The hepatitis C virus replicon presents a higher barrier to resistance to nucleoside analogs than to nonnucleoside polymerase or protease inhibitors

Abstract

Specific inhibitors of hepatitis C virus (HCV) replication that target the NS3/4A protease (e.g., VX-950) or the NS5B polymerase (e.g., R1479/R1626, PSI-6130/R7128, NM107/NM283, and HCV-796) have advanced into clinical development. Treatment of patients with VX-950 or HCV-796 rapidly selected for drug-resistant variants after a 14-day monotherapy treatment period. However, no viral resistance was identified after monotherapy with R1626 (prodrug of R1479) or NM283 (prodrug of NM107) after 14 days of monotherapy. Based upon the rapid selection of resistance to the protease and nonnucleoside inhibitors during clinical trials and the lack of selection of resistance to the nucleoside inhibitors, we used the replicon system to determine whether nucleoside inhibitors demonstrate a higher genetic barrier to resistance than protease and nonnucleoside inhibitors. Treatment of replicon cells with nucleoside inhibitors at 10 and 15 times the 50% effective concentration resulted in clearance of the replicon, while treatment with a nonnucleoside or protease inhibitor selected resistant colonies. In combination, the presence of a nucleoside inhibitor reduced the frequency of colonies resistant to the other classes of inhibitors. These results indicate that the HCV replicon presents a higher barrier to the selection of resistance to nucleoside inhibitors than to nonnucleoside or protease inhibitors. Furthermore, the combination of a nonnucleoside or protease inhibitor with a nucleoside polymerase inhibitor could have a clear clinical benefit through the delay of resistance emergence.

FIG. 1.

Replicon cells treated with a nucleoside inhibitor clear the replicon, while VX-950 or HCV-796 select for resistant colonies. Huh7 cells that stably maintain a GT 1b subgenomic replicon carrying the neoR gene were incubated with a specific HCV inhibitor at 1, 10, and 15 times the EC₅₀ in the presence of G418. After selection for 3 weeks, the remaining cells were fixed and stained with crystal violet. The replicon cells were treated with the nucleoside inhibitor R1479 (A), NM107 (B), or PSI-6130 (C); the nonnucleoside inhibitor HCV-796 (D); or the protease inhibitor VX-950 (E). A representative example of experiments is shown (n = 6 for R1479, n = 4 for NM107 and PSI-6130, n = 7 for VX-950, and n = 10 for HCV-796).

FIG. 2.

Combination treatment with a nucleoside inhibitor reduces the emergence of resistant colonies. Huh7 cells that stably maintain a GT 1b subgenomic replicon carrying the neoR gene were incubated with one or two specific HCV inhibitors at 1, 10, and 15 times the EC₅₀ in the presence of G418. After selection for 3 weeks, the remaining cells were fixed and stained with crystal violet. The replicon cells were treated with R1479 and HCV-796 (A), R1479 and VX-950 (B), PSI-6130 and HCV-796 (C), or PSI-6130 and VX-950 (D), and a representative example of at least four independent experiments is shown.

FIG. 3.

Combination of a nucleoside inhibitor with either VX-950 or HCV-796 results in additive inhibitory activity against the replicon. Huh7 cells that stably maintain a GT 1b subgenomic replicon were treated with various concentrations of a nucleoside inhibitor, either R1479 (A and B) or PSI-6130 (C and D), and either HCV-796 (A and C) or VX-950 (B and D). After 72 h, the inhibitory activity of the compounds was determined by measuring the reduction in luciferase activity. The activity of the inhibitors in combination was determined from at least three experiments and is reported as percent synergy.

FIG. 4.

Transient replicons show lack of cross-resistance to other classes of HCV inhibitors. Huh7-Lunet cells were transfected with either a wild-type GT 1b replicon or one engineered to contain mutations resulting in reduced sensitivity to VX-950 (A), HCV-796 (B), R1479 (C), or PSI-6130 (D). The transfected cells were incubated with various HCV inhibitors for 72 h, and then the replicon luciferase activity was measured. The EC₅₀ determinations were obtained from at least three independent experiments: Con1, n = 6 for R1479, n = 8 for VX-950, n = 5 for PSI-6130, and n = 6 for HCV-796; V36M, n = 5 for R1479, n = 6 for VX-950, and n = 4 for PSI-6130; T54A, n = 4 for R1479 and VX-950 and n = 3 for PSI-6130; R155K, n = 3 for R1479 and PSI-6130 and n = 4 for VX-950; V36M+R155K, n = 5 for R1479 and n = 3 for VX-950 and PSI-6130; A156S, n = 5 for R1479 and PSI-6130 and n = 6 for VX-950; A156T, n = 4 for R1479 and n = 3 for VX-950 and PSI-6130; V36M+A156T, n = 3 for R1479, VX-950, and PSI-6130; A156V, n = 6 for R1479, n = 4 for VX-950, and n = 3 for PSI-6130; S96T, n = 6 for R1479, n = 8 for VX-950, and n = 4 for HCV-796; S96T+N142T, n = 6 for R1479, n = 7 for VX-950, and n = 4 for HCV-796; C316Y, n = 4 for R1479, n = 5 for PSI-6130, and n = 6 for HCV-796; S365T, n = 3 for R1479 and PSI-6130 and n = 5 for HCV-796; M414I, n = 4 for R1479 and n = 3 for PSI-6130 and HCV-796; and S282T, n = 6 for VX-950, n = 4 for PSI-6130, and HCV-796. The fold change of the inhibitor was determined as the EC₅₀ of the inhibitor against the mutant replicon compared to the wild-type replicon and is presented as mean ± standard error of the mean.

FIG. 5.

Replication capacity of replicons containing NS3 or NS5B mutations. Huh7-Lunet cells were transfected with either a wild-type GT 1b replicon or one engineered to contain mutations resulting in reduced sensitivity to VX-950, HCV-796, R1479, or PSI-6130. The replication capacity of the mutants was determined from at least four independent experiments (n = 8 for Con1 and S282T; n = 6 for T54A, R155K, A156S, and V36M+R155K; n = 5 for V36M+A156T, V201A, C316Y, S365T; and n = 4 for S156T, A156V, V36M, S96T, N142T, S96T+N142T, C316Y, and M414I) and is expressed as their normalized replication efficiency (mean ± standard error of the mean) compared to that of the wild-type, which was set at 1.