In vitro phenotypic characterization of hepatitis C virus NS3 protease variants observed in clinical studies of telaprevir

Abstract

Telaprevir is a linear, peptidomimetic small molecule that inhibits hepatitis C virus (HCV) replication by specifically inhibiting the NS3·4A protease. In phase 3 clinical studies, telaprevir in combination with peginterferon and ribavirin (PR) significantly improved sustained virologic response (SVR) rates in genotype 1 chronic HCV-infected patients compared with PR alone. In patients who do not achieve SVR after treatment with telaprevir-based regimens, variants with mutations in the NS3·4A protease region have been observed. Such variants can contribute to drug resistance and limit the efficacy of treatment. To gain a better understanding of the viral resistance profile, we conducted phenotypic characterization of the variants using HCV replicons carrying site-directed mutations. The most frequently observed (significantly enriched) telaprevir-resistant variants, V36A/M, T54A/S, R155K/T, and A156S, conferred lower-level resistance (3- to 25-fold), whereas A156T and V36M+R155K conferred higher-level resistance (>25-fold) to telaprevir. Rarely observed (not significantly enriched) variants included V36I/L and I132V, which did not confer resistance to telaprevir; V36C/G, R155G/I/M/S, V36A+T54A, V36L+R155K, T54S+R155K, and R155T+D168N, which conferred lower-level resistance to telaprevir; and A156F/N/V, V36A+R155K/T, V36M+R155T, V36A/M+A156T, T54A+A156S, T54S+A156S/T, and V36M+T54S+R155K, which conferred higher-level resistance to telaprevir. All telaprevir-resistant variants remained fully sensitive to alpha interferon, ribavirin, and HCV NS5B nucleoside and nonnucleoside polymerase inhibitors. In general, the replication capacity of telaprevir-resistant variants was lower than that of the wild-type replicon.

Fig 1

Comparison of the activities of HCV NS3 protease inhibitors on NS3 variant replicons. Fold change in EC₅₀ was determined in the G1b 48-h assay.

Fig 2

Comparison of fold change of replicon EC₅₀ and enzyme IC₅₀ of NS3 protease inhibitors against NS3 variants. The fold change of replicon EC₅₀ was determined in the G1b 48-h assay. Data for WT (n = 4) and NS3 variants with actual or estimated values (n = 20) (V36L/M, R155I/K/S/T, A156S, and V36M+R155K for telaprevir; V36M, R155K, A156T, and V36M+R155K for boceprevir; V36M, R155I/T, and A156S for ciluprevir; and V36M, R155K, A156T, and V36M+R155K for danoprevir) were included in the comparison. A linear regression was performed on a log-transformed scale, with the resulting coefficient of determination (R²) of 0.66 having a P value < 0.0001.

Fig 3

Telaprevir EC₅₀ fold change and in vitro replicative fitness of NS3 variants in G1b replicon cells. The fold change in mean EC₅₀ and the SD were derived from Table 1 and plotted in the black bars corresponding to the y axis on the left. The replicative fitness (replication capacity) and the SD were derived from Table 5 and plotted in the gray bars corresponding to the y axis on the right. The upper dashed line represents the maximum change in EC₅₀ (i.e., 93-fold) that could be detected in the 96-h EC₅₀ assay. The lower dashed line represents the maximum change in EC₅₀ (i.e., 62-fold) that could be detected in the 48-h EC₅₀ assay. The fold changes in EC₅₀ above the maximum detection limits are represented with the “>” sign.