Mechanistic Characterization of GS-9190 (Tegobuvir), a Novel Nonnucleoside Inhibitor of Hepatitis C Virus NS5B Polymerase

Abstract

GS-9190 (Tegobuvir) is a novel imidazopyridine inhibitor of hepatitis C virus (HCV) RNA replication in vitroand has demonstrated potent antiviral activity in patients chronically infected with genotype 1 (GT1) HCV. GS-9190 exhibits reduced activity against GT2a (JFH1) subgenomic replicons and GT2a (J6/JFH1) infectious virus, suggesting that the compound's mechanism of action involves a genotype-specific viral component. To further investigate the GS-9190 mechanism of action, we utilized the susceptibility differences between GT1b and GT2a by constructing a series of replicon chimeras where combinations of 1b and 2a nonstructural proteins were encoded within the same replicon. The antiviral activities of GS-9190 against the chimeric replicons were reduced to levels comparable to that of the wild-type GT2a replicon in chimeras expressing GT2a NS5B. GT1b replicons in which the ␤-hairpin region (amino acids 435 to 455) was replaced by the corresponding sequence of GT2a were markedly less susceptible to GS-9190, indicating the importance of the thumb subdomain of the polymerase in this effect. Resistance selection in GT1b replicon cells identified several mutations in NS5B (C316Y, Y448H, Y452H, and C445F) that contributed to the drug resistance phenotype. Reintroduction of these mutations into wild-type replicons conferred resistance to GS-9190, with the number of NS5B mutations correlating with the degree of resistance. Analysis of GS-9190 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of GS-9190 is different from other nonnucleoside inhibitors. Collectively, these data demonstrate that GS-9190 represents a novel class of nonnucleoside polymerase inhibitors that interact with NS5B likely through involvement of the ␤-hairpin in the thumb subdomain.Hepatitis C virus (HCV) is a major cause of morbidity, affecting approximately 170 million people worldwide with an estimated 3 to 4 million additional new infections occurring each year (36). HCV is a positive-strand RNA virus with six major genotypes that are further divided into multiple subtypes. Due to the error-prone nature of its replication enzyme, a myriad of different viral quasispecies exists within an infected individual (32). With this high degree of viral variability, the current treatment regimen, which consists of weekly injections of pegylated alpha interferon (PEG-IFN) and twice-daily oral doses of ribavirin (RBV), is of limited efficacy and, in addition, carries significant side effects (8, 23). Although the HCV NS3/4A protease inhibitors telaprevir and boceprevir for treatment of chronic HCV infection will soon be available, these compounds will still need to be combined with the current standard of care (PEG-IFN/RBV) to be efficacious and will not cure all infected individuals (10,14,30). Therefore, the development of additional direct antiviral agents with diverse resistance profiles is necessary, with the ultimate goal of developing all-oral antiviral combinations that can achieve superior ...

The emergence of NS5B resistance associated substitution S282T after sofosbuvir‐based treatment

“…RASs were introduced into the GT1a or GT1b replicon by site‐directed mutagenesis and tested in transient transfections as described previously 23. Briefly, NS5B mutations were introduced into a plasmid encoding the PI‐hRluc replicon using a QuikChange II XL mutagenesis kit, following the manufacturer's instructions (Stratagene, La Jolla, CA).…”

Hepatitis C Virus Genetic Variability and the Presence of NS5B Resistance-Associated Mutations as Natural Polymorphisms in Selected Genotypes Could Affect the Response to NS5B Inhibitors

“…1). According to the level of resistance, NI/NNI resistance-associated variants have been divided into major (high-intermediate level of resistance) and minor (low level of resistance) variants (7,(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46). Only variants with prevalences of Ͼ1% and found in Ն2 patients were considered.…”

Clinical and In Vitro Resistance to GS-9669, a Thumb Site II Nonnucleoside Inhibitor of the Hepatitis C Virus NS5B Polymerase

“…Resistance mutations were introduced into the GT1a (15) or GT1b replicon (16) by site-directed mutagenesis and tested in transient-transfection assays, as previously described (14). Briefly, NS5B mutations were introduced into a plasmid carrying the gene encoding the PI-hRluc replicon using a QuikChange II XL mutagenesis kit, according to the manufacturer's instructions (Stratagene, La Jolla, CA).…”

“…In vitro resistance selection in replicons. Resistance selections were performed as previously described (14). Briefly, GT1a-or GT1b-containing replicon cells were cultured in the presence of 5ϫ or 20ϫ the EC 50 of GS-9669 until small colonies formed.…”