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.

Fig 1

Selection of patients for phenotypic analysis of plasma HCV RNA. Baseline, on-therapy, and follow-up isolates were analyzed (black boxes), or else the baseline sample alone was analyzed (shaded boxes). FLV, filibuvir; RAM, resistance-associated mutation, i.e., a variant residue at NS5B position 419, 422, 423, 426, 482, or 494.

Fig 2

Phenotypic analysis of NS5B clinical isolates selected from those encoding an M423 mutation on therapy (M423) and from patients receiving placebo (Placebo). BL, baseline; OT, on therapy; FUp, follow-up. The horizontal lines indicate the geometric means. (a) Susceptibility to filibuvir in a transient-replicon assay indicated as EC₅₀ (molar). Clinical isolates with a calculated EC₅₀ greater than the penultimate concentration used in the phenotypic assay are plotted as that concentration: 1.33 × 10⁻⁵ M. (b) RCs of the clinical-isolate replicons. The RC was determined as the ratio of luciferase activity in untreated cells at 96 h to the 4-h input time point and was normalized to the Con1 WT replicon. The black symbols indicate isolates for which filibuvir susceptibility could not be determined. Most symbols represent single point determinations (Table 2).

Fig 3

Effects of sequence mixtures on phenotypic detection of filibuvir resistance. Replicon RNA containing SDMs in the polymerase that affect filibuvir susceptibility were mixed with Con1 WT replicon RNA at set ratios and transiently electroporated into Huh7.5 cells for filibuvir susceptibility determination (molar). Black crosses, 100% Con1; green circles, 90% Con1 plus 10% SDM; red crosses, 75% Con1 plus 25% SDM; blue stars, 50% Con1 plus 50% SDM; orange squares, 25% Con1 plus 75% SDM; gray diamonds, 10% Con1 plus 90% SDM; pink triangles, 100% SDM. Both single-sigmoid and biphasic-sigmoid dose-response curves were fitted to each filibuvir titration data set, the best statistical fit was selected, and EC₅₀s were determined. (A) Mixture of Con1 plus M423T. (B) Mixture of Con1 plus M423V. (C) Mixture of Con1 plus I482S. Panels A to C show representative experiments in which n was equal to 2 for M423T and I482S and n was equal to 3 for M423V. (D) Forward (top) and reverse (bottom) DNA-sequencing electropherograms of a 25% Con1 plus 75% M423V RNA mixture prior to electroporation into Huh7.5 cells (Input) and extracted from cells at the 96-h assay endpoint. The predominant base at each position is indicated below each electropherogram. Orange rectangle, NS5B residue 423 codon (valine, GTG; methionine, ATG).

Fig 4

Model of the NS5B thumb 2 pocket with filibuvir bound and residues key to filibuvir resistance highlighted. Residues M423 and R422 are critical in the formation of the hydrophobic pocket in which the filibuvir cyclopentyl ring sits. M426 resides in a secondary ring of residues around the filibuvir binding pocket.