Characterization of Resistance to the Nonnucleoside NS5B Inhibitor Filibuvir in Hepatitis C Virus-Infected Patients

Abstract

Filibuvir (PF-00868554) is an investigational nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural 5B (NS5B) RNA-dependent RNA polymerase currently in development for treating chronic HCV infection. The aim of this study was to characterize the selection of filibuvir-resistant variants in HCV-infected individuals receiving filibuvir as short (3-to 10-day) monotherapy. We identified amino acid M423 as the primary site of mutation arising upon filibuvir dosing. Through bulk cloning of clinical NS5B sequences into a transient-replicon system, and supported by site-directed mutagenesis of the Con1 replicon, we confirmed that mutations M423I/T/V mediate phenotypic resistance. Selection in patients of an NS5B mutation at M423 was associated with a reduced replicative capacity in vitro relative to the pretherapy sequence; consistent with this, reversion to wild-type M423 was observed in the majority of patients following therapy cessation. Mutations at NS5B residues R422 and M426 were detected in a small number of patients at baseline or the end of therapy and also mediate reductions in filibuvir susceptibility, suggesting these are rare but clinically relevant alternative resistance pathways. Amino acid variants at position M423 in HCV NS5B polymerase are the preferred pathway for selection of viral resistance to filibuvir in vivo. Hepatitis C virus (HCV) is a leading cause of chronic liver disease, liver cancer, and cirrhosis, with an infection prevalence of 2 to 3% worldwide (9). Approximately 70% of people who become infected with HCV are unable to clear the virus naturally and develop a chronic infection. The current treatment regimen requires 6 to 12 months of combination therapy with pegylated interferon (pegIFN) (weekly injections) and ribavirin (RBV) (daily oral dosing). This standard of care results in cure rates of ϳ50% in patients with the most common genotype virus (genotype 1) and is associated with multiple side effects (2, 6, 13). Therefore, new, improved treatments are urgently needed.Multiple candidate small-molecule therapeutics that specifically target the virus (direct-acting antivirals [DAAs]) are currently in development, including inhibitors of the HCV NS3 protease, NS5A protein, and NS5B RNA-dependent RNA polymerase (10,22). The most advanced compounds, telaprevir and boceprevir, are NS3 protease inhibitors and have been approved for use in combination with pegIFN and RBV. In phase 3 clinical studies, these compounds significantly increased rates of sustained viral response (SVR) compared to the standard of care alone in both treatment-naïve and treatment-experienced patient populations (16). However, both compounds have significant side effects, including severe rash and anemia, which contribute to the overall adverse-event burden associated with treatment. Patients failing therapy also typically develop drug class resistance that may prevent effective retreatment with other HCV protease inhibitors (19). Therefore, more tolerable and efficacious DAAs with unique mechanisms of a...

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

“…For the NS5A inhibitor daclatasvir (BMS-790052), the primary resistant variants selected in vitro were NS5A M28T, Q30E/H/R, L31M/V, P32L, and Y93C/H/N for genotype 1a and L31F/V, P32L, and Y93H/N for genotype 1b, with L23F, R30Q, and P58S selected as secondary resistance substitutions (7). Although the subtype 1b variants conferred lower-level resistance (a 5-to 28-fold shift in EC 50 ), the variants in subtype 1a conferred higher levels of resistance (233-to 5,367-fold shift in EC 50 ) (7). The reported clinical resistance pattern appears similar to that seen in vitro; patients with viral breakthrough on a dual combination of protease inhibitor asunaprevir and NS5A inhibitor daclatasvir had NS5A Q30E/R, L31M/V, and Y93C/N variants (29).…”

“…Baseline prevalence of NS5B allosteric thumb site inhibitor variants. Clinical candidates VCH-759, VX-222, and filibuvir (PF-00868554), bind allosterically in the thumb region of NS5B, and the resistance profiles, including early clinical resistance data, have been reported (10,49,50). 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).…”

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

“…The frequencies of RAVs at this residue were similar between the subtype 1a and 1b viruses. RAVs at NS5B residues R422 (R422K), M426 (M426A), and V494 (V494A) were also detected in a small number of patients at baseline or the end of therapy and were found to mediate reductions in filibuvir susceptibility (13). GS-9669 has reduced in vitro activity against known resistance variants associated with thumb site II inhibitors (L419M, R422K, F429L, and I482L in GT1b, and L419M and I482L in GT1a) (11).…”

“…Other NNIs currently in phase II clinical studies include BI-207127 and BMS-791325 (binding to thumb site I), filibuvir and lomibuvir (binding to thumb site II), setrobuvir, ABT-072, and ABT-333 (binding to palm site I), and tegobuvir (also binding in the palm) (12). In a phase Ib study of filibuvir, resistance-associated variants (RAVs) at NS5B residue M423 (M423I/T/V) were observed in 76% of the patients following treatment (13). The frequencies of RAVs at this residue were similar between the subtype 1a and 1b viruses.…”

Viral Factors Associated with Response to Antiviral Therapy for Chronic Hepatitis C Virus Infection

“…The anti-viral potency of the non-nucleoside type is markedly lower when compared to that of PI, and efficacy may be HCV genotype specific [59]. Because of the lower potency of antiviral effects with high risk of developing resistant mutants, non-nucleoside-type DAAs may not be promising.…”