RESUMEN
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.
Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Pirrolidinas/farmacología , Tiazoles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Amidas , Carbamatos , Línea Celular Tumoral , Ciclopropanos , Farmacorresistencia Viral/efectos de los fármacos , Quimioterapia Combinada/métodos , Genotipo , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/virología , Humanos , Inhibidores de Proteasas/farmacología , Quinoxalinas/farmacología , Replicón/efectos de los fármacos , Sulfonamidas , Uridina/análogos & derivados , Uridina/farmacologíaRESUMEN
BACKGROUND: In HCV-infected people who fail to achieve sustained virological response after receiving a direct-acting antiviral regimen, virological failure is almost always accompanied by the presence of resistance-associated substitutions (RASs) in the target protein(s). The aim of this long-term observational study was to evaluate the persistence of NS3/4A and NS5A RASs in participants with genotype (GT) 1 infection who relapsed following treatment with a grazoprevir-containing treatment regimen. METHODS: RASs were evaluated at baseline (that is, pre-dose on day 1 of the original treatment), at the time of virological failure, and up to follow-up week 96. A total of 58 participants were included. RESULTS: In participants treated with elbasvir/grazoprevir ± ribavirin, observed baseline NS3 RASs included 56F, 80K/L, 122N and 170V/I, and observed treatment-emergent NS3 RASs included 36M, 56F/H, 122G, 132I, 156G/I/L/P/T, 168A/E/G/V/Y and 170T. Observed baseline NS5A RASs included 28M/T/V, 30H/R, 31M/V and 93H/N, and treatment-emergent NS5A RASs included 28A/G/S/T, 30H/R, 31M/V and 93H/N/S. Baseline NS3 and NS5A RASs present at time of failure tended to persist during follow-up, and most were detectable for more than 2 years following virological failure. Treatment-emergent NS5A RASs present at time of failure also tended to persist for more than 2 years following virological failure (93%). By contrast, >80% of treatment-emergent NS3 RASs detected at failure had been supplanted by wild type by week 36. CONCLUSIONS: Treatment-emergent NS5A RASs can persist for extended periods of time. Retreatment strategies should take account of the presence of these RASs.
Asunto(s)
Antivirales/uso terapéutico , Benzofuranos/uso terapéutico , Hepacivirus/efectos de los fármacos , Hepatitis C Crónica/tratamiento farmacológico , Imidazoles/uso terapéutico , Quinoxalinas/uso terapéutico , Serina Proteasas/genética , Proteínas no Estructurales Virales/genética , Adulto , Sustitución de Aminoácidos , Esquema de Medicación , Combinación de Medicamentos , Farmacorresistencia Viral/genética , Femenino , Estudios de Seguimiento , Expresión Génica , Genotipo , Hepacivirus/clasificación , Hepacivirus/genética , Hepatitis C Crónica/etnología , Hepatitis C Crónica/virología , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Recurrencia , Ribavirina/uso terapéutico , Insuficiencia del Tratamiento , Carga Viral/efectos de los fármacosRESUMEN
We describe the discovery of MK-6169, a potent and pan-genotype hepatitis C virus NS5A inhibitor with optimized activity against common resistance-associated substitutions. SAR studies around the combination of changes to both the valine and aminal carbon region of elbasvir led to the discovery of a series of compounds with substantially improved potency against common resistance-associated substitutions in the major genotypes, as well as good pharmacokinetics in both rat and dog. Through further optimization of key leads from this effort, MK-6169 (21) was discovered as a preclinical candidate for further development.
Asunto(s)
Antivirales/farmacología , Descubrimiento de Drogas , Farmacorresistencia Viral/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/farmacocinética , Perros , Genotipo , Hepacivirus/genética , Hepacivirus/metabolismo , Masculino , Ratas , Distribución TisularRESUMEN
Although genotype 4 (GT4)-infected patients represent a minor overall percentage of the global hepatitis C virus (HCV)-infected population, the high prevalence of the genotype in specific geographic regions coupled with substantial sequence diversity makes it an important genotype to study for antiviral drug discovery and development. We evaluated two direct-acting antiviral agents-grazoprevir, an HCV NS3/4A protease inhibitor, and elbasvir, an HCV NS5A inhibitor-in GT4 replicons prior to clinical studies in this genotype. Following a bioinformatics analysis of available GT4 sequences, a set of replicons bearing representative GT4 clinical isolates was generated. For grazoprevir, the 50% effective concentration (EC50) against the replicon bearing the reference GT4a (ED43) NS3 protease and NS4A was 0.7 nM. The median EC50 for grazoprevir against chimeric replicons encoding NS3/4A sequences from GT4 clinical isolates was 0.2 nM (range, 0.11 to 0.33 nM; n = 5). The difficulty in establishing replicons bearing NS3/4A resistance-associated substitutions was substantially overcome with the identification of a G162R adaptive substitution in NS3. Single NS3 substitutions D168A/V identified from de novo resistance selection studies reduced grazoprevir antiviral activity by 137- and 47-fold, respectively, in the background of the G162R replicon. For elbasvir, the EC50 against the replicon bearing the reference full-length GT4a (ED43) NS5A gene was 0.0002 nM. The median EC50 for elbasvir against chimeric replicons bearing clinical isolates from GT4 was 0.0007 nM (range, 0.0002 to 34 nM; n = 14). De novo resistance selection studies in GT4 demonstrated a high propensity to suppress the emergence of amino acid substitutions that confer high-potency reductions to elbasvir. Phenotypic characterization of the NS5A amino acid substitutions identified (L30F, L30S, M31V, and Y93H) indicated that they conferred 15-, 4-, 2.5-, and 7.5-fold potency losses, respectively, to elbasvir. The activity profiles of grazoprevir and elbasvir supported the testing of the direct-acting antivirals in clinical studies.
