Mutations Conferring Resistance to a Potent Hepatitis C Virus Serine Protease Inhibitor In Vitro

Abstract

BILN 2061 is a novel, specific hepatitis C virus (HCV) NS3 serine protease inhibitor discovered by Boehringer Ingelheim that has shown potent activity against HCV replicons in tissue culture and is currently under clinical investigation for the treatment of HCV infection. The poor fidelity of the HCV RNA-dependent RNA polymerase will likely lead to the development of drug-resistant viruses in treated patients. The development of resistance to BILN 2061 was studied by the in vitro passage of HCV genotype 1b replicon cells in the presence of a fixed concentration of the drug. Three weeks posttreatment, four colonies were expanded for genotypic and phenotypic characterization. The 50% inhibitory concentrations of BILN 2061 for these colonies were 72-to 1,228-fold higher than that for the wild-type replicon. Sequencing of the individual colonies identified several mutations in the NS3 serine protease gene. Molecular clones containing the single amino acid substitution A156T, R155Q, or D168V resulted in 357-fold, 24-fold, and 144-fold reductions in susceptibility to BILN 2061, respectively, compared to the level of susceptibility shown by the wild-type replicon. Modeling studies indicate that all three of these residues are located in close proximity to the inhibitor binding site. These findings, in addition to the three-dimensional structure analysis of the NS3/NS4A serine protease inhibitor complex, provide a strategic guide for the development of next-generation inhibitors of HCV NS3/NS4A serine protease.Hepatitis C virus (HCV) infection is believed to be the leading cause of chronic hepatitis, end-stage cirrhosis, and hepatocellular carcinoma, affecting over 4 million Americans and about 170 million people worldwide. Currently, the most effective treatment of HCV infection involves a combination of the nucleoside analog ribavirin with alpha interferon (IFN-␣). However, the regimen is prolonged and not well tolerated, and only approximately half of the genotype 1 HCV-infected individuals have a sustained virological response, although the response rate improves significantly (ϳ80%) when genotypes 2 and 3 are treated (7,29). An oral agent that offers promise as an efficacious alternative to IFN or that may be used in IFNcontaining regimens and that improves efficacy and/or the side effect profile is in great demand.The HCV genome is a 9.6-kb single-stranded RNA of positive polarity encoding a large polyprotein that is posttranslationally cleaved into structural and nonstructural proteins (3,12,23). The N-terminal domain (approximately 180 amino acids) of NS3 and the small hydrophobic NS4A protein compose a heterodimeric enzyme catalyzing the posttranslational processing of the HCV nonstructural proteins (1, 2, 23). Its structure has been extensively studied by X-ray crystallography (9, 30, 31) and nuclear magnetic resonance spectroscopy (1, 6). The proteolytic activity of NS3/NS4A serine protease is known to be essential for viral RNA replication (10,14). A recent study indicated that the NS3/NS4A serine protease a...

Anti‐HCV therapies in chimeric scid‐Alb/uPA mice parallel outcomes in human clinical application†

“…Mean HCV RNA dropped by 1.05 log in BILN 2061-treated animals after 7 days' treatment (6.55 Ϯ Despite the initial decrease in HCV titer in all mice treated with BILN 2061, four of six mice had varying degrees of rebound in viral titers at the 14-day point, although they remained on therapy. Because a series of mutations in NS3 (A156T, R155Q, D168V) were previously reported to result in a protease-resistant phenotype in replicons exposed to BILN2061, 10,11 we sequenced the plasma RNA from the two most pronounced rebounders in an attempt to detect these mutations in vivo. The HCV sequence in all samples (including the inoculum) had the expected wild-type residues at all three positions.…”

“…Sequencing, however, did not reveal the A156T, R155Q, or D168V mutations previously reported to result in resistance in replicons. 10,11 We did identify a Q80K mutation that existed in the inoculum before exposure to the drug, and that attenuated the response to BILN by a factor of 3 to 4 when inserted into a replicon system. This mutation may well have contributed to the rebound seen in some of the mice.…”

Preexisting drug-resistance mutations reveal unique barriers to resistance for distinct antivirals

“…Although all these protease inhibitors lose potency against A156T and R155K mutations, the overall fold of resistance conferred by R155K is smaller for VX-950 (∼10 fold) than for MK-7009, BILN-2061, and INTM-191 (70-to 250-fold) (9,25). VX-950 also retains activity against mutations at position D168 that confer very high levels of resistance to MK-7009, BILN-2061, and INTM-191 (up to >100-fold) (9,(26)(27)(28). Because the R155K and D168V mutations are more fit than A156T, they probably are present in the replicon quasispecies at higher frequencies (9).…”

Tracking the Evolution of MultipleIn VitroHepatitis C Virus Replicon Variants under Protease Inhibitor Selection Pressure by 454 Deep Sequencing

“…EC 50 s of the mutants were compared to those of the parental wild-type HCV replicon, and fold changes were calculated. Replication capacity was determined in the absence of inhibitors as previously described (28,35) by measuring luciferase levels 48 h postelectroporation and normalizing to the luciferase levels obtained 4 h postelectroporation.…”

“…The replication capacities of the different mutant replicons were determined as a measure of viral fitness using standard procedures (28,35). The luciferase signal at 48 h posttransfection was normalized to the signal obtained at 4 h to correct for differences in transfection efficiencies, and the replication capacities of mutants were calculated relative to that of the wild type.…”