Susceptibility of Treatment-Naive Hepatitis C Virus (HCV) Clinical Isolates to HCV Protease Inhibitors

In order to assess the natural variation in susceptibility to hepatitis C virus (HCV) NS3 protease inhibitors (PIs) among untreated HCV patient samples, the susceptibilities of 39 baseline clinical isolates were determined using a transient-replication assay on a panel of HCV PIs, including two α-ketoamides (VX-950 and SCH-503034) and three macrocyclic inhibitors (MK-7009, ITMN-191, and TMC-435350). Some natural variation in susceptibility to all HCV PIs tested was observed among the baseline clinical isolates. The susceptibility to VX-950 correlated strongly with the susceptibility to SCH-503034. A moderate correlation was observed between the susceptibilities to ITMN-191 and MK-7009. In contrast, the phenotypic correlations between the α-ketoamides and macrocyclic inhibitors were significantly lower. This difference is partly attributable to reduced susceptibility of the HCV variants containing the NS3 polymorphism Q80K (existing in 47% of genotype 1a isolates) to the macrocyclic compounds but no change in the sensitivity of the same variants to the α-ketoamides tested. Our results suggest that the natural variation in baseline susceptibility may contribute to different degrees of antiviral response among patients in vivo, particularly at lower doses.Hepatitis C virus (HCV) is characterized by a high degree of genetic diversity because of its rapid replication rate and turnover, combined with the poor fidelity of the HCV RNA-dependent RNA polymerase (RdRp) (3, 5, 32). The nucleotide sequences among the six different genotypes (GTs) differ at 30 to 35% of nucleotide sites (25, 26). Each of the six major GTs of HCV contains a series of closely related subtypes whose nucleotide sequences typically differ from each other by 20 to 25%. Furthermore, 5 to 8% sequence divergence is present between individual strains (variants) of HCV within a given subtype. A comprehensive analysis of HCV NS3 sequences from a larger number of GT-1 isolates found that amino acid polymorphisms were detected all along the protease sequence, including residues associated either with resistance to HCV protease inhibitors (PIs) (V36, I170, and D168) or with compensatory mutations (I72, T72, Q86, and I153) (1, 2, 30). However, many questions remain, including whether natural variation in the NS3 protease sequence impacts the susceptibility of HCV to PIs currently in development and whether there are any relationships among the chemotypes of the PIs and their baseline susceptibilities.Hepatitis C virus NS3/4A serine PIs have demonstrated potent antiviral activity in subjects infected with HCV GT-1 by specifically blocking NS3/4A protease-dependent HCV polyproprotein processing. Among these PIs, VX-950 (telaprevir) and SCH-503034 (boceprevir), the two most clinically advanced NS3/4A serine PIs, are both α-ketoamide compounds that covalently bind to the active-site serine of the protease (6, 9, 10, 14, 16, 22). These drugs also have similar resistance profiles. Mutations V36A/M, T54A, R155K, and A156S in the NS3 protease gene, conferring a low level of resistance to VX-950, were identified in HCV GT-1-infected patients treated with VX-950 monotherapy or in combination with alpha interferon (IFN-α) and ribavirin (11, 21). In addition, the single mutant A156T or A156V or the double mutant at positions 36/155 or 36/156, all conferring high-level resistance, were observed in some VX-950-treated patients. Similarly, samples from SCH-503034-treated patients revealed the emergence of the low- to moderate-level resistance mutations T54A, V170A, and A156S and, less commonly, the high-level resistance mutant A156T (27, 28).More recently, other HCV PIs, including ITMN-191, TMC-435350, and MK-7009, have progressed to the early stages of clinical evaluation (12, 15, 19, 20, 23, 29). These compounds are structurally related macrocyclic inhibitors that are chemically distinct from VX-950 and SCH-503034. The mutations that display a reduced susceptibility to ITMN-191 include D168A/E/V, A156S/V, F43S, Q41R, and S138T in the NS3 protease; S489L in the NS3 helicase; and V23A in NS4A (24). S489L in the helicase domain buttresses the P2 pocket, and the substitution V23A is at a site in the NS4A peptide cofactor that borders the P1′ pocket (24). The F43S, Q41R, R155K, A156T, and D168Y mutations in NS3 also displayed low- to high-level reduced sensitivity to MK-7009 (12). Since these PIs had some overlapping and some nonoverlapping resistance profiles, it would be of interest to compare their potencies against the natural HCV variants derived from treatment-naïve patient isolates.Standard replicon assays limit the assessment of the inhibitory activities of anti-HCV compounds to a few laboratory-optimized strains. These assays may not reflect the range of activities of a compound against the heterogeneous viral population that exists in HCV-infected patients. Previously, we have reported the development of a replicon-based shuttle vector to allow rapid phenotypic analysis of the NS3 protease domain from samples of a large number of HCV-infected patients (18). We demonstrated a high success rate and reproducibility of the novel replicon-based phenotypic assay. However, the assay sensitivity for detection of mutants in a mixed population may be reduced for resistance mutations that are less fit or with lower levels of resistance or samples containing only a small proportion of a mutant in a population (18). For example, reduced susceptibility to BILN-2061 was observed for mutant/wild-type mixtures of 5%/95% for the D168V mutation but was observed for only 50%/50% mixtures for the A156T resistance mutation. Using this phenotypic-analysis assay, the susceptibilities of a panel of HCV treatment-naïve clinical isolates to VX-950 (telaprevir), SCH-503034 (boceprevir), ITMN-191, MK-7009, and TMC-435350, as well as GS-9132 (also known as ACH-806, an NS4A antagonist [31]), were evaluated in the present study. GS-9132 was included in the study, as mutations that conferred resistance mapped in the NS4A-binding region of NS3, although the precise mechanism of action of GS-9132 has not been fully elucidated. Overall, the baseline clinical isolate susceptibilities were compared among this panel of drugs. Finally, the effects of natural substitutions in the HCV protease gene on drug susceptibilities were investigated.