Hepatitis C virus (HCV) genotype (GT) 2 represents approximately 9% of all viral infections globally. While treatment outcomes for GT2-infected patients have improved substantially with direct-acting antiviral agents (DAAs) compared to interferon-α, the presence of polymorphisms in NS5A can impact efficacy of NS5A inhibitor-containing regimens. Thus, pathways of NS5A resistance were explored in GT2 subtypes using elbasvir, an NS5A inhibitor with broad genotype activity. Resistance selection studies, resistance analysis in NS5A-inhibitor treated virologic failures, antiviral activities in replicons bearing a panel of GT2 subtype sequences and amino acid substitutions introduced by site-directed mutagenesis were performed to define determinants of inhibitor susceptibility. Elbasvir showed differential antiviral activity in replicons bearing GT2 sequences. The EC50 values for replicons bearing reference NS5A sequences for GT2a and GT2b were 0.003 and 3.4 nanomolar (nM) respectively. Studies with recombinant replicons demonstrated crosstalk between amino acid positions 28 and 31. The combination of phenylalanine and methionine at positions 28 and 31 respectively, conferred the highest potency reduction for elbasvir in GT2a and GT2b. This combination was observed in failures from the C-SCAPE trial. Addition of grazoprevir, an NS3/4A protease inhibitor, to elbasvir more effectively suppressed the emergence of resistance in GT2 at modest inhibitor concentrations (3X EC90). Ruzasvir, a potent, pan-genotype NS5A inhibitor successfully inhibited replicons bearing GT2 resistance-associated substitutions (RASs) at positions 28 and 31. The studies demonstrate crosstalk between amino acids at positions 28 and 31 in NS5A modulate inhibitor potency and may impact treatment outcomes in some HCV GT2-infected patients.

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.

Purpose: A detailed analysis of hepatitis C virus (HCV) resistance-associated substitutions (RASs) is required to understand why people fail direct-acting antiviral therapies. This study was conducted to assess RASs in an analysis of 2 trials evaluating the second-generation NS3/4A protease inhibitor grazoprevir (GZR) in combination with peginterferon/ribavirin. Patients and methods: From a total of 113 participants with HCV genotype 1 infection, RASs were evaluated in 25 patients who relapsed and 6 patients with on-treatment virologic breakthrough using consensus Sanger and clonal sequence analysis of NS3/NS4a genes, with in vitro testing of replicon mutants. Next-generation sequencing (NGS) was used in a subset of participants to assess minority variants and the evolution of the whole viral genome. Results: Baseline RASs did not predict treatment failure. Relapse was most commonly associated with RASs at D168, although additional RASs (Y56, R155 and A156) were also detected, particularly in participants with on-treatment breakthrough. Treatment-emergent RASs usually reverted to wild-type (WT), suggesting these mutations were associated with a negative fitness cost (confirmed using in vitro assays). NGS was the most sensitive assay for the detection of minor variants. Significant viral sequence divergence (up to 5.9% codons) was observed across whole genomes in association with the acquisition and reversion of RASs. Conclusion: Relapse with GZR and peginterferon/ribavirin is commonly associated with single RASs in NS3 that generally revert to WT, while breakthrough follows more complex patterns of viral resistance. NGS suggests that large diverse pools of viral quasispecies that emerge with RASs facilitate rapid viral evolution.

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.

Grazoprevir is a potent pan-genotype and macrocyclic inhibitor of hepatitis C virus (HCV) NS3/4A protease and was developed for treating chronic HCV infection. In HCV genotype (GT) 1a, grazoprevir maintains potent activity against a majority of NS3 resistance-associated amino acid substitutions, including the highly prevalent and naturally occurring Q80K polymorphism that impacts simeprevir, another NS3/4A protease inhibitor. The basis for an unexpected difference in the clinical impact of some NS3 substitutions was investigated. Phenotypic analysis of resistance-associated substitutions identified in NS3 from GT1a-infected patients who failed therapy with grazoprevir (in combination with elbasvir, an inhibitor of HCV NS5A protein) showed that positions 56, 156, and 168 in NS3 were most impactful because they diminished protein-inhibitor interactions. Although an amino acid substitution from aspartic acid to alanine at position 168 (D168A) reduced the potency of grazoprevir, its combination with R155K unexpectedly nullified this effect. Molecular dynamics and free-energy surface studies indicated that Asp-168 is important in anchoring Arg-155 for ligand binding but is not critical for Lys-155 because of the inherent flexibility of its side chain. Moreover, modeling studies supported a strong direct cation-heterocycle interaction between the Lys-155 side chain of the double substitution, R155K/D168A, and the lone pair on the quinoxaline in grazoprevir. This unique interaction provides a structural basis for grazoprevir's higher potency than simeprevir, an inhibitor to which the double substitution confers a significant reduction in potency. Our findings are consistent with the detection of R155K/D168A in NS3 from virologic failures treated with simeprevir but not grazoprevir.

