynn; Marsh, Kennan C.; Stoll, Vincent; Mamo, Mulugeta; Morfitt, David C.; Nguyen, Bach; Schmidt, James M.; Swanson, Sue J.; Mo, Hongmei; Kati, Warren M.; Molla, Akhteruzzaman; Kempf, Dale J.; (Abbott)

    2009-06-25

    A series of symmetry-based HIV protease inhibitors was designed and synthesized. Modification of the core regiochemistry and stereochemistry significantly affected the potency, metabolic stability, and oral bioavailability of the inhibitors, as did the variation of a pendent arylmethyl P3 group. Optimization led to the selection of two compounds, 10c (A-790742) and 9d (A-792611), for advancement to preclinical studies. Both compounds displayed low nanomolar potency against wild type HIV in the presence of human serum, low rates of metabolism in human liver microsomes, and high oral bioavailability in animal models. The compounds were examined in a preclinical model for the hyperbilirubinemia observed with some HIV PIs, and both exhibited less bilirubin elevation than comparator compounds. X-ray crystallographic analyses of the new cores were used to examine differences in their binding modes. The antiviral activity of the compounds against protease inhibitor resistant strains of HIV was also determined.

  6. 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

  7. 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.

  8. Pharmacological treatment with inhibitors of nuclear export enhances the antitumor activity of docetaxel in human prostate cancer.

    Science.gov (United States)

    Gravina, Giovanni Luca; Mancini, Andrea; Colapietro, Alessandro; Marampon, Francesco; Sferra, Roberta; Pompili, Simona; Biordi, Leda Assunta; Iorio, Roberto; Flati, Vincenzo; Argueta, Christian; Landesman, Yosef; Kauffman, Michael; Shacham, Sharon; Festuccia, Claudio

    2017-12-19

    Docetaxel (DTX) modestly increases patient survival of metastatic castration-resistant prostate cancer (mCRPC) due to insurgence of pharmacological resistance. Deregulation of Chromosome Region Maintenance (CRM-1)/ exportin-1 (XPO-1)-mediated nuclear export may play a crucial role in this phenomenon. Here, we evaluated the effects of two Selective Inhibitor of Nuclear Export (SINE) compounds, selinexor (KPT-330) and KPT-251, in association with DTX by using 22rv1, PC3 and DU145 cell lines with their. DTX resistant derivatives. We show that DTX resistance may involve overexpression of β-III tubulin (TUBB3) and P-glycoprotein as well as increased cytoplasmic accumulation of Foxo3a. Increased levels of XPO-1 were also observed in DTX resistant cells suggesting that SINE compounds may modulate DTX effectiveness in sensitive cells as well as restore the sensitivity to DTX in resistant ones. Pretreatment with SINE compounds, indeed, sensitized to DTX through increased tumor shrinkage and apoptosis by preventing DTX-induced cell cycle arrest. Basally SINE compounds induce FOXO3a activation and nuclear accumulation increasing the expression of FOXO-responsive genes including p21, p27 and Bim causing cell cycle arrest. SINE compounds-catenin and survivin supporting apoptosis. βdown-regulated Cyclin D1, c-myc, Nuclear sequestration of p-Foxo3a was able to reduce ABCB1 and TUBB3 H2AX levels, prolonged γ expression. Selinexor treatment increased DTX-mediated double strand breaks (DSB), and reduced the levels of DNA repairing proteins including DNA PKc and Topo2A. Our results provide supportive evidence for the therapeutic use of SINE compounds in combination with DTX suggesting their clinical use in mCRPC patients.

  9. The EGFR Inhibitor Gefitinib Enhanced the Response of Human Oral Squamous Cell Carcinoma to Cisplatin In Vitro.

    Science.gov (United States)