Asunto(s)
Antivirales/farmacología , Benzofuranos/farmacología , Hepatitis C Crónica/enzimología , Imidazoles/farmacología , Inhibidores de Proteasas/farmacología , Quinoxalinas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Amidas , Carbamatos , Ciclopropanos , Genotipo , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Replicón/genética , Serina Proteasas , SulfonamidasRESUMEN
We describe the research that led to the discovery of compound 40 (ruzasvir, MK-8408), a pan-genotypic HCV nonstructural protein 5A (NS5A) inhibitor with a "flat" GT1 mutant profile. This NS5A inhibitor contains a unique tetracyclic indole core while maintaining the imidazole-proline-valine Moc motifs of our previous NS5A inhibitors. Compound 40 is currently in early clinical trials and is under evaluation as part of an all-oral DAA regimen for the treatment of chronic HCV infection.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Polimorfismo Genético , Pirrolidinas/química , Pirrolidinas/farmacología , Tiazoles/química , Tiazoles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/farmacocinética , Línea Celular , Perros , Haplorrinos , Hepacivirus/genética , Compuestos Heterocíclicos de 4 o más Anillos/farmacocinética , Humanos , Pirrolidinas/farmacocinética , Ratas , Relación Estructura-Actividad , Tiazoles/farmacocinéticaRESUMEN
PTC725 is a small molecule NS4B-targeting inhibitor of hepatitis C virus (HCV) genotype (gt) 1 RNA replication that lacks activity against HCV gt2. We analyzed the Los Alamos HCV sequence database to predict susceptible/resistant HCV gt's according to the prevalence of known resistance-conferring amino acids in the NS4B protein. Our analysis predicted that HCV gt3 would be highly susceptible to the activity of PTC725. Indeed, PTC725 was shown to be active against a gt3 subgenomic replicon with a 50% effective concentration of â¼5 nM. De novo resistance selection identified mutations encoding amino acid substitutions mapping to the first predicted transmembrane region of NS4B, a finding consistent with results for PTC725 and other NS4B-targeting compounds against HCV gt1. This is the first report of the activity of an NS4B targeting compound against HCV gt3. In addition, we have identified previously unreported amino acid substitutions selected by PTC725 treatment which further demonstrate that these compounds target the NS4B first transmembrane region.
Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Indoles/farmacología , Sulfonamidas/farmacología , Proteínas no Estructurales Virales/genética , Sustitución de Aminoácidos , Línea Celular Tumoral , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Genoma Viral , Genotipo , Humanos , Mutación , Replicón/efectos de los fármacos , Proteínas no Estructurales Virales/metabolismoRESUMEN
We describe the impact of proline modifications, in our tetracyclic-indole based series of nonstructural protein 5A (NS5A) inhibitors, to their replicon profiles. This work identified NS5A inhibitors with an improved and flattened resistance profiles.