Direct‐acting antiviral agents have not been studied exclusively in patients with inherited blood disorders and hepatitis C virus (HCV) infection. The objective of the randomized, placebo‐controlled, phase III C‐EDGE IBLD study was to assess the safety and efficacy of elbasvir/grazoprevir (EBR/GZR) in patients with inherited bleeding disorders and HCV infection. One hundred fifty‐nine adults with HCV infection and sickle cell anemia, thalassemia, or hemophilia A/B or von Willebrand disease were enrolled at 31 study sites in the United States, Europe, Australia, Canada, Israel, and Thailand. Patients were given an oral, once‐daily, fixed‐dose combination of EBR/GZR 50 mg/100 mg for 12 weeks and randomized to the immediate‐treatment group (ITG) or deferred‐treatment group (DTG; placebo followed by active treatment). The primary endpoints were the proportion of patients in the ITG with unquantifiable HCV RNA 12 weeks posttreatment (sustained virological response 12 weeks after completion of study treatment; SVR12) and the comparison of safety in the ITG and DTG. In the ITG, 100 of 107 patients (93.5%) achieved SVR12, 6 relapsed, and 1 was lost to follow‐up. SVR12 was achieved in 94.7% (18 of 19), 97.6% (40 of 41), and 89.4% (42 of 47) of patients with sickle cell disease, β‐thalassemia, and hemophilia A/B or von Willebrand disease, respectively. Serious adverse events were reported by 2.8% (n = 3) and 11.5% (n = 6) of patients in the ITG and DTG, respectively. Hemoglobin levels and international normalized ratio values were similar in patients receiving EBR/GZR and placebo; among patients with hemoglobinopathies, change in mean hemoglobin levels was similar in those receiving EBR/GZR compared to those receiving placebo. Conclusion: These results add to the expanding pool of data available for EBR/GZR, indicating a high level of efficacy and favorable tolerability in patients with HCV infection. (Hepatology 2017;66:736–745)