    Khalil, Ashraf; Jameson, Mark J

    2017-12-01

    The epidermal growth factor receptor (EGFR) is highly expressed in a variety of solid tumors including oral cavity squamous cell carcinoma (OSCC) and has been implicated in the resistance of these tumors to cisplatin. This study was performed to determine if the EGFR tyrosine kinase inhibitor gefitinib could enhance the cytotoxic effect of cisplatin on OSCC cells in vitro. The expression of EGFR and the phosphorylation of its downstream signaling to ERK, and AKT pathway were detected by Western blotting. Cell proliferation and survival were determined by AlamarBlue and colony formation assay respectively. Cells apoptosis were determined by Western blotting for cleaved PARP protein and by flowcytometry of cells stained with Annexin V and PI. Cal27, OSC19, and SCC25 cells treated with gefitinib 1 μM demonstrated reduced phosphorylation of EGFR, AKT, and ERK proteins with very limited inhibition of proliferation. Cisplatin inhibited proliferation of the same cell lines in a dose-dependent manner. The concentration producing 50% inhibition (IC50) for cisplatin decreased in the presence of gefitinib 1 μM, and a combination of cisplatin 5 µM and gefitinib 1 µM caused synergistic growth inhibition and synergistic reduction in cell survival. The growth inhibitory effect of the combination was associated with reduced ERK and AKT activation, increased poly ADP ribose polymerase (PARP) cleavage, and increased apoptosis. Thus, in OSCC cells in vitro, inhibition of EGFR activity with gefitinib enhances the apoptotic effect of cisplatin. This has potential implications for enhancement of cisplatin effectiveness in tumors that over-express the EGFR.

  10. Safety of postoperative administration of human urinary trypsin inhibitor in lung cancer patients with idiopathic pulmonary fibrosis.

    Directory of Open Access Journals (Sweden)

    Yoshikane Yamauchi

    Full Text Available Patients with idiopathic pulmonary fibrosis (IPF undergoing pulmonary resection for lung cancer carry risks of acute exacerbations of IPF (AE postoperatively. Currently, agents which may attenuate AE are actively sought. Urinary trypsin inhibitor, ulinastatin, is a synthetic glycoprotein which may potentially inhibit various inflammatory factors associated with the development and progression of IPF. The present study was done to evaluate the effects of administration of high dose ulinastatin in lung cancer patients with IPF immediately following lung resection.Patients with IPFs radiologically diagnosed on high resolution CT, and histologically diagnosed resectable lung cancers, were eligible for the study. The effects of escalating doses of ulinastatin 3×10(5, 6×10(5, and 9×10(5 units/body/day, administered postoperatively for 3 days were evaluated. The endpoints were safety and feasibility.Nine patients were evaluated, in cohorts of 3 patients per dosage. Postoperative follow up ranged from 3 to 12 months (median 9 months. The postoperative courses were uneventful in all patients. No subjective adverse events such as abdominal symptoms or skin rashes, or objective adverse events as per serum laboratory tests, such as liver or kidney dysfunctions potentially attributable to ulinastatin administration were observed. AE was seen in one patient at 3 months after surgery, but since this occurred shortly after administration of chemotherapy, it was considered to be attributable to the chemotherapy rather than surgery.Ulinastatin administration after lung resection in lung cancer patients with IPF was considered to be safe and feasible. Further study is planned at the highest dose of this study to evaluate efficacy.UMIN.ac.jp/ctr/UMIN000002410.

  11. 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.

  12. Genetic Mapping and Functional Studies of a Natural Inhibitor of the Insulin Receptor Tyrosine Kinase: The Mouse Ortholog of Human α2-HS Glycoprotein

    Science.gov (United States)

    Cintrón, Vivian J.; Ko, Minoru S. H.; Chi, Kenneth D.; Gross, Jason P.; Srinivas, Pothur R.; Goustin, Anton Scott

    2000-01-01

    Fetuin/α2-HS glycoprotein (α2-HSG) homologs have been identified in several species including rat, sheep, pig, rabbit, guinea pig, cattle, mouse and human. Multiple physiological roles for these homologs have been suggested, including ability to bind to hydroxyapatite crystals and to specifically inhibit the tyrosine kinase (TK) activity of the insulin receptor (IR). In this study we report the identification, cloning, and characterization of the mouse Ahsg gene and its function as an IR-TK inhibitor. Genomic clones derived from a mouse Svj 129 genomic library were sequenced in order to characterize the intron–exon organization of the mouse Ahsg gene, including an 875 bp subclone containing 154 bp upstream from the transcription start site, the first exon, and part of the first intron. A second genomic subclone harboring a 3.45 kb Bgl II fragment contained exons 2, 3 and 4 in addition to two adjacent elements within the first intron-a repetitive element of the B1 family (92 bp) and a 271 bp tract of (T,C)n * (A,G)n. We have mapped mouse Ahsg at 16 cM adjacent to the Diacylglycerol kinase 3 (Dagk3) gene on chromosome 16 by genotyping interspecific backcross panels between C57BL/6J and Mus spretus. The position is syntenic with human chromosome 3q27, where the human AHSG gene resides. Using recombinant mouse α2-HSG expressed from a recombinant baculovirus, we demonstrate that mouse α2-HSG inhibits insulin–stimulated IR autophosphorylation and IR-TKA in vitro. In addition, mouse α2-HSG (25μg/ml) completely abolishes insulin-induced DNA synthesis in H-35 rat hepatoma cells. Based on the sequence data and functional analysis, we conclude that the mouse Ahsg gene is the true ortholog of the human AHSG gene. PMID:11467416

