RESUMO
11beta-hydroxysteroid dehydrogenase 1 regulates the tissue availability of cortisol by interconverting cortisone and cortisol. It is capable of functioning as both a reductase and a dehydrogenase depending upon the surrounding milieu. In this work, we have studied the reaction mechanism of a soluble form of human 11beta-hydroxysteroid dehydrogenase 1 and its mode of inhibition by potent and selective inhibitors belonging to three different structural classes. We found that catalysis follows an ordered addition with NADP(H) binding preceding the binding of the steroid. While all three inhibitors tested bound to the steroid binding pocket, they differed in their interactions with the cofactor NADP(H). Compound A, a pyridyl amide bound more efficiently to the NADPH-bound form of 11beta-hydroxysteroid dehydrogenase 1. Compound B, an adamantyl triazole, was unaffected by NADP(H) binding and the sulfonamide, Compound C, showed preferential binding to the NADP+ -bound form of 11beta-hydroxysteroid dehydrogenase 1. These differences were found to augment significant selectivity towards inhibition of the reductase reaction versus the dehydrogenase reaction. This selectivity may translate to differences in the in vivo effects of 11beta-hydroxysteroid dehydrogenase 1 inhibitors.
Assuntos
11-beta-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenases/metabolismo , Piridinas/farmacologia , Sulfonamidas/farmacologia , Triazóis/farmacologia , Humanos , Cinética , NADP/metabolismoRESUMO
INTRODUCTION: The time required to obtain Institutional Review Board (IRB) approval is a frequent subject of efforts to reduce unnecessary delays in initiating clinical trials. This study was conducted by and for IRB directors to better understand factors affecting approval times as a first step in developing a quality improvement framework. METHODS: 807 IRB-approved clinical trials from 5 University of California campuses were analyzed to identify operational and clinical trial characteristics influencing IRB approval times. RESULTS: High workloads, low staff ratios, limited training, and the number and types of ancillary reviews resulted in longer approval times. Biosafety reviews and the need for billing coverage analysis were ancillary reviews that contributed to the longest delays. Federally funded and multisite clinical trials had shorter approval times. Variability in between individual committees at each institution reviewing phase 3 multisite clinical trials also contributed to delays for some protocols. Accreditation was not associated with shorter approval times. CONCLUSIONS: Reducing unnecessary delays in obtaining IRB approval will require a quality improvement framework that considers operational and study characteristics as well as the larger institutional regulatory environment.
RESUMO
Reversible tetrapeptide-based compounds have been shown to effectively inhibit the hepatitis C virus NS3.4A protease. Inhibition of viral replicon RNA production in Huh-7 cells has also been demonstrated. We show herein that the inclusion of hydrogen bond donors on the P4 capping group of tetrapeptide-based inhibitors result in increased binding potency to the NS3.4A protease. The capping groups also impart significant effects on the pharmacokinetic profile of these inhibitors.
Assuntos
Antivirais/farmacocinética , Hepacivirus/efeitos dos fármacos , Inibidores de Proteases/farmacocinética , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/síntese química , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Desenho de Fármacos , Hepacivirus/enzimologia , Ligação de Hidrogênio , Camundongos , Testes de Sensibilidade Microbiana , Oligopeptídeos/antagonistas & inibidores , Inibidores de Proteases/síntese química , Relação Estrutura-Atividade , Replicação Viral/fisiologiaRESUMO
VX-950 is a potent, selective, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3-4A serine protease, and it demonstrated excellent antiviral activity both in genotype 1b HCV replicon cells (50% inhibitory concentration [IC50] = 354 nM) and in human fetal hepatocytes infected with genotype 1a HCV-positive patient sera (IC50 = 280 nM). VX-950 forms a covalent but reversible complex with the genotype 1a HCV NS3-4A protease in a slow-on, slow-off process with a steady-state inhibition constant (K(i)*) of 7 nM. Dissociation of the covalent enzyme-inhibitor complex of VX-950 and genotype 1a HCV protease has a half-life of almost an hour. A >4-log10 reduction in the HCV RNA levels was observed after a 2-week incubation of replicon cells with VX-950, with no rebound of viral RNA observed after withdrawal of the inhibitor. In several animal species, VX-950 exhibits a favorable pharmacokinetic profile with high exposure in the liver. In a recently developed HCV protease mouse model, VX-950 showed excellent inhibition of HCV NS3-4A protease activity in the liver. Therefore, the overall preclinical profile of VX-950 supports its candidacy as a novel oral therapy against hepatitis C.
Assuntos
Hepacivirus/enzimologia , Oligopeptídeos/farmacologia , Oligopeptídeos/farmacocinética , Inibidores de Serina Proteinase/farmacologia , Inibidores de Serina Proteinase/farmacocinética , Administração Oral , Animais , Área Sob a Curva , Sítios de Ligação , Disponibilidade Biológica , Linhagem Celular , Células Cultivadas , Cães , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Hepacivirus/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos SCID , Oligopeptídeos/administração & dosagem , RNA Viral/fisiologia , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Replicon/fisiologia , Inibidores de Serina Proteinase/administração & dosagem , Especificidade por SubstratoRESUMO
VX-950 is a potent, small molecule, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease and has recently been shown to possess antiviral activity in a phase I trial in patients chronically infected with genotype 1 HCV. In a previous study, we described in vitro resistance mutations against either VX-950 or another HCV NS3.4A protease inhibitor, BILN 2061. Single amino acid substitutions that conferred drug resistance (distinct for either inhibitor) were identified in the HCV NS3 serine protease domain. The dominant VX-950-resistant mutant (A156S) remains sensitive to BILN 2061. The major BILN 2061-resistant mutants (D168V and D168A) are fully susceptible to VX-950. Modeling analysis suggested that there are different mechanisms of resistance for these mutations induced by VX-950 or BILN 2061. In this study, we identified mutants that are cross-resistant to both HCV protease inhibitors. The cross-resistance conferred by substitution of Ala(156) with either Val or Thr was confirmed by characterization of the purified enzymes and reconstituted replicon cells containing the single amino acid substitution A156V or A156T. Both cross-resistance mutations (A156V and A156T) displayed significantly diminished fitness (or replication capacity) in a transient replicon cell system.