Abstract Background: Complications from chronic hepatitis C virus (HCV) infection are a major cause of morbidity and mortality among individuals with inherited blood disorders (IBLD). Inability to tolerate ribavirin and frequent comorbidities have limited HCV treatment options in these patients. The aim of the C-EDGE IBLD study was to evaluate the efficacy and safety of a once-daily, fixed-dose combination of elbasvir 50 mg (EBR, an NS5A inhibitor) and grazoprevir 100 mg (GZR, an NS3/4A protease inhibitor) in patients with HCV infection and IBLD, including those with hemoglobinopathies. Methods: C-EDGE-IBLD was a randomized, double-blind, placebo-controlled study of treatment-naïve and treatment-experienced patients with HCV genotype (GT)1, 4, or 6 infection and IBLD (hemophilia A/B, von Willebrand disease, β-thalassemia, or sickle cell anemia). Patients were randomized in a 2:1 ratio to an immediate-treatment group (ITG; 12 weeks of EBR/GZR) or deferred-treatment group (DTG; 12 weeks of placebo, followed by EBR/GZR for 12 weeks). Randomization was stratified according to the presence of cirrhosis and IBLD diagnosis. The primary endpoints were the proportion of patients in the ITG who achieved sustained virologic response (SVR12, HCV RNA <15 IU/mL 12 weeks after completion of treatment) and a comparison of safety and tolerability between patients receiving EBR/GZR in the ITG vs those receiving placebo in the DTG. Results: One hundred fifty-nine patients were randomized (ITG, n = 107; DTG, n = 52). Three patients in the DTG did not commence deferred active treatment; therefore, 156 patients received ≥1 dose of EBR/GZR. Mean age was 44 years. Other baseline patient demographics were as follows: 75% male; 18% black; 41% GT1a-infected; 46% GT1b-infected; 11% GT4-infected; 24% cirrhotic; 6% HIV/HCV co-infected; 43% with hemophilia A/B or von Willebrand disease; 38% with β-thalassemia; 18% with sickle cell anemia. SVR12 was achieved by 92.9% (145/156) of patients receiving EBR/GZR (ITG 93.5% [100/107]; DTG 91.8% [45/49]). Of the 11 patients who did not achieve SVR12, 2 discontinued treatment for reasons unrelated to study medication and 9 relapsed, including 7 who had NS5A resistance-associated variants present at baseline. One patient achieved SVR12 but relapsed between follow-up weeks 12 and 24. SVR12 was achieved by 90.8% (59/65), 95.7% (67/70), and 94.4% (17/18) of patients with HCV GT1a, GT1b, and GT4 infection, respectively (2 patients with GT1 non-a,b achieved SVR and the 1 patient with GT6 infection relapsed). SVR 12 was achieved by 96.3% (26/27) of patients with sickle cell anemia, 95% (57/60) with β-thalassemia, and 89.9% (62/69) with hemophilia A/B or von Willebrand disease. During the initial treatment period (EBR/GZR vs placebo), serious adverse events were reported in 3 (2.9%) patients in the ITG (1 drug-related, 2 related to IBLD) and 6 (11.5%) patients receiving placebo in the DTG (1 drug-related, 3 related to IBLD). In the DTG placebo phase, 1 patient discontinued treatment due to an adverse event and 1 patient withdrew consent. No patient in either arm discontinued treatment due to worsening of underlying IBLD. There was 1 hepatic event of clinical interest in each arm (ALT >3× baseline and >100 U/L). In the EBR/GZR treatment phase of the DTG (n = 49), 3 patients reported serious adverse events; none were drug-related and 2 were related to IBLD. Conclusions: Final data from the C-EDGE IBLD study indicate that EBR/GZR is well tolerated and effective in patients with HCV GT1 or 4 infection with IBLD. Disclosures Hézode: BMS: Consultancy; Janssen: Consultancy; MSD: Consultancy; AbbVie: Consultancy; Gilead: Consultancy. Fried:NIH: Research Funding; Gilead: Consultancy, Research Funding; TARGET PharmaSolutions: Equity Ownership; Merck: Consultancy, Research Funding; BMS: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding. Colombo:Merck: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead Science: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Tibotec: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Vertex: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen Cilag: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Achillion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Lundbeck: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GSK: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GenSpera: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AlfaWasserman: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jennerex: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Intercept: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Bourlière:MSD: Consultancy; AbbVie: Consultancy; Gilead: Consultancy; Janssen: Consultancy; GSK: Consultancy; BMS: Consultancy. Ben-Ari:Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Strasser:MSD/Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Norgine: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Honoraria. Perumalswami:Merck: Research Funding; Gilead: Research Funding. Zamor:Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Lee:Eli Lilly: Consultancy; BMS: Research Funding; Gilead: Research Funding; Sanofi: Consultancy; Merck: Research Funding; Novartis: Consultancy. Satoskar:Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Research Funding. Sherman:AbbVie: Research Funding; Gilead: Research Funding; BMS: Research Funding; Merck: Consultancy, Research Funding. Morgan:Merck & Co., Inc.: Employment, Equity Ownership. Qiu:Merck: Employment. Hwang:Merck: Employment, Equity Ownership. Robertson:Merck: Employment, Equity Ownership. Nguyen:Merck: Employment. Barr:Merck: Employment, Equity Ownership. Wahl:Merck: Employment. Haber:Merck: Employment. Chase:Merck: Employment. Talwani:Merck & Co., Inc: Employment. Di Marco:Gilead: Research Funding.