  13. AMG 900, a potent inhibitor of aurora kinases causes pharmacodynamic changes in p-Histone H3 immunoreactivity in human tumor xenografts and proliferating mouse tissues.

    Science.gov (United States)

    Juan, Gloria; Bush, Tammy L; Ma, Connie; Manoukian, Raffi; Chung, Grace; Hawkins, Jennifer M; Zoog, Stephen; Kendall, Richard; Radinsky, Robert; Loberg, Robert; Friberg, Greg; Payton, Marc

    2014-11-04

    The Aurora family of serine-threonine kinases are essential regulators of cell division in mammalian cells. Aurora-A and -B expression and kinase activity is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis. AMG 900 is a highly potent and selective pan-aurora kinase inhibitor that has entered clinical evaluation in adult patients with advanced cancers. In mice, oral administration of AMG 900 blocks the phosphorylation of histone H3 on serine-10 (p-Histone H3), a proximal substrate of aurora-B and inhibits the growth of multiple human tumor xenografts, including multidrug-resistant models. In order to establish a preclinical pharmacokinetic-pharmacodynamic (PK-PD) relationship for AMG 900 that could be translated to the clinic, we used flow cytometry and laser scanning cytometry detection platforms to assess the effects on p-Histone H3 inhibition in terms of sensitivity, precision, and specificity, in human tumor xenografts in conjunction with mouse skin and bone marrow tissues. Mice with established COLO 205 tumors were administered AMG 900 at 3.75, 7.5, and 15 mg/kg and assessed after 3 hours. Significant suppression of p-Histone H3 in mouse skin was only observed at 15 mg/kg (p Histone H3 in tumors and surrogate tissues (although tissues such as skin may be less sensitive for assessing PD effects). To further extend our work, we evaluated the feasibility of measuring p-Histone H3 using fine-needle aspirate (FNA) tumor xenograft biopsies. Treatment with AMG 900 significantly inhibited p-Histone H3 (>99% inhibition, p Histone H3 positive cells using mock FNAs from primary human breast tumor tissues. Phosphorylation of histone H3 is a useful biomarker to determine the pharmacodynamics (PD) activity of AMG 900. FNA biopsies may be a viable approach for assessing AMG 900 PD effects in the clinic.

  14. 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 plate...... correlation between these metabolic and functional activities indicates that there exist, besides cAMP-phosphodiesterase inhibition, additional mechanisms of action for the platelet aggregation inhibitory effect of dihydropyrano- and dihydrofuranocoumarins....... 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...

  15. 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

    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...... 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...

  16. Factor J, an inhibitor of the complement classical pathway: the quantitation by an ELISA inhibition assay in normal human serum.

    Science.gov (United States)

    Jiménez-Clavero, M A; González-Rubio, C; Fontán, G; López-Trascasa, M

    1994-06-01

    Factor J (FJ) is a protein present in human serum, with inhibitory activity against C1. Here we describe the quantitation of FJ in human serum by means of an ELISA inhibition assay. We have purified FJ from the urine of a normal donor following a previously published method with slight modifications. Polyclonal anti-FJ antibodies have been raised in rabbits immunized with a single dose of purified antigen injected in multiple sites. IgG from polyclonal FJ antiserum, coupled to a solid matrix (Affi-Prep gel) was able to adsorb purified FJ antigenically and functionally. Furthermore, anti-FJ specifically retained serum components antigenically related with urine FJ. Taking into account this reactivity, we have developed an inhibition enzyme-linked immunosorbent assay (ELISA) useful for measuring FJ levels in normal human serum. This immunoassay involves preincubating polyclonal anti-FJ with different dilutions of normal human serum to quantitatively reduce the antibody available to bind to purified FJ-coated microtiter plates. Binding of remaining antibody to the microtiter plate is measured spectrophotometrically using peroxidase-conjugated secondary antibody. Quantitation is accomplished by comparison with a known quantity of purified FJ. Conditions for optimization of this quantitative assay have been assessed, including trials with different blocking agents, of which nonfat milk gave the best results. Preliminary experiments showed the existence of paradoxical effects, that is, high nonspecific binding at high serum dilutions. We have eliminated these effects by including high ionic strength (0.4 M NaCl) in the sample incubation solution. Sensitivity and reproducibility parameters have also been established. FJ levels have been measured for the first time in sera from 86 healthy donors.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. 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.