Assuntos
Carbamatos/farmacologia , Farmacorresistência Viral , Hepacivirus/enzimologia , Compostos Macrocíclicos/farmacologia , Mutação , Oligopeptídeos/farmacologia , Quinolinas/farmacologia , Inibidores de Serina Proteinase/farmacologia , Tiazóis/farmacologia , Proteínas não Estruturais Virais/farmacologia , Substituição de Aminoácidos , Aminoácidos/química , Ácido Aspártico/química , Sítios de Ligação , Genes Dominantes , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Humanos , Técnicas In Vitro , Concentração Inibidora 50 , Cinética , Modelos Químicos , Modelos Moleculares , RNA Viral/fisiologia , Replicon/fisiologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
20-fluoro-17(20)-pregnenolone derivatives were designed as enol mimics of pregnenolone. All of the targeted, novel fluoroolefins were potent inhibitors of C17(20) lyase.
Assuntos
Esteroide 17-alfa-Hidroxilase/antagonistas & inibidores , Esteroides Fluorados/síntese química , Compostos de Vinila/farmacologia , Animais , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Macaca fascicularis , Pregnenolona/análogos & derivados , Esteroide 17-alfa-Hidroxilase/metabolismo , Esteroides Fluorados/farmacologia , Relação Estrutura-Atividade , Compostos de Vinila/síntese química , Compostos de Vinila/químicaRESUMO
Novel 21,21-difluorovinyl steroids, designed as difluorinated C20(21) enol mimics of pregnenolone, were targeted as potential mechanism-based inhibitors of C17(20) lyase, a crucial enzyme in the biosynthesis of testosterone. Addition of (difluoromethyl)diphenylphosphine oxide reagent to 17-acetyl steroids was the approach chosen for the construction of these compounds. Of particular interest were the abnormal Wittig products which formed during attempted preparation of the triene 9. The target difluoroolefin 3 was found to be a moderately potent, time-dependent inhibitor of the enzyme.
Assuntos
Pregnenolona/química , Pregnenolona/farmacologia , Esteroide 17-alfa-Hidroxilase/antagonistas & inibidores , Esteroides Fluorados/química , Esteroides Fluorados/farmacologia , Animais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Macaca fascicularis , Masculino , Relação Estrutura-Atividade , Testículo/enzimologia , Fatores de TempoRESUMO
We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3.4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3.4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3.4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. The major BILN 2061-resistant mutations at Asp(168) are fully susceptible to VX-950, and the dominant resistant mutation against VX-950 at Ala(156) remains sensitive to BILN 2061. Modeling analysis suggests that there are different mechanisms of resistance to VX-950 and BILN 2061.
Assuntos
Carbamatos/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/enzimologia , Compostos Macrocíclicos , Oligopeptídeos/farmacologia , Quinolinas , Inibidores de Serina Proteinase/farmacologia , Tiazóis/farmacologia , Proteínas não Estruturais Virais/química , Aminoácidos/química , Ácido Aspártico/química , Sítios de Ligação , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Genes Dominantes , Concentração Inibidora 50 , Cinética , Modelos Químicos , Modelos Moleculares , Mutação , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Fatores de Tempo , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
Tetrapeptide-based peptidomimetic compounds have been shown to effectively inhibit the hepatitis C virus NS3.4A protease without the need of a charged functionality. An aldehyde is used as a prototype reversible electrophilic warhead. The SAR of the P1 and P2 inhibitor positions is discussed.
Assuntos
Hepacivirus/enzimologia , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Inibidores de Proteases/síntese química , Hepacivirus/efeitos dos fármacos , Cinética , Modelos Moleculares , Inibidores de Proteases/farmacologia , Conformação Proteica , Relação Estrutura-Atividade , Difração de Raios XRESUMO
The alpha-ketoamide warhead (e.g., 15) was found to be a practical replacement for aliphatic aldehydes in a series of HCV NS3.4A protease inhibitors. Structure-activity relationships and prime side optimization are discussed.
Assuntos
Hepacivirus/enzimologia , Compostos Macrocíclicos , Quinolinas , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Carbamatos/química , Carbamatos/metabolismo , Hepacivirus/efeitos dos fármacos , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
We recently described the identification of an optimized alpha-ketoamide warhead for our series of HCV NS3.4A inhibitors. We report herein a series of HCV protease inhibitors incorporating 3-alkyl-substituted prolines in P(2). These compounds show exceptional enzymatic and cellular potency given their relatively small size. The marked enhancement of activity of these 3-substituted proline derivatives relative to previously reported 4-hydroxyproline derivatives constitutes additional evidence for the importance of the S(2) binding pocket as the defining pharmacophore for inhibition of the NS3.4A enzyme.