In vitro resistance profile of the hepatitis C virus NS3/4A protease inhibitor TMC435

Abstract

TMC435 is a small-molecule inhibitor of the NS3/4A serine protease of hepatitis C virus (HCV) currently in phase 2 development. The in vitro resistance profile of TMC435 was characterized by selection experiments with HCV genotype 1 replicon cells and the genotype 2a JFH-1 system. In 80% (86/109) of the sequences from genotype 1 replicon cells analyzed, a mutation at NS3 residue D168 was observed, with changes to V or A being the most frequent. Mutations at NS3 positions 43, 80, 155, and 156, alone or in combination, were also identified. A transient replicon assay confirmed the relevance of these positions for TMC435 inhibitory activity. The change in the 50% effective concentrations (EC(50)s) observed for replicons with mutations at position 168 ranged from <10-fold for those with the D168G or D168N mutation to approximately 2,000-fold for those with the D168V or D168I mutation, compared to the EC(50) for the wild type. Of the positions identified, mutations at residue Q80 had the least impact on the activity of TMC435 (<10-fold change in EC(50)s), while greater effects were observed for some replicons with mutations at positions 43, 155, and 156. TMC435 remained active against replicons with the specific mutations observed after in vitro or in vivo exposure to telaprevir or boceprevir, including most replicons with changes at positions 36, 54, and 170 (<3-fold change in EC(50)s). Replicons carrying mutations affecting the activity of TMC435 remained fully susceptible to alpha interferon and NS5A and NS5B inhibitors. Finally, combinations of TMC435 with alpha interferon and NS5B polymerase inhibitors prevented the formation of drug-resistant replicon colonies.

FIG. 1.

Summary of results from in vitro selection experiments. (A) Frequency of changes in the protease domain of NS3 (amino acids 1 to 181) observed in experiments conducted with different replicon-containing cell lines in the presence or the absence of TMC435. NS3 sequences from 120 control replicon colonies or cell pools (21 of genotype 1a and 99 of genotype 1b) and 109 TMC435-treated replicon colonies or cell pools (46 of genotype 1a and 63 of genotype 1b) were assessed. Positions at which mutations were present in more than three sequences are shown. Dark gray bars, TMC435-treated cells; white bars, control cells. (B) Frequency of mutations at one or more of NS3 positions 43, 80, 155, 156, and 168 in cells selected with TMC435. Experiments were performed with genotype 1a (Huh7-SG1a H77) or genotype 1b (Huh7-Luc and Huh7-con1b) replicon-containing cells. Mutations containing a mixture of the wild-type residue with a mutated residue are counted as containing the mutated residue only. The frequency was calculated for each subtype. Black bars, genotype 1a; light gray bars, genotype 1b.

FIG. 2.

Selection experiments with JFH-1. Huh7.5 cells were transfected with RNA-encoding JFH-1wt, and the cells were passaged in the presence of 500 nM or 4,000 nM TMC435 or in the absence of inhibitor (control). Cells were harvested at each passage, and the JFH-1 RNA was quantified by using quantitative RT-PCR and normalized against the quantity of the host RPL13A gene. The JFH-1 RPL13A levels 3 days after transfection (after the first passage and at the start of TMC435 treatment) were set equal to 100%, and the changes over time are shown. White diamonds, cells cultured in the absence of inhibitor; gray squares, cells cultured in the presence of 500 nM TMC435; black triangles, cells cultured in the presence of 4,000 nM TMC435. Time points at which a CPE was observed and no cells could be harvested are indicated with a dashed line.

FIG. 3.

Influence of anti-HCV inhibitors used alone or in combination on the formation of replicon colonies. The images are of cells treated with TMC435 or thiophene-2 alone and in combination. Concentrations are expressed as multiples of the respective EC₅₀s determined in a genotype 1b replicon assay with a luciferase readout.

FIG. 4.

Structural perspective on selected mutations. (A) Overall view of the NS3/4A-TMC435 complex with the key in vitro resistance-conferring mutation positions highlighted (5). (B) Close-up of the extended S2 binding region occupied by the substituted quinoline ring of bound TMC435. The figure was prepared with the PyMol program (Delano Scientific, LLC).