  18. Modeling of cooked starch digestion process using recombinant human pancreatic α-amylase and maltase-glucoamylase for in vitro evaluation of α-glucosidase inhibitors.

    Science.gov (United States)

    Cao, Xiaofang; Zhang, Chen; Dong, Yangyang; Geng, Peng; Bai, Fang; Bai, Gang

    2015-09-23

    In human, digestion of cooked starch mainly involves breaking down of α-amylase to α-limit dextrins and small linear malto-oligosaccharides, which are in turn hydrolyzed to glucose by the gut mucosal maltase-glucoamylase (MGAM). Human pancreatic α-amylase (HPA), amino- and carboxyl-terminal portions of MGAM (ntMGAM and ctMGAM) catalyze the hydrolysis of α-D-(1,4) glycosidic linkages in starch, playing a crucial role in the production of glucose in the human lumen. Accordingly, these enzymes are effective drug targets for the treatments of type 2 diabetes and obesity. In this study, a Plackett-Burman based statistical screening procedure was adopted to determine the most critical factors affecting cooked starch digestion by the combination of HPA, ctMGAM and ntMGAM. Six factors were tested and experimental results showed that pH and temperature were the major influencing factors, with optimal pH and temperature at 6.0 and 50 °C, respectively. Surprisingly, ntMGAM had no significant contribution to the glucose production from starch digestion compared to the HPA and ctMGAM. The optimal proportion of HPA and ctMGAM in a starch digestion system was further determined by response surface methodology. Results showed a maximum starch digestion (88.05%) within 0.5 h when used HPA:ctMGAM=1:9 (U). The inhibitory effects of various inhibitors on the cooked starch digestion by HPA1/ctMGAM9 were evaluated by determining their half maximal inhibitory concentration (IC50) values. Acarviostatin II03 showed the highest inhibitory activity, with 67 times higher potency than acarbose. Moreover, acarviostatin II03 could significantly depress postprandial blood glucose levels in mice, better than that by acarbose. These findings suggest that our in vitro enzymatic system can simulate in vivo starch digestion process, and thus can be used to screen and evaluate α-glucosidase inhibitors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Simultaneous quantitative determination of the HIV protease inhibitors amprenavir, indinavir, nelfinavir, ritonavir and saquinavir in human plasma by ion-pair high-performance liquid chromatography with ultraviolet detection

    NARCIS (Netherlands)

    van Heeswijk, R. P.; Hoetelmans, R. M.; Harms, R.; Meenhorst, P. L.; Mulder, J. W.; Lange, J. M.; Beijnen, J. H.

    1998-01-01

    A reversed-phase high-performance liquid chromatographic assay for the simultaneous quantitative determination of five HIV protease inhibitors (i.e. amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) in human plasma is described. Sample pretreatment consisted of solid-phase extraction

  20. Phase I Trial of the Human Double Minute 2 Inhibitor MK-8242 in Patients With Advanced Solid Tumors.

    Science.gov (United States)

    Wagner, Andrew J; Banerji, Udai; Mahipal, Amit; Somaiah, Neeta; Hirsch, Heather; Fancourt, Craig; Johnson-Levonas, Amy O; Lam, Raymond; Meister, Amy K; Russo, Giuseppe; Knox, Clayton D; Rose, Shelonitda; Hong, David S

    2017-04-20

    profile. The observed clinical activity (partial response and prolonged progression-free survival) provides an impetus for further study of HDM2 inhibitors in liposarcoma.

  1. 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

  2. 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.

  3. On-Line Restricted Access Molecularly Imprinted Solid-Phase Extraction of Selective Serotonin Reuptake Inhibitors Directly from Untreated Human Plasma Samples Followed by HPLC-UV Analysis.