Asunto(s)
Antivirales/farmacología , Benzofuranos/farmacología , Imidazoles/farmacología , Indoles/química , Prolina/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/química , Benzofuranos/química , Imidazoles/química , Relación Estructura-ActividadRESUMEN
The selection of resistance-associated variants (RAVs) against single agents administered to patients chronically infected with hepatitis C virus (HCV) necessitates that direct-acting antiviral agents (DAAs) targeting multiple viral proteins be developed to overcome failure resulting from emergence of resistance. The combination of grazoprevir (formerly MK-5172), an NS3/4A protease inhibitor, and elbasvir (formerly MK-8742), an NS5A inhibitor, was therefore studied in genotype 1a (GT1a) replicon cells. Both compounds were independently highly potent in GT1a wild-type replicon cells, with 90% effective concentration (EC90) values of 0.9 nM and 0.006 nM for grazoprevir and elbasvir, respectively. No cross-resistance was observed when clinically relevant NS5A and NS3 RAVs were profiled against grazoprevir and elbasvir, respectively. Kinetic analyses of HCV RNA reduction over 14 days showed that grazoprevir and elbasvir inhibited prototypic NS5A Y93H and NS3 R155K RAVs, respectively, with kinetics comparable to those for the wild-type GT1a replicon. In combination, grazoprevir and elbasvir interacted additively in GT1a replicon cells. Colony formation assays with a 10-fold multiple of the EC90 values of the grazoprevir-elbasvir inhibitor combination suppressed emergence of resistant colonies, compared to a 100-fold multiple for the independent agents. The selected resistant colonies with the combination harbored RAVs that required two or more nucleotide changes in the codons. Mutations in the cognate gene caused greater potency losses for elbasvir than for grazoprevir. Replicons bearing RAVs identified from resistant colonies showed reduced fitness for several cell lines and may contribute to the activity of the combination. These studies demonstrate that the combination of grazoprevir and elbasvir exerts a potent effect on HCV RNA replication and presents a high genetic barrier to resistance. The combination of grazoprevir and elbasvir is currently approved for chronic HCV infection.
Asunto(s)
Antivirales/farmacología , Inhibidores de Proteasas/farmacología , Quinoxalinas/farmacología , Amidas , Benzofuranos/farmacología , Carbamatos , Ciclopropanos , Quimioterapia Combinada , Genotipo , Hepacivirus/efectos de los fármacos , Imidazoles/farmacología , Mutación/genética , Replicón/efectos de los fármacos , Replicón/genética , Ribavirina/farmacología , SulfonamidasRESUMEN
BACKGROUND & AIMS: All-oral regimens combining different classes of direct-acting antivirals (DAA) are highly effective for treatment of patients with chronic hepatitis C. NS5A inhibitors will likely form a component of future interferon-sparing treatment regimens. However, despite their potential, the detailed mechanism of action of NS5A inhibitors is unclear. To study their mechanisms, we compared their kinetics of antiviral suppression with those of other classes of DAA, using the hepatitis C virus genotype 1a cell culture-infectious virus H77S.3. METHODS: We performed detailed kinetic analyses of specific steps in the hepatitis C virus life cycle using cell cultures incubated with protease inhibitors, polymerase inhibitors, or NS5A inhibitors. Assays were designed to measure active viral RNA synthesis and steady-state RNA abundance, polyprotein synthesis, virion assembly, and infectious virus production. RESULTS: Despite their high potency, NS5A inhibitors were slow to inhibit viral RNA synthesis compared with protease or polymerase inhibitors. By 24 hours after addition of an NS5A inhibitor, polyprotein synthesis was reduced <50%, even at micromolar concentrations. In contrast, inhibition of virus release by NS5A inhibitors was potent and rapid, with onset of inhibition as early as 2 hours. Cells incubated with NS5A inhibitors were rapidly depleted of intracellular infectious virus and RNA-containing hepatitis C virus particles, indicating a block in virus assembly. CONCLUSIONS: DAAs that target NS5A rapidly inhibit intracellular assembly of genotype 1a virions. They also inhibit formation of functional replicase complexes, but have no activity against preformed replicase, thereby resulting in slow shut-off of viral RNA synthesis.
Asunto(s)
Antivirales/farmacología , Inhibidores Enzimáticos/farmacología , Hepacivirus/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Ensamble de Virus/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Farmacorresistencia Viral , Genotipo , Hepacivirus/enzimología , Hepacivirus/genética , Hepacivirus/crecimiento & desarrollo , Humanos , Cinética , Mutación , Inhibidores de Proteasas/farmacología , ARN Viral/biosíntesis , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
While new direct-acting antiviral agents for the treatment of chronic hepatitis C virus (HCV) infection have been approved, there is a continued need for novel antiviral agents that act on new targets and can be used in combination with current therapies to enhance efficacy and to restrict the emergence of drug-resistant viral variants. To this end, we have identified a novel class of small molecules, exemplified by PTC725, that target the nonstructural protein 4B (NS4B). PTC725 inhibited HCV 1b (Con1) replicons with a 50% effective concentration (EC50) of 1.7 nM and an EC90 of 9.6 nM and demonstrated a >1,000-fold selectivity window with respect to cytotoxicity. The compounds were fully active against HCV replicon mutants that are resistant to inhibitors of NS3 protease and NS5B polymerase. Replicons selected for resistance to PTC725 harbored amino acid substitutions F98L/C and V105M in NS4B. Anti-replicon activity of PTC725 was additive to synergistic in combination with alpha interferon or with inhibitors of HCV protease and polymerase. Immunofluorescence microscopy demonstrated that neither the HCV inhibitors nor the F98C substitution altered the subcellular localization of NS4B or NS5A in replicon cells. Oral dosing of PTC725 showed a favorable pharmacokinetic profile with high liver and plasma exposure in mice and rats. Modeling of dosing regimens in humans indicates that a once-per-day or twice-per-day oral dosing regimen is feasible. Overall, the preclinical data support the development of PTC725 for use in the treatment of chronic HCV infection.
