). Blockage of NS3 protease activity therefore is expected to inhibit HCV replication by both direct suppression of viral protein production as well as by restoring host responsiveness to IFN. Using structure-assisted design, a ketoamide inhibitor, SCH 503034, was generated which demonstrated potent (overall inhibition constant, 14 nM) time-dependent inhibition of the NS3 protease in cell-free enzyme assays as well as robust in vitro activity in the HCV replicon system, as monitored by immunofluorescence and real-time PCR analysis. Continuous exposure of repliconbearing cell lines to six times the 90% effective concentration of SCH 503034 for 15 days resulted in a greater than 4-log reduction in replicon RNA. The combination of SCH 503034 with IFN was more effective in suppressing replicon synthesis than either compound alone, supporting the suggestion of Foy and coworkers that combinations of IFN with protease inhibitors would lead to enhanced therapeutic efficacy.

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 (HCV) genotype 1a cell culture-infectious virus H77S.3. Methods We performed detailed kinetic analyses of specific steps in the HCV 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 to protease or polymerase inhibitors. By 24 hrs after addition of an NS5A inhibitor, polyprotein synthesis was reduced less than 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 hrs. Cells incubated with NS5A inhibitors were rapidly depleted of intracellular infectious virus and RNA-containing HCV particles, indicating a block in virus assembly. Conclusions DAAs that target NS5A rapidly inhibit intracellular assembly of gentoype 1a virions. They also inhibit formation of functional replicase complexes, but have no activity against pre-formed replicase, thereby resulting in slow shut-off of viral RNA synthesis.

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 (EC 90 ) 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 EC 90 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. Hepatitis C virus (HCV) is a leading cause of chronic liver disease, with an estimated 130 to 170 million people infected globally. WHO estimates that more than 350,000 people die every year from hepatitis C-related liver diseases (1, 2, 3). The introduction of direct-acting antiviral agents (DAAs) as add-ons to the previous standard of care (SOC) consisting of pegylated interferon alpha plus ribavirin (PR) significantly improved sustained virologic response (SVR) rates from 40 to 50% to 65 to 70% in the previously hard-to-cure genotype 1 (GT1) patients after a 24-to 48-week treatment course. Further treatment advancements have been achieved with the introduction of interferon-free all-oral DAAs, with SVR rates now in excess of 90% after 12 weeks of therapy for GT1 patients (4, 5, 6). Recent reports indicate that therapy can be further simplified and likely shortened to Ͻ12 weeks in some cases while maintaining high SVR rates. Preexisting baseline resistance-associated variants (RAVs) and resistance selection remain contributory rea...

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 (EC 50 ) of 1.7 nM and an EC 90 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.C hronic hepatitis C virus (HCV) infection is a worldwide epidemic disease with an estimate of over 170 million people chronically infected worldwide (1). Approximately 60 to 85% of HCV infections result in chronic hepatitis that can lead to liver fibrosis, cirrhosis, and hepatocellular carcinoma (2). The current standard of care (SOC) for chronic hepatitis C infection, pegylated alpha interferon in combination with ribavirin, has serious side effects and limited efficacy, especially for infection with HCV genotype 1, which is the most prevalent HCV genotype (3, 4). Two HCV protease inhibitors, boceprevir (Victrelis) and telaprevir (Incivek), for the therapy of HCV genotype 1 infection in combination with the SOC were approved for use nearly 2 years ago. In addition, a number of other direct-acting antivirals (DAAs) in clinical trials have demonstrated encouraging efficacy in combination therapies (5). Currently, the HCV antivirals in preclinical and clinical development are inhibitors of the viral protease, polymerase, or nonstructural protein 5A (NS5A) (6). Due to the emergence of viral variants resistant to the DAAs, even in combination therapy with the SOC (7-9) and the potential for viral rebound after cessation of antiviral therapy, it is essential to discover and develop novel HCV inhibitors that act on new targets and can be used in combination with the SOC and/or DAAs to enhance efficacy and to delay or possibly prevent the emergence of drug-resistant...

A novel, potent, and orally bioavailable inhibitor of hepatitis C RNA replication targeting NS4B, compound 4t (PTC725), has been identified through chemical optimization of the 6-(indol-2-yl)pyridine-3-sulfonamide 2 to improve DMPK and safety properties. The focus of the SAR investigations has been to identify the optimal combination of substituents at the indole N-1, C-5, and C-6 positions and the sulfonamide group to limit the potential for in vivo oxidative metabolism and to achieve an acceptable pharmacokinetic profile. Compound 4t has excellent potency against the HCV 1b replicon, with an EC50 = 2 nM and a selectivity index of >5000 with respect to cellular GAPDH. Compound 4t has an overall favorable pharmacokinetic profile with oral bioavailability values of 62%, 78%, and 18% in rats, dogs, and monkeys, respectively, as well as favorable tissue distribution properties with a liver to plasma exposure ratio of 25 in rats.