    Science.gov (United States)

    da Silva, Katrine Kyona Muniz Sirgom; Boralli, Vanessa Bergamin; Wisniewski, Célio; Figueiredo, Eduardo Costa

    2016-03-01

    In this article, we proposed a new restricted access molecularly imprinted polymer coated with bovine serum albumin (RAMIP-BSA) to be used in a multidimensional chromatographic system applied for the analysis of six selective serotonin reuptake inhibitors (SSRIs) directly from untreated human plasma samples. Fluoxetine, methacrylic acid and ethylene glycol dimethacrylate were used as the template, functional monomer and cross-linker, respectively. The imprinted polymer was covered with a bovine serum albumin (BSA) layer via the interconnections between the amine groups of the BSA using glutaraldehyde as a cross-linker. The obtained RAMIP-BSA was able to extract the SSRIs directly from the human plasma, while ∼100% of the proteins were excluded from the sample. Selectivity coefficients were calculated for fluoxetine (template) in comparison with venlafaxine, duloxetine, citalopram, fluvoxamine, paroxetine and sertraline, and the values were >1 in all cases, attesting to the presence of binding sites in the imprinted polymer. The method presented analytical ranges from 20 to 500 µg/L and correlation coefficients >0.99 for all of the SSRIs (fluoxetine, venlafaxine, duloxetine, citalopram, fluvoxamine and sertraline). Precision and accuracy presented variation coefficients and relative errors 85%. The proposed method was able to analyze three samples per hour, and each column was used at least 50 times without any significant changes in its performance. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  4. Low concentrations of lithium and cyclooxygenase inhibitors enhance endothelin-1 (ET-1)-induced contractions in human temporal artery, but not in porcine ophthalmic artery.

    Science.gov (United States)

    Bakken, I J; Vincent, M B; White, L R; Cappelen, J; Skaanes, K O; Sjaastad, O

    1992-11-01

    Endothelins are a recently discovered group of potent vasoconstrictor peptides synthesized by endothelial cells and other tissues in various species, which seem to participate in the regulation of vascular tonus. Abnormalities in vasoactivity in the head may be an important event in headache pathophysiology, although the mechanisms responsible for such constrictions and/or dilations are not known. The endothelium and its constrictor peptide, endothelin, may play a key role in such mechanisms. Of the various drugs used in the treatment of headache, lithium is an accepted treatment for cluster headache, and indomethacin is the drug of choice for the associated condition chronic paroxysmal hemicrania. The mechanism of action of these drugs in these headaches is not known. Due to the possible involvement of endothelin in headache disorders, the objective of this study was to verify the effects of lithium and cyclooxygenase inhibitors (indomethacin, acetylsalicylic acid and naproxen) on endothelin-1 (ET-1)-induced contractions in isolated human temporal arteries and porcine ophthalmic arteries. It was found that all drugs increased the (ET-1)-induced contractions in human temporal arteries. Conversely, there were no significant changes induced by the drugs in porcine ophthalmic arteries. These results are consistent with the variation of activity often seen in different vascular beds and between species. The potential importance of such reactions for the understanding of vascular changes putatively involved in headache development and treatment is discussed.

  5. 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.

  6. A novel inhibitor of human La protein with anti-HBV activity discovered by structure-based virtual screening and in vitro evaluation.

    Directory of Open Access Journals (Sweden)

    Jing Tang

    Full Text Available BACKGROUND: Over 350 million people worldwide are infected with hepatitis B virus (HBV, a major cause of liver failure and hepatocellular carcinoma. Current therapeutic agents are highly effective, but are also associated with development of viral resistance. Therefore, strategies for identifying other anti-HBV agents with specific, but distinctive mechanisms of action are needed. The human La (hLa protein, which forms a stabilizing complex with HBV RNA ribonucleoprotein to promote HBV replication, is a promising target of molecular therapy. AIMS: This study aimed to discover novel inhibitors of hLa that could inhibit HBV replication and expression. METHODS: A multistage molecular docking approach was used to screen a Specs database and an in-house library against hLa binding sites. Sequential in vitro evaluations were performed to detect potential compounds with high scores in HepG2.2.15 cells. RESULTS: Of the 26 potential compounds with high scores chosen for experimental verification, 12 had HBV DNA inhibition ratios of less than 50% with P<0.05. Six had significant inhibition of HBV e antigen (HBeAg levels, and 13 had significant inhibition of HBV surface antigen (HBsAg levels by in vitro assays. Compounds HBSC-11, HBSC-15 and HBSC-34 (HBSC is system prefix for active compounds screened by the library were selected for evaluation. HBSC-11 was found to have an obvious inhibitory effect on hLa transcription and expression. CONCLUSIONS: Our findings suggest that anti-HBV activity of HBSC-11 may be mediated by a reduction in hLa levels. In addition, our data suggest the potential clinical use of hLa inhibitors, such as HBSC-11, for treating HBV infection.

