Dataset Information

Abundant drug-resistant NS3 mutants detected by deep sequencing in hepatitis C virus-infected patients undergoing NS3 protease inhibitor monotherapy.

ABSTRACT: The high genetic variation of hepatitis C virus (HCV) results in rapid selection of drug resistance mutations (DRMs) during monotherapy with direct-acting antivirals (DAAs). It has been proposed that each possible single mutant preexists in infected individuals; however, the levels of preexisting DRMs are too low to be directly quantified in most patients using current techniques. In this study, we evaluated the presence of DRMs in HCV-infected patients treated with the HCV protease inhibitors GS-9256 or GS-9451 as monotherapy using deep sequencing in 137 longitudinal samples from 45 patients. Software was developed to analyze deep-sequencing results with an assay cutoff of 0.25%. No NS3 DRMs that confer resistance to GS-9256 and GS-9451 (R155K, A156T, and D168V/E) were observed in 33 baseline samples at >0.25%. In contrast, these and other substitutions at NS3 positions 155, 156, and 168 were detected in 19/27 patients at day 2 (24 h) and 21/21 at day 4 (84 h) of monotherapy but not in placebo-treated patients. Based on the DRM growth kinetics during drug treatment, pretreated NS3 mutations at amino acids 155, 156, and 168 were estimated on average at 0.025% and 0.015% per genotype 1a and 1b HCV-infected patients, respectively. Relative fitness of the DRM viruses was shown to be significantly lower than the wild type. Deep-sequencing analyses of NS3 protease inhibitor-treated HCV-infected patients suggest a limit of HCV viral load suppression of 3.6 to 3.8 log(10) with NS3 protease inhibitor monotherapy that does not suppress the identified preexisting NS3 DRMs and thus a need for a combination therapy.

SUBMITTER: Svarovskaia ES

PROVIDER: S-EPMC3457457 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Abundant drug-resistant NS3 mutants detected by deep sequencing in hepatitis C virus-infected patients undergoing NS3 protease inhibitor monotherapy.

Svarovskaia Evguenia S ES Martin Ross R McHutchison John G JG Miller Michael D MD Mo Hongmei H

Journal of clinical microbiology 20120725 10

The high genetic variation of hepatitis C virus (HCV) results in rapid selection of drug resistance mutations (DRMs) during monotherapy with direct-acting antivirals (DAAs). It has been proposed that each possible single mutant preexists in infected individuals; however, the levels of preexisting DRMs are too low to be directly quantified in most patients using current techniques. In this study, we evaluated the presence of DRMs in HCV-infected patients treated with the HCV protease inhibitors G ...[more]

PMID: 22837328

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Project description:Asunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) NS3 protease inhibitor demonstrating efficacy in alfa interferon-sparing, direct-acting antiviral dual-combination regimens (together with the NS5A replication complex inhibitor daclatasvir) in patients chronically infected with HCV genotype 1b. Here, we describe a comprehensive in vitro genotypic and phenotypic analysis of asunaprevir-associated resistance against genotypes 1a and 1b using HCV replicons and patient samples obtained from clinical studies of short-term asunaprevir monotherapy. During genotype 1a resistance selection using HCV replicons, the primary NS3 protease substitutions identified were R155K, D168G, and I170T, which conferred low- to moderate-level asunaprevir resistance (5- to 21-fold) in transient-transfection susceptibility assays. For genotype 1b, a higher level of asunaprevir-associated resistance was observed at the same selection pressures, ranging from 170- to 400-fold relative to the wild-type control. The primary NS3 protease substitutions identified occurred predominantly at amino acid residue D168 (D168A/G/H/V/Y) and were associated with high-level asunaprevir resistance (16- to 280-fold) and impaired replication capacity. In asunaprevir single-ascending-dose and 3-day multiple-ascending-dose studies in HCV genotype 1a- or 1b-infected patients, the predominant pre-existing NS3 baseline polymorphism was NS3-Q80K. This substitution impacted initial virologic response rates in a single-ascending-dose study, but its effects after multiple doses were more ambiguous. Interestingly, for patient NS3 protease sequences containing Q80 and those containing K80, susceptibilities to asunaprevir were comparable when tested in an enzyme assay. No resistance-associated variants emerged in these clinical studies that significantly impacted susceptibility to asunaprevir. Importantly, asunaprevir-resistant replicons remained susceptible to an NS5A replication complex inhibitor, consistent with a role for asunaprevir in combination therapies.

Project description:BACKGROUND: Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed. RESULTS: The atomic coordinates of crystallographic structure 1CU1 and 1DY9 were used as starting model for modeling of the NS3 protease variant structures. The NS3 protease variant structures are composed of six subdomains, which occur in sequence along the polypeptide chain. The protease domain exhibits the dual beta-barrel fold that is common among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related beta-alpha-beta subdomains and a third subdomain of seven helices and three short beta strands. The latter domain is usually referred to as the helicase alpha-helical subdomain. The rmsd value of bond lengths and bond angles, the average G-factor and Verify 3D values are presented for NS3 protease variant structures. CONCLUSIONS: This project increases the certainty that homology modeling is an useful tool in structural biology and that it can be very valuable in annotating genome sequence information and contributing to structural and functional genomics from virus. The structural models will be used to guide future efforts in the structure-based drug design of a new generation of NS3 protease variants inhibitors. All models in the database are publicly accessible via our interactive website, providing us with large amount of structural models for use in protein-ligand docking analysis.

Dataset Information

Abundant drug-resistant NS3 mutants detected by deep sequencing in hepatitis C virus-infected patients undergoing NS3 protease inhibitor monotherapy.

Publications

Abundant drug-resistant NS3 mutants detected by deep sequencing in hepatitis C virus-infected patients undergoing NS3 protease inhibitor monotherapy.

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