Characterization of Hepatitis C Virus Resistance from a Multiple-Dose Clinical Trial of the Novel NS5A Inhibitor GS-5885

Abstract

c GS-5885 is a novel hepatitis C virus (HCV) NS5A inhibitor. In a 3-day monotherapy study in treatment-naive genotype 1a (GT1a) and GT1b HCV-infected subjects, median viral load reductions ranged from 2.3 to 3.3 log 10 HCV RNA IU/ml across dosing cohorts (1, 3, 10, 30, or 90 mg once daily). Here, we report viral sequencing and phenotypic analysis of clinical isolates from this study. Detection of baseline NS5A amino acid substitutions at positions 28, 30, 31, or 93 in GT1a was associated with a reduced treatment response. In the GT1b cohort, Y93H was detected in 100% of subjects at day 4 or 14. In the Gt1a cohort, population sequencing detected NS5A resistance-associated mutations at day 4 or 14 for 3/10 subjects at the 1-mg dose and for all subjects dosed at >3 mg. A subset of mutants that confer a low level of reduced susceptibility to GS-5885 was not detected by population sequencing at the 30-and 90-mg doses. Subject-derived M28T, Q30R, L31M, and Y93C mutations all conferred >30-fold reductions in GS-5885 and daclatasvir susceptibilities in vitro. Site-directed NS5A mutants also showed reduced susceptibility to GS-5885. However, all NS5A mutants tested remained fully susceptible to other classes of direct-acting antivirals (DAAs), interferon alpha, and ribavirin. Importantly, the nonoverlapping resistance profile and high potency of GS-5885 support its further development with other direct-acting antivirals for the treatment of chronic HCV. (This study has been registered at ClinicalTrials.gov under registration number NCT01193478.) H epatitis C virus (HCV) infection is a global health issue, with approximately 170 million people infected worldwide (1).The standard of care (SOC) has been pegylated alpha interferon and ribavirin until the more recent approval of two NS3 protease inhibitors, telaprevir and boceprevir, for use in conjunction with pegylated alpha interferon and ribavirin (1-3). The last several years have seen a great expansion of new direct-acting antivirals (DAAs) in clinical development to augment or supplant treatment with pegylated alpha interferon and ribavirin. HCV nonstructural protein 5A (NS5A) has emerged as a viable and attractive viral target for small-molecule inhibition. Although there is no known enzymatic activity for NS5A, it is essential for viral replication (4). The first NS5A replication complex inhibitor to show efficacy in the clinic was daclatasvir (BMS-790052) (5). This compound elicited rapid and profound reductions in HCV RNA and validated NS5A as a clinical target. Sequence analysis of clinical isolates following daclatasvir monotherapy identified the main resistanceassociated mutations (RAMs) at NS5A amino acid positions 28, 30, 31, and 93 (6).In addition to daclatasvir, several other NS5A replication complex inhibitors have entered the clinic, including GS-5885, PPI-461, ABT-267, and GSK2336805 (7-10). In addition, preclinical data have recently been described for several other NS5A replication complex inhibitors, including EDP-239, IDX719, MK-4882, , highlighting ...

Clinical Resistance to Velpatasvir (GS-5816), a Novel Pan-Genotypic Inhibitor of the Hepatitis C Virus NS5A Protein

“…More substantial suppression of wild-type virus by larger doses resulted in more frequent detection of resistant variants, although RASs may be generated during treatment because of the high error rate of HCV RNA replication. This is consistent with other phase 1 HCV DAA monotherapy studies (28,43,44) and suggests that RASs preexist among patients with pretreatment RASs included Y93H (n ϭ 3 [GT1a, n ϭ 2; GT1b, n ϭ 1]), L31M (n ϭ 11 [GT1a, n ϭ 7; GT2b, n ϭ 4]), H58D (GT3a, n ϭ 3), Q30H (GT1a, n ϭ 2), M28T (GT1a, n ϭ 3), and Q30R (GT1a, n ϭ 2). (B) Changes in frequencies among patients without pretreatment RASs included Y93H (n ϭ 38 [GT1a, n ϭ 18; GT1b, n ϭ 6; GT2b, n ϭ 2; GT3, n ϭ 12]), L31M (n ϭ 16 [GT1a, n ϭ 11; GT1b, n ϭ 4; GT2b, n ϭ 1]), L31V (n ϭ 25 [GT1a, n ϭ 18; GT1b, n ϭ 5; GT2b, n ϭ 1; GT3, n ϭ 1]), H58D (GT1a, n ϭ 5), M28T (GT1a, n ϭ 17), Q30H (GT1a, n ϭ 6), Q30R (GT1a, n ϭ 10), Y93N (n ϭ 21 [GT1a, n ϭ 14; GT1b, n ϭ 2; GT3a, n ϭ 5]).…”

“…Deep-sequencing reads were aligned and processed with internally developed software via a multistep method to identify the substitutions present at levels of Ͼ1% (17). Sequencing analysis included NS5A class RASs that were summarized by the HCV Drug Resistance Advisory Group (18) and/or recently observed in clinical trials with LDV, VEL, DCV, ABT-267, ABT-530, and MK-8742 (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29) and including positions 24,28,30,31,32,38,58,92, and 93. (34) replicons (backbone GT1a H77, GT1b Con1, GT2a JFH-1, GT3a S52, and GT4a ED43, respectively) by site-directed mutagenesis and tested in transient transfections as previously described (35).…”

Resistance to Direct-Acting Antiviral Agents in Treatment of Hepatitis C Virus Infections

“…Primary substitutions in NS5A sequences of genotype 1a that are associated with resistance to LDV involves residues K24, M28, Q30, L31, P32, H58, and Y93, while genotype 1b includes mainly L31, P58, A92, and Y93 [43,44]. In genotype 4, mutations at residues L28 and L30 are associated with relapse after DCV-based treatment (unpublished data from our laboratory).…”

Ledipasvir/Sofosbuvir

“…1 In vitro resistance modeling shows decreased susceptibility to ledipasvir against HCV with Q30E and Y93H mutations in the genotype 1a strain and Y93H in genotype 1b strain. 18 Those mutations along with M28T, Q30R, L31M, and Y93C mutations were found to have reduced susceptibility to ledipasvir, although all mutants tested remained fully susceptible to other classes of direct-acting antiretrovirals, such as the protease inhibitors used for HCV, interferon alfa, and ribavirin. 18 When ledipasvir was examined in HCV genotype 3, there were indications that multiple strains had reduced susceptibility to the medication's antiviral effects.…”

“…18 Those mutations along with M28T, Q30R, L31M, and Y93C mutations were found to have reduced susceptibility to ledipasvir, although all mutants tested remained fully susceptible to other classes of direct-acting antiretrovirals, such as the protease inhibitors used for HCV, interferon alfa, and ribavirin. 18 When ledipasvir was examined in HCV genotype 3, there were indications that multiple strains had reduced susceptibility to the medication's antiviral effects. 19 Sofosbuvir is a prodrug that has an active metabolite with a direct-acting antiviral agent that inhibits HCV NS5B RNA-dependent polymerase, a vital component in viral replication.…”