  7. 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.

  8. Overcoming bortezomib resistance in human B cells by anti-CD20/rituximab-mediated complement-dependent cytotoxicity and epoxyketone-based irreversible proteasome inhibitors

    Directory of Open Access Journals (Sweden)

    Verbrugge Sue Ellen

    2013-01-01

    Full Text Available Abstract Background In clinical and experimental settings, antibody-based anti-CD20/rituximab and small molecule proteasome inhibitor (PI bortezomib (BTZ treatment proved effective modalities for B cell depletion in lymphoproliferative disorders as well as autoimmune diseases. However, the chronic nature of these diseases requires either prolonged or re-treatment, often with acquired resistance as a consequence. Methods Here we studied the molecular basis of acquired resistance to BTZ in JY human B lymphoblastic cells following prolonged exposure to this drug and examined possibilities to overcome resistance by next generation PIs and anti-CD20/rituximab-mediated complement-dependent cytotoxicity (CDC. Results Characterization of BTZ-resistant JY/BTZ cells compared to parental JY/WT cells revealed the following features: (a 10–12 fold resistance to BTZ associated with the acquisition of a mutation in the PSMB5 gene (encoding the constitutive β5 proteasome subunit introducing an amino acid substitution (Met45Ile in the BTZ-binding pocket, (b a significant 2–4 fold increase in the mRNA and protein levels of the constitutive β5 proteasome subunit along with unaltered immunoproteasome expression, (c full sensitivity to the irreversible epoxyketone-based PIs carfilzomib and (to a lesser extent the immunoproteasome inhibitor ONX 0914. Finally, in association with impaired ubiquitination and attenuated breakdown of CD20, JY/BTZ cells harbored a net 3-fold increase in CD20 cell surface expression, which was functionally implicated in conferring a significantly increased anti-CD20/rituximab-mediated CDC. Conclusions These results demonstrate that acquired resistance to BTZ in B cells can be overcome by next generation PIs and by anti-CD20/rituximab-induced CDC, thereby paving the way for salvage therapy in BTZ-resistant disease.

  9. Proteinase inhibitors in Brazilian leguminosae

    Directory of Open Access Journals (Sweden)

    C. A. M. Sampaio

    1991-01-01

    Full Text Available Serine proteinase inhitors, in the seeds of several Leguminosae from the Pantanal region (West Brazil, were studied using bovine trypsin, a digestive enzyme, Factor XIIa and human plasma Kallikrein, two blood clotting factors. The inhibitors were purified from Enterolobium contortisiliquum (Mr=23,000, Torresea cearensis (Mr = 13,000, Bauhinia pentandra (Mr = 20,000 and Bauhinia bauhinioides (Mr = 20,000. E. contortisiliquum inhibitor inactivates all three enzymes, whereas the T. cearensis inhibitor inactivates trypsin and Factor XSSa, but does nor affect plasma kallikrein; both Bauhinia inhibitors, on the other hand, inactivate trypsin and plasma kallikrein but only the Bpentandra inhibitor affects Factor XIIa. Ki values were calculated between 10 [raised to the power of] -7 and 10 [raised to the power of] -8 M.

  10. 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.

  11. Enhanced neurite outgrowth of human model (NT2) neurons by small-molecule inhibitors of Rho/ROCK signaling.

    Science.gov (United States)

    Roloff, Frank; Scheiblich, Hannah; Dewitz, Carola; Dempewolf, Silke; Stern, Michael; Bicker, Gerd

