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.

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.