Asunto(s)
Antivirales/metabolismo , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Indoles/farmacología , Sulfonamidas/farmacología , Proteínas no Estructurales Virales/metabolismo , Sustitución de Aminoácidos , Animales , Antivirales/farmacocinética , Línea Celular Tumoral , Farmacorresistencia Viral/genética , Sinergismo Farmacológico , Humanos , Indoles/metabolismo , Indoles/farmacocinética , Interferón-alfa/farmacología , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , Ratas , Ratas Sprague-Dawley , Sulfonamidas/metabolismo , Sulfonamidas/farmacocinética , Proteínas no Estructurales Virales/genética , Replicación Viral/efectos de los fármacosRESUMEN
Current assays for the activity of viral RNA-dependent RNA polymerases (RdRps) are inherently end-point measurements, often requiring the use of radiolabeled or chemically modified nucleotides to detect reaction products. In an effort to improve the characterization of polymerases that are essential to the life cycle of RNA viruses and develop antiviral therapies that target these enzymes, a continuous nonradioactive assay was developed to monitor the activity of RdRps by measuring the release of pyrophosphate (PP(i)) generated during nascent strand synthesis. A coupled-enzyme assay method based on the chemiluminescent detection of PP(i), using ATP sulfurylase and firefly luciferase, was adapted to monitor poliovirus 3D polymerase (3D(pol)) and the hepatitis C virus nonstructural protein 5B (NS5B) RdRp reactions. Light production was dependent on RdRp and sensitive to the concentration of oligonucleotide primer directing RNA synthesis. The assay system was found to be amenable to sensitive kinetic studies of RdRps, requiring only 6nM 3D(pol) to obtain a reliable estimate of the initial velocity in as little as 4 min. The assay can immediately accommodate the use of both homopolymer and heteropolymer RNA templates lacking uridylates and can be adapted to RNA templates containing uridine by substituting alpha-thio ATP for ATP. The low background signal produced by other NTPs can be corrected from no enzyme (RdRp) controls. The effect of RdRp/RNA template preincubation was assessed using NS5B and a homopolymer RNA template and a time-dependent increase of RdRp activity was observed. Progress curves for a chain terminator (3(')-deoxyguanosine 5(')-triphosphate) and an allosteric NS5B inhibitor demonstrated the predicted time- and dose-dependent reductions in signal. This assay should facilitate detailed kinetic studies of RdRps and their potential inhibitors using either standard or single-nucleotide approaches.
Asunto(s)
ARN Polimerasa Dependiente del ARN/análisis , Regulación Alostérica , Luciferina de Luciérnaga , Hepacivirus/metabolismo , Luciferasas , Conformación de Ácido Nucleico , Poliovirus/enzimología , ARN/metabolismo , Factores de Tiempo , Proteínas no Estructurales Virales/análisis , Proteínas no Estructurales Virales/metabolismoRESUMEN
Inspection of over 250 hepatitis C virus (HCV) genome sequences shows that a threonine is strictly conserved at the P1 position in the NS3-NS4A (NS3-4A) autoproteolysis junction, while a cysteine is maintained as the P1 residue in all of the putative trans cleavage sites (NS4A-4B, NS4B-5A, and NS5A-5B). To understand why T631 is conserved at the NS3-4A junction of HCV, a series of in vitro transcription-translation studies were carried out using wild-type and mutant (T631C) NS3-4A constructs bearing native, truncated, and mutant NS4A segments. The autocleavage of the wild-type junction was found to be dependent on the presence of the central cofactor domain of NS4A (residues 21 to 34). In contrast, all NS3-4A T631C mutant proteins underwent self-cleavage even in the absence of the cofactor. Subgenomic replicons derived from the Con1 strain of HCV and bearing the T631C mutation showed reduced levels of colony formation in transfection studies. Similarly, replicons derived from a second genotype 1b virus, HCV-N, demonstrated a comparable reduction in replication efficiency in transient-transfection assays. These data suggest that the threonine is conserved at position 631 because it serves two functions: (i) to slow processing at the NS3-4A cleavage site, ensuring proper intercalation of the NS4A cofactor with NS3 prior to polyprotein scission, and (ii) to prevent subsequent product inhibition by the NS3 C terminus.