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

Abstract

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 (ECs) 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. 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.

HCV Replicon Systems: Workhorses of Drug Discovery and Resistance

“…These high fitness RASs to NS5A-inhibitors (e.g., daclatasvir, ledipasvir, and ombitasvir) confer a low barrier to resistance, and can emerge early and persist in patients following administration of NS5A-based treatments (Krishnan et al, 2015b ; Zeuzem et al, 2017 ; Dietz et al, 2018 ; Fourati et al, 2018 ). The majority of these RAS are also selected in replicon cells and confer resistance to recently recommended pangenotypic NS5A-inhibitors (Cheng et al, 2013 ; Liu et al, 2015 ; Ng et al, 2017a ; Asante-Appiah et al, 2018 ). In contrast, the NS5B RAS S282T exhibited reduced fitness and does not appear to emerge in patients following administration of sofosbuvir (NPI) (Lam et al, 2012 ; Wyles et al, 2017 ).…”

“…The RASs R155W, A156T, and D168K/L/R conferred 20- to 100-fold resistance, whereas A156/L/V exhibited >100-fold resistance to voxilaprevir (Han et al, 2019 ). The NS5A amino acid substitutions M28T/V and Q30D/E/H/R/K in the GT1a replicon yielded moderate to high resistance to ruzasvir, daclatasvir, ledipasvir, ombitasvir, elbasvir, and pibrentasvir, while L31M/V to GT1b, whereas Y93H/N/C in both the GT1 replicons conferred negligible to significantly high resistance (Krishnan et al, 2015a ; Liu et al, 2015 ; Asante-Appiah et al, 2018 ). HCV GT1 replicons carrying amino acid substitutions that confer > 2-fold resistance to various DAAs are listed in the Table 1 .…”

“…The efficient replication by the JFH-1 replicon makes it among the most extensively used replicon systems for identifying pangenotypic HCV inhibitors. The JFH-1 replicon carrying NS5A-F28S confers resistance to declatasvir, lidipasvir, and velpatasvir while L31V confers resistance to ombitasvir and Y93H to ruzasvir (Asante-Appiah et al, 2018 ). Several NS3 RASs confer significant resistance, including R155W to voxilaprevir and A156T/V to volxilaprevir and glecaprevir (Ng et al, 2017b ; Han et al, 2019 ).…”

“…The inclusion of NS3 T235I, NS5A-T210A, and NS5B-R465K increased the replication potential of the parental replicon, rendering the replicon amenable for screening and evaluation of DAAs (Saeed et al, 2013 ). An intergenotypic GT3 replicon cloned in the Con1 genetic background that carried the RAS NS5A-Y93H mutation conferring resistance to different NS5A inhibitors, including daclatasvir, ombitasvir, and elbasvir, also conferred significant loss in susceptibility to the recently discovered pibrentasvir and ruzasvir (Ng et al, 2017a ; Asante-Appiah et al, 2018 ). In addition, a stably replicating GT3a replicon (PR87A7) harboring NS3-168Q, which is a highly conserved amino acid among GT3a isolates, was found to impart resistance to several anti-NS3 DAAs (Guo et al, 2019 ).…”

“…Incorporation of the mutation R465G (NS5B), which was also identified in Con1, combined with NS5A S232I yielded few G418 resistant colonies (Saeed et al, 2012 ). In the ED43 genetic background (carries adaptive mutations NS3 G162R) inclusion of the NS5A RASs L28V or L30H do not confer significant resistance against pibrentasvir, nor do Y93H or L30H against ruzasvir (Ng et al, 2017a ; Asante-Appiah et al, 2018 ). However, incorporation of NS3-R156T/V and D168H/V in the stable chimeric GT4a replicon in a Con1 genetic background conferred resistance to glecaprevir, while NS5A L28V conferred resistance to ombitasvir (Krishnan et al, 2015a ; Ng et al, 2017b ).…”