The Molecular Basis of Drug Resistance against Hepatitis C Virus NS3/4A Protease Inhibitors

Abstract

Hepatitis C virus (HCV) infects over 170 million people worldwide and is the leading cause of chronic liver diseases, including cirrhosis, liver failure, and liver cancer. Available antiviral therapies cause severe side effects and are effective only for a subset of patients, though treatment outcomes have recently been improved by the combination therapy now including boceprevir and telaprevir, which inhibit the viral NS3/4A protease. Despite extensive efforts to develop more potent next-generation protease inhibitors, however, the long-term efficacy of this drug class is challenged by the rapid emergence of resistance. Single-site mutations at protease residues R155, A156 and D168 confer resistance to nearly all inhibitors in clinical development. Thus, developing the next-generation of drugs that retain activity against a broader spectrum of resistant viral variants requires a comprehensive understanding of the molecular basis of drug resistance. In this study, 16 high-resolution crystal structures of four representative protease inhibitors – telaprevir, danoprevir, vaniprevir and MK-5172 – in complex with the wild-type protease and three major drug-resistant variants R155K, A156T and D168A, reveal unique molecular underpinnings of resistance to each drug. The drugs exhibit differential susceptibilities to these protease variants in both enzymatic and antiviral assays. Telaprevir, danoprevir and vaniprevir interact directly with sites that confer resistance upon mutation, while MK-5172 interacts in a unique conformation with the catalytic triad. This novel mode of MK-5172 binding explains its retained potency against two multi-drug-resistant variants, R155K and D168A. These findings define the molecular basis of HCV N3/4A protease inhibitor resistance and provide potential strategies for designing robust therapies against this rapidly evolving virus.

Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants: In Vitro Selection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle

“…A98T was identified in HCV genotype 3 patients, either treatment naïve or failing telaprevir therapy without apparent resistance (55,78). Interactions between residues 155 and 168 have been described previously (83,84), and substitutions at position 168 might compensate for the loss of replication induced by substitutions at position 155 by restoring such interactions.…”

Substitutions at NS3 Residue 155, 156, or 168 of Hepatitis C Virus Genotypes 2 to 6 Induce Complex Patterns of Protease Inhibitor Resistance

“…Single HCV antigen-positive cells were counted automatically, counts from treated wells were related to means of counts from infected, nontreated wells, and sigmoidal concentrationresponse curves were fitted [Y ϭ Top/(1 ϩ 10 [log10(EC50) Ϫ X] ϫ Hill slope )] to obtained data following transformation of X values. Log 10 median effective concentration (EC 50 ) and standard errors (SE) of log 10 (EC 50 ) from replicate experiments were used to calculate inverse-variance weighted means of log 10 (EC 50 ) with SE and 95% confidence intervals (CI). Mean differences between log 10 (EC 50 ) of variants versus original recombinants with SE and 95% CI were calculated from the inversevariance weighted-mean log 10 (EC 50 ) values.…”

“…Log 10 median effective concentration (EC 50 ) and standard errors (SE) of log 10 (EC 50 ) from replicate experiments were used to calculate inverse-variance weighted means of log 10 (EC 50 ) with SE and 95% confidence intervals (CI). Mean differences between log 10 (EC 50 ) of variants versus original recombinants with SE and 95% CI were calculated from the inversevariance weighted-mean log 10 (EC 50 ) values. Inverse logarithmic transformation rendered median EC 50 with 95% CI and median fold differences with 95% CI.…”

Guidelines for the successful generation of protein–ligand complex crystals

“…In the case of HCV NS3 protease, the activity of NS3 increases upon binding of the HCV NS4 peptide by anchoring the active-site residues, and crystal structures of NS3 bound to different protease inhibitors have been generated using the NS4 peptide, either by addition of the NS4 peptide to the NS3 protein prior to crystallization (Yan et al, 1998) or by constructing an NS3/NS4 fusion protein (Romano et al, 2012).…”