RESUMO
Inhibition of NS5A has emerged as an attractive strategy to intervene in hepatitis C virus (HCV) replication. Ruzasvir (formerly MK-8408) was developed as a novel NS5A inhibitor to improve upon the potency and barrier to resistance of early compounds. Ruzasvir inhibited HCV RNA replication with 50% effective concentrations (EC50s) of 1 to 4 pM in Huh7 or Huh7.5 cells bearing replicons for HCV genotype 1 (GT1) to GT7. The antiviral activity was modestly (10-fold) reduced in the presence of 40% normal human serum. The picomolar potency in replicon cells extended to sequences of clinical isolates available in public databases that were synthesized and tested as replicons. In GT1a, ruzasvir inhibited common NS5A resistance-associated substitutions (RASs), with the exception of M28G. De novo resistance selection studies identified pathways with certain amino acid substitutions at residues 28, 30, 31, and 93 across genotypes. Substitutions at position 93 were more common in GT1 to -4, while changes at position 31 emerged frequently in GT5 and -6. With the exception of GT4, the reintroduction of selected RASs conferred a ≥100-fold potency reduction in the antiviral activity of ruzasvir. Common RASs from other classes of direct-acting antiviral agents (DAAs) did not confer cross-resistance to ruzasvir. The interaction of ruzasvir with an NS3/4A protease inhibitor (grazoprevir) and an NS5B polymerase prodrug (uprifosbuvir) was additive to synergistic, with no evidence of antagonism or cytotoxicity. The antiviral profile of ruzasvir supported its further evaluation in human trials in combination with grazoprevir and uprifosbuvir.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Pirrolidinas/farmacologia , Tiazóis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Amidas , Carbamatos , Linhagem Celular Tumoral , Ciclopropanos , Farmacorresistência Viral/efeitos dos fármacos , Quimioterapia Combinada/métodos , Genótipo , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/virologia , Humanos , Inibidores de Proteases/farmacologia , Quinoxalinas/farmacologia , Replicon/efeitos dos fármacos , Sulfonamidas , Uridina/análogos & derivados , Uridina/farmacologiaRESUMO
(2'R)-Ethynyl uridine 3, and its (2'S)-diastereomer 10, are synthesised in a divergent fashion from the inexpensive parent nucleoside. Both nucleoside analogues are obtained from a total of 5 simple synthetic steps and 3 trivial column chromatography purifications. To evaluate their effectiveness against HCV NS5B polymerase, the nucleosides were converted to their respective 5'-O-triphosphates. Subsequently, this lead to the discovery of the 2'-ß-ethynyl 18 and -propynyl 20 nucleotides having significantly improved potency over Sofosbuvir triphosphate 24.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Nucleosídeos/farmacologia , Uridina/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Conformação Molecular , Nucleosídeos/síntese química , Nucleosídeos/química , Relação Estrutura-Atividade , Uridina/análogos & derivados , Uridina/químicaRESUMO
HCV infections are the leading causes for hepatocellular carcinoma and liver transplantation in the United States. Recent advances in drug discovery have identified direct acting antivirals which have significantly improved cure rates in patients. Current efforts are directed towards identification of novel direct acting antiviral targeting different mechanism of actions which could become part of all oral therapies. We recently disclosed the identification of a novel tricyclic indole derived inhibitors of HCV NS5B polymerase that bound to the enzyme close to the active site. In this manuscript we describe further optimization of potency and pharmacokinetics (PK) of these inhibitors to identify compounds in low nM potency against gt-1b. These analogs also demonstrate excellent PK in rats and monkeys when administered as a dimethyl ethyl amino ester prodrug.
Assuntos
Ésteres/farmacocinética , Hepacivirus/efeitos dos fármacos , Indóis/farmacocinética , Proteínas não Estruturais Virais/antagonistas & inibidores , Administração Oral , Animais , Descoberta de Drogas , Ésteres/química , Haplorrinos , Hepacivirus/enzimologia , Humanos , Indóis/química , Pró-Fármacos/farmacologia , Ratos , Relação Estrutura-AtividadeRESUMO
The discovery of lead compound 2e was described. Its covalent binding to HCV NS5B polymerase enzyme was investigated by X-ray analysis. The results of distribution, metabolism and pharmacokinetics were reported. Compound 2e was demonstrated to be potent (replicon GT-1b EC50 = 0.003 µM), highly selective, and safe in in vitro and in vivo assays.
Assuntos
Inibidores Enzimáticos/química , Hepacivirus/enzimologia , Indóis/química , Quinolinas/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Cristalografia por Raios X , Cães , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Haplorrinos , Humanos , Indóis/síntese química , Indóis/farmacocinética , Indóis/farmacologia , Masculino , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Quinolinas/síntese química , Quinolinas/farmacocinética , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas não Estruturais Virais/metabolismoRESUMO
The characterization of HCV genome has identified various vital functional proteins involved in the life cycle of hepatitis C virus. This has resulted in many novel enzymatic targets that are potential for development of therapeutic agents. The HCV RNA dependent RNA polymerase (HCV NS5B) is one such essential enzyme for HCV replication that has been well characterized and studied by various groups to develop novel therapies for hepatitis C. In this paper, we describe our efforts towards the identification and structure-activity relationship (SAR) of novel tricyclic indole derivatives that bind close to the palm site of the NS5B polymerase. X-ray crystal structure of an inhibitor bound to the polymerase is also described.
