In vitro characterization of GSK2485852, a novel hepatitis C virus polymerase inhibitor

Abstract

GSK2485852 (referred to here as GSK5852) is a hepatitis C virus (HCV) NS5B polymerase inhibitor with 50% effective concentrations (EC50s) in the low nanomolar range in the genotype 1 and 2 subgenomic replicon system as well as the infectious HCV cell culture system. We have characterized the antiviral activity of GSK5852 using chimeric replicon systems with NS5B genes from additional genotypes as well as NS5B sequences from clinical isolates of patients infected with HCV of genotypes 1a and 1b. The inhibitory activity of GSK5852 remained unchanged in these intergenotypic and intragenotypic replicon systems. GSK5852 furthermore displays an excellent resistance profile and shows a <5-fold potency loss across the clinically important NS5B resistance mutations P495L, M423T, C316Y, and Y448H. Testing of a diverse mutant panel also revealed a lack of cross-resistance against known resistance mutations in other viral proteins. Data from both the newer 454 sequencing method and traditional population sequencing showed a pattern of mutations arising in the NS5B RNA-dependent RNA polymerase in replicon cells exposed to GSK5852. GSK5852 was more potent than HCV-796, an earlier inhibitor in this class, and showed greater reductions in HCV RNA during long-term treatment of replicons. GSK5852 is similar to HCV-796 in its activity against multiple genotypes, but its superior resistance profile suggests that it could be an attractive component of an all-oral regimen for treating HCV.

Fig 1

Structures of GSK5852 and HCV-796.

Fig 2

Cross-resistance profiling of GSK5852 or HCV-796 on HCV mutants. Genotype 1b replicons containing the indicated amino acid changes in NS3, NS4B, NS5A, and NS5B were constructed by site-directed mutagenesis. The mean EC₅₀s for GSK5852 or HCV-796 were determined by transient-transfection assays (n ≥ 4) and compared to the Con1 EC₅₀ to calculate fold changes.

Fig 3

Inter- and intragenotypic activity of GSK5852. The mean EC₅₀s for genotype 1a, 1b, 4a, and 5a patient sequence replicons and genotype 2a, 2b, and 3a consensus sequence replicons are represented by open circles (○), and the Con1 replicon is represented by the filled circles (●). EC₅₀s were determined by transient transfection for genotype 1a and 1b and with stable cell lines for the non-genotype 1 chimeric replicons (n ≥ 4).

Fig 4

HCV RNA reduction assay. Genotype 1a (A) or 1b (B) replicon cells were treated with compounds and reductions in replicon RNA determined as described in Materials and Methods. ●, DMSO control; ■, 3× EC₉₀ GSK5852; □, 10× EC₉₀ GSK5852; ▲, 10× EC₉₀ HCV-796.