Natural Variation in Drug Susceptibility to HCV Polymerase Inhibitors in Treatment-naïve HCV Patient Isolates

S.-C. C. Sun; A. Bae; X. Qi; J. Harris; K. A. Wong; M. D. Miller; H. Mo

Disclosures

J Viral Hepat. 2011;18(12):861-870. 

In This Article

Abstract and Introduction

Abstract

To assess the natural variation in drug susceptibility among treatment-naïve hepatitis C virus (HCV) patient isolates, the susceptibilities of chimeric replicons carrying the HCV NS5B polymerase from up to 51 patient isolates against a panel of diverse HCV nonnucleoside polymerase inhibitors were evaluated using a replicon-based transient replication assay. Some patient to patient variation in susceptibility to the panel of three HCV nonnucleoside polymerase inhibitors was observed. Linear regression and correlation analyses revealed no correlations among the susceptibilities to the polymerase inhibitors tested. Our results suggest that variable antiviral responses to HCV nonnucleoside polymerase inhibitors may be observed because of the natural variation in baseline susceptibility. In addition, the lack of correlation among the susceptibilities to three classes of HCV polymerase inhibitors evaluated here supports their possible combined use in a combination therapy strategy

Introduction

Hepatitis C virus (HCV) is a positive-strand RNA virus and is estimated to infect over 170 million people worldwide.[1,2] To date pegylated interferon-α plus ribavirin, the current standard of therapy for HCV infection, is associated with incomplete efficacy and various side effects.[3–6] Therefore, intense effort and time have been devoted to the discovery and development of novel and selective small-molecule inhibitors of HCV. Although no direct small-molecule antivirals have yet been approved for therapeutic use, the NS3 protease and NS5B polymerase are considered to be prime targets, and inhibitors of each enzyme have shown strong antiviral activity in early clinical trials.[7–11]

HCV RNA-dependent RNA polymerase is the essential enzyme for replication of HCV RNA. A number of HCV polymerase inhibitors have been discovered; some have advanced to phase I/II clinical trials and have demonstrated antiviral activity in HCV-infected subjects in monotherapy.[9,10,12] Among these HCV polymerase inhibitors, a number of nucleoside analogues (2'-Me-C, R1479, NM283, R1626 and R7128) bind to the active site of the HCV polymerase.[13–15] In vitro resistance selection in the presence of nucleoside analogues identified a S282T resistance mutation for 2'-Me-C, and two (S96T and N142T) for R1479.[16] The S282T mutation has also been reported in virologic breakthrough in patients on an NM283 (Idenix Pharmaceuticals, Cambridge, MA, USA) clinical trial.[17,18] In addition to the active site, there are four allosteric binding sites for nonnucleoside inhibitors (NNIs) within the NS5B polymerase including: palm I (palm domain near the active site), palm II (partially overlapping palm I and towards the active site), thumb I (thumb domain near the fingertips) and thumb II (the outer surface of the thumb domain).[19–21] First, the benzothiadiazine and acylpyrrolidine class of NNIs bind to the palm I domain of the polymerase.[7,20] Resistance selection in the presence of benzothiadiazines revealed amino acid substitutions at residues 411, 414, 448, 451, 553, 554, 555, 556 and 559.[22–25] Second, the benzofuran class of NNIs (HCV-796; Wyeth/Viropharma) binds to the palm II domain. HCV-796 (benzofuran) has been reported to select a major resistance mutation at amino acid residue 316 in an in vitro replicon system, and in early clinical trials.[26,27] Distinct from the above HCV polymerase inhibitors, benzimidazoles and indoles bind to the thumb I site.[28] Reduced susceptibility to this class of compounds was associated with the selection of mutations at residues 495 and 496 of the NS5B polymerase.[28] Lastly, the thiophene carboxylic acids bind to the thumb II site.[20,29,30] The most frequent mutations observed in vitro and in vivo for this class are located at amino acid position 423 (M423T/V/I) followed by positions 419 (L419M) and 482 (I482L).[20,30,31]

HCV is characterized by a high degree of genetic diversity.[32,33] The nucleotide sequences among the six different genotypes differ at 30–35% of the nucleotide sites.[34,35] Each of the six major genotypes of HCV contains a series of more closely related subtypes that typically differ from each other by 20–25% in nucleotide sequences. Furthermore, 5–8% sequence divergence was observed between individual strains (variants) of HCV within a given subtype (GT-1a and GT-1b). It is possible that this high degree of natural sequence variation may have an impact on the susceptibility of patient isolates to HCV NS5B inhibitors, possibly, influencing the clinical response to direct antivirals. Therefore, it is of interest to test drug candidates against circulating genotypes from a variety of clinical isolates. In the present study, the susceptibilities of a panel of patient-derived NS5B polymerase to a number of nonnucleoside polymerase inhibitors were evaluated using a transient replication assay. The correlations in drug susceptibility among the different polymerase inhibitors were analysed.

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