Assuntos
Antivirais/química , Antivirais/farmacologia , Hepacivirus/enzimologia , Indóis/química , Indóis/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Hepacivirus/química , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Simulação de Acoplamento Molecular , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Relação Estrutura-AtividadeRESUMO
Development of SAR at the C2 position of indole lead 1, a palm site inhibitor of HCV NS5B polymerase (NS5B IC(50)=0.053µM, replicon EC(50)=4.8µM), is described. Initial screening identified an acyl sulfonamide moiety as an isostere for the C2 carboxylic acid group. Further SAR investigation resulted in identification of acyl sufonamide analog 7q (NS5B IC(50)=0.039µM, replicon EC(50)=0.011µM) with >100-fold improved replicon activity.
Assuntos
Antivirais/farmacologia , Indóis/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Química Farmacêutica/métodos , Cristalografia por Raios X/métodos , Desenho de Fármacos , Humanos , Ligação de Hidrogênio , Concentração Inibidora 50 , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
SAR development of indole-based palm site inhibitors of HCV NS5B polymerase exemplified by initial indole lead 1 (NS5B IC(50)=0.9 µM, replicon EC(50)>100 µM) is described. Structure-based drug design led to the incorporation of novel heterocyclic moieties at the indole C3-position which formed a bidentate interaction with the protein backbone. SAR development resulted in leads 7q (NS5B IC(50)=0.032 µM, replicon EC(50)=1.4 µM) and 7r (NS5B IC(50)=0.017 µM, replicon EC(50)=0.3 µM) with improved enzyme and replicon activity.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos/farmacologia , Indóis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Ácidos Carboxílicos , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/química , Indóis/síntese química , Indóis/química , Modelos Moleculares , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismoRESUMO
SAR exploration from an initial hit, (S)-N-(2-cyclohexenylethyl)-2-fluoro-6-(2-(1-hydroxy-3-phenylpropan-2-ylamino)-2-oxoethoxy)benzamide (1), identified using our proprietary automated ligand identification system (ALIS),(1) has led to a novel series of selective hepatitis C virus (HCV) NS5B polymerase inhibitors with improved in vitro potency as exemplified by (S)-2-fluoro-6-(2-(1-hydroxy-3-phenylpropan-2-ylamino)-2-oxoethoxy)-N-isopentyl-N-methylbenzamidecarboxamide (41) (IC(50)=0.5 microM). The crystal structure of an analogue (44) was solved and provided rationalization of the SAR of this series, which binds in a distinct manner in the palm domain of NS5B, consistent with biochemical analysis using enzyme mutant variants. These data warrant further lead optimization efforts on this novel series of non-nucleoside inhibitors targeting the HCV polymerase.
Assuntos
Benzamidas/química , Benzamidas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hepacivirus/enzimologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Antivirais/química , Antivirais/farmacologia , Cristalografia , Desenho de Fármacos , Hepatite C/tratamento farmacológico , Humanos , Modelos Moleculares , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/químicaRESUMO
The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase is essential for viral replication and a clinically validated antiviral target. Discovery of HCV polymerase inhibitors is greatly facilitated by the availability of a suitable biochemical assay using purified NS5B. We describe here a novel NS5B polymerase assay of de novo initiated RNA synthesis directed from a synthetic heteropolymeric RNA template modified with dideoxycytidine at its 3'-end. This assay has been successfully used for screening and characterization of both initiation and elongation inhibitors of the HCV NS5B polymerase.
Assuntos
Ensaios Enzimáticos/métodos , Hepacivirus/genética , Hepacivirus/metabolismo , RNA Viral/biossíntese , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Antivirais/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Humanos , Moldes Genéticos , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
Pyridine carboxamide-based inhibitors of the hepatitis C virus (HCV) NS5B polymerase were diversified and optimized to a variety of topologically related scaffolds. In particular, the 2-methyl nicotinic acid scaffold was developed into inhibitors with improved biochemical (IC50-GT1b = 0.014 µM) and cell-based HCV replicon potency (EC50-GT1b = 0.7 µM). Biophysical and biochemical characterization identified this novel series of compounds as palm site binders to HCV polymerase.
RESUMO
The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) essential for replication of the viral RNA genome. Purified NS5B has been reported to exhibit multiple activities in vitro. Using a synthetic heteropolymeric RNA template with dideoxycytidine at its 3'-end, we examined de novo initiation and primer extension in a system devoid of self-priming and terminal nucleotide transferase activities. Products predominantly of template size and its multiples were detected. High concentrations of nucleoside triphosphates (K(app)(m) approximately 100-400 mum) corresponding to the first three incorporated nucleotides were found to be required for efficient de novo RNA synthesis. In the presence of initiating di- or trinucleotides, however, the amount of NTP needed to achieve maximal activity dropped 10(3)- to 10(4)-fold, revealing a much reduced nucleotide requirement for elongation (K(app)(m) approximately 0.03-0.09 microm). Accordingly, single round extension from an exogenous primer following preincubation of the enzyme with template and primer could also be supported by <0.1 microm levels of NTP. De novo synthesis at high NTP concentrations was shown to be preferred over primer extension. On a dideoxycytidine-blocked synthetic RNA template derived from the 3'-end of the HCV(-)UTR, the addition of the corresponding initiating trinucleotide also dramatically reduced the NTP levels needed to achieve efficient RNA synthesis. Thus, distinct nucleotide requirements exist for initiation and elongation steps catalyzed by the HCV NS5B polymerase.