We have been focused on identifying a structurally different next generation inhibitor to MK-5172 (our Ns3/4a protease inhibitor currently under regulatory review), which would achieve superior pangenotypic activity with acceptable safety and pharmacokinetic profile. These efforts have led to the discovery of a novel class of HCV NS3/4a protease inhibitors containing a unique spirocyclic-proline structural motif. The design strategy involved a molecular-modeling based approach, and the optimization efforts on the series to obtain pan-genotypic coverage with good exposures on oral dosing. One of the key elements in this effort was the spirocyclization of the P2 quinoline group, which rigidified and constrained the binding conformation to provide a novel core. A second focus of the team was also to improve the activity against genotype 3a and the key mutant variants of genotype 1b. The rational application of structural chemistry with molecular modeling guided the design and optimization of the structure-activity relationships have resulted in the identification of the clinical candidate MK-8831 with excellent pan-genotypic activity and safety profile.

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.

Background Genotype (gt)6 HCV is common amongst HCV-positive populations of the Asia–Pacific region but cell culture models for this gt have only recently been developed. Boceprevir (SCH503034) is a clinically available inhibitor of the HCV NS3 protein. We investigated the efficacy of boceprevir for inhibiting replication of a chimeric gt1b replicon encoding a gt6a NS3 protease and defined the development of mutations in the protease when boceprevir treatment was applied. Methods We constructed a chimeric gt1b subgenomic replicon encoding a gt6 NS3 protease (NS3p) sequence (gt6NS3p_gt1b). The boceprevir EC 50 value against replication of this replicon was determined using quantitative reverse transcriptase PCR. Next-generation sequencing was used to identify nucleotide changes associated with boceprevir resistance. The replication capacities of chimeric replicons containing mutations associated with boceprevir resistance were determined by colony formation efficiency assays. Results The boceprevir EC 50 value for the gt6NS3p_gt1b replicon was 535 ±79 nM. Boceprevir-resistant gt6NS3p_gt1b replicon cell lines could be selected and they demonstrated drug-associated amino acid changes that have previously been reported in other HCV gts. Interestingly, no mutations were observed at A156, a position defined for boceprevir resistance in gt1 NS3p, while mutation at N122, which is rarely reported in boceprevir-resistant gt1 proteases, was frequently observed. Re-introduction of these mutations into the chimeric replicon altered their replication capacity, ranging from complete abolishment of replication (A156T) to increasing replication capacity (V36A, N122S). This report provides the first characterization of gt6 HCV resistance to boceprevir. Conclusions A chimeric HCV replicon encoding gt6 NS3 protease is sensitive to boceprevir and develops drug-resistant mutations at amino acid sites previously reported for other gts. Mutation at N122 also appears to be associated with boceprevir resistance in the gt6 NS3 protease.

With the goal of identifying inhibitors of hepatitisC virus (HCV) NS3/4a protease that are potent against a wide range of genotypes and clinically relevant mutant viruses, several subseries of macrocycles were investigated based on observations made during the discovery of MK-5172. Quinazolinone-containing macrocycles were identified as promising leads, and optimization for superior cross-genotype and mutant enzyme potency as well as rat liver and plasma concentrations following oral dosing, led to the development of MK-2748. Additional investigation of a series of bis-macrocycles containing a fused 18- and 15-membered ring system were also optimized for the same properties, leading to the discovery of MK-6325. Both compounds display the broad genotype and mutant potency necessary for clinical development as next-generation HCV NS3/4a protease inhibitors.

