In Vitro Resistance Study of AG-021541, a Novel Nonnucleoside Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase

Abstract

A novel class of nonnucleoside hepatitis C virus (HCV) polymerase inhibitors characterized by a dihydropyrone core was identified by high-throughput screening. Crystallographic studies of these compounds in complex with the polymerase identified an allosteric binding site close to the junction of the thumb and finger domains, approximately 30 Å away from the catalytic center. AG-021541, a representative compound from this series, displayed measurable in vitro antiviral activity against the HCV genotype 1b subgenomic replicon with a mean 50% effective concentration of 2.9 M. To identify mutations conferring in vitro resistance to AG-021541, resistance selection was carried out using HCV replicon cells either by serial passages in increasing concentrations of AG-021541 or by direct colony formation at fixed concentrations of the compound. We identified several amino acid substitutions in the AG-021541-binding region of the polymerase, including M423(T/V/I), M426T, I482(S/T), and V494A, with M423T as the predominant change observed. These mutants conferred various levels of resistance to AG-021541 and structurally related compounds but remained sensitive to interferon and HCV polymerase inhibitors known to interact with the active site or other allosteric sites of the protein. In addition, dihydropyrone polymerase inhibitors retained activity against replicons that contain signature resistance changes to other polymerase inhibitors, including S282T, C316N, M414T, and P495(S/L), indicating their potential to be used in combination therapies with these polymerase inhibitors. AG-021541-resistant replicon cell lines provide a valuable tool for mechanism-of-action studies of dihydropyrone polymerase inhibitors. The clinical relevance of in vitro resistance to HCV polymerase inhibitors remains to be investigated.Hepatitis C virus (HCV) has emerged as one of most important etiological factors for blood-transmitted chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (34, 38). The infection becomes persistent in about 85% of infected individuals, despite the presence of a strong humoral and cellular immune response (3). Currently, about 4.5 million individuals in the United States and more than 170 million worldwide are infected with HCV, which represents an important public health problem. A combination of pegylated forms of alpha interferon (IFN-␣) and ribavirin is the only therapy available against HCV, but the success rate observed in individuals infected with genotype 1, which is the most prevalent genotype in the United States and worldwide, is only about 40% to 50% (7,8,25). In addition, IFN-␣ therapy is associated with significant side effects including fatigue, headache, myalgia, fever, nausea, and insomnia in more than 30% of patients. Ribavirin also causes hemolytic anemia in 10% to 20% of patients (22,36). Consequently, there remains a significant unmet medical need for more effective and safer HCV therapy.The HCV genome is a single-stranded, positive-sense RNA of approximately 9.6 kb (5). The genom...

Association of hepatitis C virus NS5B variants with resistance to new antiviral drugs among untreated patients

“…Two other codons (282 and 316) implicated with resistance to new NS5B inhibitors were also within the NS5B fragment sequenced in this study (Dutartre et al 2006, Shi et al 2008, McCown et al 2009). Codon 282 was implicated with mutations related to 2me-cytosine resistance (mutation S282T) and all isolates analysed coded for serine in this position (Dutartre et al 2006).…”

Phenotypic Characterization of Resistant Val 36 Variants of Hepatitis C Virus NS3-4A Serine Protease

“…It is buried in a hydrophobic environment and is not in direct contact with telaprevir. However, Val 36 does make direct contact with Phe 43 , which is part of the S1Ј substrate-binding pocket of the HCV protease (Fig. 2B).…”

Hepatitis C Virus Variants with Decreased Sensitivity to Direct-Acting Antivirals (DAAs) Were Rarely Observed in DAA-Naive Patients prior to Treatment

“…Variants selected clinically during a 3-day dosing study of VX-222 were at NS5B 419, 422, 423, 482, 486, and 494 (51); however, in the ZENITH study in patients that had virologic breakthrough during treatment with VX-222 and telaprevir the NS5B variants observed were L419S and R422K (52). A partially overlapping resistance profile was reported for filibuvir with variants at NS5B 423, 426, 482, and 494 identified in vitro (53). Variants at NS5B 423 were the predominant treatment emergent variant in patients that failed a filibuvir-containing regimen (54).…”