    2015-01-01

    Axonal injury in the adult human central nervous system often results in loss of sensation and motor functions. Promoting regeneration of severed axons requires the inactivation of growth inhibitory influences from the tissue environment and stimulation of the neuron intrinsic growth potential. Especially glial cell derived factors, such as chondroitin sulfate proteoglycans, Nogo-A, myelin-associated glycoprotein, and myelin in general, prevent axon regeneration. Most of the glial growth inhibiting factors converge onto the Rho/ROCK signaling pathway in neurons. Although conditions in the injured nervous system are clearly different from those during neurite outgrowth in vitro, here we use a chemical approach to manipulate Rho/ROCK signalling with small-molecule agents to encourage neurite outgrowth in cell culture. The development of therapeutic treatments requires drug testing not only on neurons of experimental animals, but also on human neurons. Using human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA (Ras homolog gene family, member A GTPase) activation and promotes neurite growth. Inhibition of the downstream effector Rho kinase by the drug Y-27632 results in a strong increase in neurite outgrowth. Conversely, activation of the Rho pathway by lysophosphatidic acid results in growth cone collapse and eventually to neurite retraction. Finally, we show that blocking of Rho kinase, but not RhoA results in an increase in neurons bearing neurites. Due to its anti-inflammatory and neurite growth promoting action, the use of a pharmacological treatment of damaged neural tissue with Ibuprofen should be explored.

  12. Enhanced neurite outgrowth of human model (NT2 neurons by small-molecule inhibitors of Rho/ROCK signaling.

    Directory of Open Access Journals (Sweden)

    Frank Roloff

    Full Text Available Axonal injury in the adult human central nervous system often results in loss of sensation and motor functions. Promoting regeneration of severed axons requires the inactivation of growth inhibitory influences from the tissue environment and stimulation of the neuron intrinsic growth potential. Especially glial cell derived factors, such as chondroitin sulfate proteoglycans, Nogo-A, myelin-associated glycoprotein, and myelin in general, prevent axon regeneration. Most of the glial growth inhibiting factors converge onto the Rho/ROCK signaling pathway in neurons. Although conditions in the injured nervous system are clearly different from those during neurite outgrowth in vitro, here we use a chemical approach to manipulate Rho/ROCK signalling with small-molecule agents to encourage neurite outgrowth in cell culture. The development of therapeutic treatments requires drug testing not only on neurons of experimental animals, but also on human neurons. Using human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA (Ras homolog gene family, member A GTPase activation and promotes neurite growth. Inhibition of the downstream effector Rho kinase by the drug Y-27632 results in a strong increase in neurite outgrowth. Conversely, activation of the Rho pathway by lysophosphatidic acid results in growth cone collapse and eventually to neurite retraction. Finally, we show that blocking of Rho kinase, but not RhoA results in an increase in neurons bearing neurites. Due to its anti-inflammatory and neurite growth promoting action, the use of a pharmacological treatment of damaged neural tissue with Ibuprofen should be explored.

  13. Raf kinase inhibitor protein (RKIP) inhibits the cell migration and invasion in human glioma cell lines in vitro.

    Science.gov (United States)

    Lei, Xuhui; Chang, Liang; Ye, Wei; Jiang, Chuanlu; Zhang, Zhiren

    2015-01-01

    To investigate the effects and the potential mechanisms of RKIP on cell migration, invasion and proliferation in human glioma cell lines in vitro. The RKIP over-expressing and RKIP knockdown human U87 glioma cells were used to reveal the effects of RKIP on human glioma cells migration, invasion and proliferation. After the recombinant plasmid pcDNA3.0-RKIP or RKIP-shRNA was transfected into the cell lines U87 by the means of liposome assay, the cells migration, invasion and proliferation were detected by wound healing, Transwell and MTT assay. Then, the levels of RKIP, MMP-3, MMP-9 and HMGA2 mRNA transcription were measured by means of RT-qPCR and levels of proteins expressions were determined using Western blot. The results of MTT assay suggested that the PKIP have little inhibitive effects on glioma cells proliferation (P>0.05). The present paper showed that the migration distances in the group of RKIP-shRNA were markedly increased compared to the pcDNA3.0-RKIP and control. Similarly, the results showed that the numbers of invasion cells in RKIP-shRNA were remarkably increased than the pcDNA3.0-RKIP group and control group. Western blot and RT-qPCR suggested that over-expressions of RKIP lessened the MMP-2, MMP-9 and HMGA2 expression, however, turning down the RKIP expression showed the inverse effects. RKIP inhibits the cells migrations and invasions. Meanwhile, RKIP might inhibit the glioma cells through inhibiting MMPs and HMAG2 expression. Therefore, we demonstrated that RKIP is an underlying target for the treatment of glioma.

