Mutations conferring resistance to a hepatitis C virus (HCV) RNA-dependent RNA polymerase inhibitor alone or in combination with an HCV serine protease inhibitor in vitro

Abstract

Compounds A-782759 (an N-1-aza-4-hydroxyquinolone benzothiadiazine) and BILN-2061 are specific anti-hepatitis C virus (HCV) agents that inhibit the RNA-dependent RNA polymerase and the NS3 serine protease, respectively. Both compounds display potent activity against HCV replicons in tissue culture. In order to characterize the development of resistance to these anti-HCV agents, HCV subgenomic 1b-N replicon cells were cultured with A-782759 alone or in combination with BILN-2061 at concentrations 10 times above their corresponding 50% inhibitory concentrations in the presence of neomycin. Single substitutions in the NS5B polymerase gene (H95Q, N411S, M414L, M414T, or Y448H) resulted in substantial decreases in susceptibility to A-782759. Similarly, replicons containing mutations in the NS5B polymerase gene (M414L or M414T), together with single mutations in the NS3 protease gene (A156V or D168V), conferred high levels of resistance to both A-782759 and BILN-2061. However, the A-782759-resistant mutants remained susceptible to nucleoside and two other classes of nonnucleoside NS5B polymerase inhibitors, as well as interferon. In addition, we found that the frequency of replicons resistant to both compounds was significantly lower than the frequency of resistance to the single compound. Furthermore, the dually resistant mutants displayed significantly reduced replication capacities compared to the wild-type replicon. These findings provide strategic guidance for the future treatment of HCV infection.

FIG. 1.

Chemical structures of the HCV polymerase inhibitors from Abbott, 1 (A-782759); Shire, 2; Merck, 3; Boehringer Ingelheim, 4; and Glaxo Smithkline, 6, as well as the HCV protease inhibitor BILN-2061, 5.

FIG. 2.

Comparison of replication capacity of wild-type replicon with those of the recombinant mutant replicons. Cells were transfected either with wild-type or with mutant replicon RNA in the absence of BILN-2061, and luciferase activity was measured at 4 hours and 4 days after transfection. The replication capacity of each mutant was calculated by comparing the firefly luciferase activity generated by the mutant to that generated by wild-type replicon at day 4, after adjusting for minor differences in transfection efficiencies (the 4-h luciferase activity). The data are averages of at least two separate experiments with six replicates in each experiment. The error bars represent standard deviations.

FIG. 3.

Locations of mutations and inhibitor binding sites for HCV polymerase enzyme. A ribbon representation (orange) of the protein is shown for Protein Database entry 1C2P (27). Mutations are shown as blue balls at the alpha carbons of the specified residues. Space-filling representations of various inhibitors are also shown: phenylalanine-amide (purple) (57), diamide (gray) (8), and nucleoside (green) (24).