Background. We analyzed the impact of pretreatment variants conferring boceprevir-resistance on sustained virologic response (SVR) rates achieved with boceprevir plus peginterferon-alpha/ribavirin (P/R) for hepatitis C virus (HCV)-genotype-1 infection.Methods. NS3-protease-polymorphisms emerging coincident with virologic failure on boceprevir/P/R regimens were identified as resistance-associated variants (RAVs). Baseline samples pooled from 6 phase II or phase III clinical trials were analyzed for RAVs by population sequencing. Interferon (IFN)-responsiveness was predefined as >1 log reduction in HCV-RNA level during the initial 4-week lead-in treatment with P/R before boceprevir was added. The effective boceprevir-concentration inhibiting RAV growth by 50% (EC50) was determined using a replicon assay relative to the wild-type referent.Results. Sequencing was performed in 2241 of 2353 patients (95.2%) treated with boceprevir. At baseline, RAVs were detected in 178 patients (7.9%), including 153 of 1498 genotype-1a infections (10.2%) and 25 of 742 genotype-1b infections (3.4%) (relative risk, 3.03; 95% confidence interval [CI], [2.01, 4.58]). For IFN-responders, SVR24 (SVR assessed 24 weeks after discontinuation of all study medications) rates were 78% and 76% with or without RAVs detected at baseline, respectively. For the 510 poor IFN-responders, SVR24 rates were 8 of 36 subjects (22.2% [11.7%, 38.1%]) when baseline RAVs were detected vs 174 of 474 subjects (36.7% [32.5%, 41.1%]) when baseline RAVs were not detected (relative likelihood of SVR24 [95% CI], 0.61 [0.32, 1.05]). Sustained virologic response was achieved in 7 of 8 (87.5%) IFN-nonresponders with baseline variants exhibiting <= 2-fold increased EC50 for boceprevir in a replicon assay, whereas only 1 of 15 (7%) IFN-nonresponders with baseline RAVs associated with >= 3-fold increased EC50 achieved SVR.Conclusions. Baseline protease-variants appear to negatively impact SVR rates for boceprevir/P/R regimens only when associated with decreased boceprevir susceptibility in vitro after a poor IFN-response during the lead-in period.

We have previously reported the discovery of our P2-P4 macrocyclic HCV NS3/4a protease inhibitor MK-5172, which in combination with the NS5a inhibitor MK-8742 recently received a breakthrough therapy designation from the US FDA for treatment of chronic HCV infection. Our goal for the next generation NS3/4a inhibitor was to achieve pan-genotypic activity while retaining the pharmacokinetic profile of MK-5172. One of the areas for follow-up investigation involved replacement of the quinoxaline moiety in MK-5172 with a quinoline and studying the effect of substitution at 4-position of the quinoline. The rationale for this effort was based on molecular modeling, which indicated that such modifications would improve interactions with the S2 subsite, in particular with D79. We wish to report herein the discovery of highly potent inhibitors with pan-genotypic activity and an improved profile over MK-5172, especially against gt-3a and A156 mutants.

Background & Aims: To examine the antiviral activity of boceprevir, a hepatitis C virus (HCV) protease inhibitor, in HCV genotype (G) 2/3-infected patients.Methods: We assessed boceprevir and telaprevir activity against an HCV G2 and G3 isolates enzyme panel, in replicon, and in phenotypic cell-based assays. Additionally, a phase I study evaluated the antiviral activity of boceprevir monotherapy (200 mg BID, 400 mg BID, or 400 mg TID) vs. placebo for 14 days in HCV G2/3 treatment-naive patients.Results: Boceprevir and telaprevir similarly inhibited G1 and G2 NS3/4A enzymes and replication in Cl and G2 replicon and cell-based assays. However, telaprevir demonstrated lower potency than boceprevir against HCV G3a enzyme (K-i = 75 nM vs. 17 nM), in the G3a replicon assay (EC50 = 953 nM vs. 159 nM), and against HCV G3a NS3 isolates (IC50 = 3312 nM vs. 803 nM) in the cell-based assay. In HCV G2/3-infected patients, boceprevir (400 mg TID) resulted in a maximum mean decrease in HCV RNA of -1.60 log vs. -0.21 log with placebo.Conclusions: In vitro, boceprevir is more active than telaprevir against the HCV G3 NS3/4A enzyme in cell-based and biochemical assays and against G3 isolates in replicon assays. In HCV G2/3-infected treatment-naive patients, decreases in HCV RNA levels with boceprevir (400 mg TID) were comparable to those observed with the same dose in HCV treatment-experienced G1-infected patients. (C) 2013 Published by Elsevier B.V. on behalf of the European Association for the Study of the Liver.

Introduction of a nitrogen atom into the benzene ring of a previously identified HCV replication (replicase) benzothiazole inhibitor 1, resulted in the discovery of the more potent pyridothiazole analogues 3. The potency and PK properties of the compounds were attenuated by the introductions of various functionalities at the R1, R2 or R3 positions of the molecule (compound 3). Inhibitors 38 and 44 displayed excellent potency, selectivity (GAPDH/MTS CC50), PK parameters in all species studied, and cross genotype activity.