  14. Synthesis and Enantiomeric Separation of a Novel Spiroketal Derivative: A Potent Human Telomerase Inhibitor with High in Vitro Anticancer Activity.

    Science.gov (United States)

    Fuggetta, Maria Pia; De Mico, Antonella; Cottarelli, Andrea; Morelli, Franco; Zonfrillo, Manuela; Ulgheri, Fausta; Peluso, Paola; Mannu, Alberto; Deligia, Francesco; Marchetti, Mauro; Roviello, Giovanni; Reyes Romero, Atilio; Dömling, Alexander; Spanu, Pietro

    2016-10-13

    The synthesis, the enantiomeric separation, and the characterization of new simple spiroketal derivatives have been performed. The synthesized compounds have shown a very high anticancer activity. Cell proliferation assay showed that they induce a remarkable inhibition of cell proliferation in all cell lines treated, depending on culture time and concentration. The compounds have also shown a potent nanomolar human telomerase inhibition activity and apoptosis induction. CD melting experiments demonstrate that spiroketal does not affect the G-quadruplex (G4) thermal stability. Docking studies showed that telomerase inhibition could be determined by a spiroketal interaction with the telomerase enzyme.

  15. High yield purification and characterization of engineered human P450 1A2 and generation of immuno-inhibitor antibodies.

    Science.gov (United States)

    Louërat-Oriou, B; Flinois, J P; Beaune, P H; Pompon, D

    1999-02-01

    P450 S12, an engineered human P450 1A2 containing the 88-first amino-acids of the P450 1A1, demonstrates particularly high expression level in yeast while exhibiting catalytic properties very similar to the moderately expressed natural human P450 1A2. To facilitate P450 purification by nickel chelate chromatography, C-terminal extensions including histidine tags were tested. The -G(H)4 extension was found to be particularly efficient for permitting high expression levels without any catalytic alteration. This engineered P450 was purified to electrophoretic homogeneity (18 nmol/mg of protein) at a very high yield (87%) without any detectable formation of P420. P450 S12 activities were reconstituted in the presence of yeast and Arabidopsis thaliana (ATR1) NADPH-P450 reductases. The plant reductase supported better ethoxyresorufin-, methoxyresorufin- and phenacetin-O-dealkylase activities than the yeast reductase in reconstituted systems. Interestingly, polyclonal antibodies raised against purified P450 S12 selectively recognized in Western blot and fully immuno-inhibited the natural or recombinant P450 1A2 with very limited or no cross-reaction with P450 1A1 and other isoenzymes.

  16. 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

  17. Regulation of proliferation in developing human tooth germs by MSX homeodomain proteins and cyclin-dependent kinase inhibitor p19INK4d.

    Science.gov (United States)

    Kero, Darko; Vukojevic, Katarina; Stazic, Petra; Sundov, Danijela; Mardesic Brakus, Snjezana; Saraga-Babic, Mirna

    2017-10-02

    Before the secretion of hard dental tissues, tooth germs undergo several distinctive stages of development (dental lamina, bud, cap and bell). Every stage is characterized by specific proliferation patterns, which is regulated by various morphogens, growth factors and homeodomain proteins. The role of MSX homeodomain proteins in odontogenesis is rather complex. Expression domains of genes encoding for murine Msx1/2 during development are observed in tissues containing highly proliferative progenitor cells. Arrest of tooth development in Msx knockout mice can be attributed to impaired proliferation of progenitor cells. In Msx1 knockout mice, these progenitor cells start to differentiate prematurely as they strongly express cyclin-dependent kinase inhibitor p19 INK4d . p19 INK4d induces terminal differentiation of cells by blocking the cell cycle in mitogen-responsive G1 phase. Direct suppression of p19 INK4d by Msx1 protein is, therefore, important for maintaining proliferation of progenitor cells at levels required for the normal progression of tooth development. In this study, we examined the expression patterns of MSX1, MSX2 and p19 INK4d in human incisor tooth germs during the bud, cap and early bell stages of development. The distribution of expression domains of p19 INK4d throughout the investigated period indicates that p19 INK4d plays active role during human tooth development. Furthermore, comparison of expression domains of p19 INK4d with those of MSX1, MSX2 and proliferation markers Ki67, Cyclin A2 and pRb, indicates that MSX-mediated regulation of proliferation in human tooth germs might not be executed by the mechanism similar to one described in developing tooth germs of wild-type mouse.