Interplay of Amino Acid Residues at Positions 28 and 31 in NS5A Defines Resistance Pathways in HCV GT2.

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.

In Vitro Antiviral Profile of Ruzasvir, a Potent and Pangenotype Inhibitor of Hepatitis C Virus NS5A.

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.

The Combination of Grazoprevir, a Hepatitis C Virus (HCV) NS3/4A Protease Inhibitor, and Elbasvir, an HCV NS5A Inhibitor, Demonstrates a High Genetic Barrier to Resistance in HCV Genotype 1a Replicons.

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.

Structure-activity relationships of proline modifications around the tetracyclic-indole class of NS5A inhibitors.

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.

Depletion of DSS1 protein disables homologous recombinational repair in human cells.

DSS1 is a small, highly acidic protein widely conserved among eukaryotes as a component of the 19S proteasome and implicated in ubiquitin-mediated proteolysis. The BRCA2 tumor suppressor protein functions in homologous recombinational repair (HRR) of DNA double-strand breaks, and does so in part through the actions of a carboxy-proximal region that binds DNA and several other proteins, including DSS1. In the unicellular eukaryote Ustilago maydis, Dss1 interacts with Brh2, a BRCA2-like protein, and regulates its function in mediating HRR. We used RNA interference to deplete DSS1 in human cells, and assayed the effects on double-strand break repair by homologous recombination. Partial depletion of DSS1 protein in human cells reduced the efficiency of HRR to small fractions of normal levels. Residual HRR activity correlated roughly with the residual level of DSS1 expression. The results imply that mammalian DSS1 makes a critical contribution to the function of BRCA2 in mediating HRR, and hence to genomic stability. Activity of the ubiquitin-proteasome system can influence HRR. However, treatment with proteasome inhibitors only partially reproduced the effects of DSS1 depletion on HRR, suggesting that the function of DSS1 in HRR involves more than proteolysis per se.

Discovery of MK-6169, a Potent Pan-Genotype Hepatitis C Virus NS5A Inhibitor with Optimized Activity against Common Resistance-Associated Substitutions.

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.

Discovery of Ruzasvir (MK-8408): A Potent, Pan-Genotype HCV NS5A Inhibitor with Optimized Activity against Common Resistance-Associated Polymorphisms.

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.