Identification of HCV protease inhibitor resistance mutations by selection pressure-based method

Abstract

A major challenge to successful antiviral therapy is the emergence of drug-resistant viruses. Recent studies have developed several automated analyses of HIV sequence polymorphism based on calculations of selection pressure (Ka/Ks) to predict drug resistance mutations. Similar resistance analysis programs for HCV inhibitors are not currently available. Taking advantage of the recently available sequence data of patient HCV samples from a Phase II clinical study of protease inhibitor boceprevir, we calculated the selection pressure for all codons in the HCV protease region (amino acid 1–181) to identify potential resistance mutations. The correlation between mutations was also calculated to evaluate linkage between any two mutations. Using this approach, we identified previously known major resistant mutations, including a recently reported mutation V55A. In addition, a novel mutation V158I was identified, and we further confirmed its resistance to boceprevir in protease enzyme and replicon assay. We also extended the approach to analyze potential interactions between individual mutations and identified three pairs of correlated changes. Our data suggests that selection pressure-based analysis and correlation mapping could provide useful tools to analyze large amount of sequencing data from clinical samples and to identify new drug resistance mutations as well as their linkage and correlations.

Resistance Analysis of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir

“…In patient 10 (600-mg cohort), emergent 1a NS3 protease substitutions detected were V55A and Q80K (Table 6). Although genotypic substitutions at these amino acid positions have been associated with resistance to other HCV NS3 PIs (36), in vitro NS3/4A enzyme susceptibility analyses of patient NS3 protease sequences revealed no changes in asunaprevir potency over time (Table 6). Similar findings have also been shown for the MAD study (34).…”

Analysis of Hepatitis C Virus Intrahost Diversity across the Coding Region by Ultradeep Pyrosequencing

“…Previously reported markers of resistance to hepatitis C virus inhibitors were analyzed in the four treatment-naïve patients, including mutations at 16 previously reported positions in the NS3/4a region (C16S, V36A/M/L/G, A39V, Q41R, F43S/I/V/C, T54S/A, V55A, Q80R/K/H/G/L, R109K, S138T, M175L, R155K/T/I/S/M/Q/L/G, V158I, A156V/T/S/I/G/F, D168A/V/E/G/N/T/Y/ I/H, V170A/T, L170T, and I170V) (13,17,24,27), 12 in the NS5A region (M28T, F28S, L23F, Q30E/H/R, L31M/F/V, Q54L, P32L, P58S, M21, Y93H/M/N, C92R, R318W, and D320E) (7,10,31), and 13 in the NS5B region (S282T/R, L314F, C316Y/F/S, M411S, M423T/I, P495S/L/A/T, V499A, M414I, C445F, Y448CH, Y452H, P496A/S, and L419M/V) (12,17,28). This analysis was based on the previously performed SNP analysis.…”

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

“…In fact, in vitro studies reveal reduced activity for ITMN-191 and TMC435 against S138T variants, while boceprevir remains fully active (30,43). The bulky P4 tert-butyl group of boceprevir extends outside the substrate envelope contacting V158; the V158I variant has lower affinity for this drug, likely due to a steric clash (45). This variant may also impact the affinity of ITMN-191, as its P4 tert-butyl also protrudes at the same location.…”