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High-throughput screening and rapid inhibitor triage using an infectious chimeric Hepatitis C virus.

Wichroski MJ, Fang J, Eggers BJ, Rose RE, Mazzucco CE, Pokornowski KA, Baldick CJ, Anthony MN, Dowling CJ, Barber LE, Leet JE, Beno BR, Gerritz SW, Agler ML, Cockett MI, Tenney DJ - PLoS ONE (2012)

Bottom Line: The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors.Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism.Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.

View Article: PubMed Central - PubMed

Affiliation: Bristol-Myers Squibb Research and Development, Wallingford, Connecticut, United States of America.

ABSTRACT
The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening). The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors. Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism. Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.

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HCV Genome Replication Inhibitors.A. The genome replication inhibitors (Inhs 6–13) were confirmed by comparing potency (EC50) against the 1a/2a-Rluc virus and a corresponding JFH1 replicon. B & C. Genotype 2a and HCV selectivity were assessed by comparing inhibitor potency against genotype 2a, genotype 1a and BVDV replicons and compounds were subsequently grouped into genotype 2a-selective (B) and non-selective (C).
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pone-0042609-g007: HCV Genome Replication Inhibitors.A. The genome replication inhibitors (Inhs 6–13) were confirmed by comparing potency (EC50) against the 1a/2a-Rluc virus and a corresponding JFH1 replicon. B & C. Genotype 2a and HCV selectivity were assessed by comparing inhibitor potency against genotype 2a, genotype 1a and BVDV replicons and compounds were subsequently grouped into genotype 2a-selective (B) and non-selective (C).

Mentions: HCV and Bovine viral diarrhea virus (BVDV) replicons were used to determine if Inhs 7–13 were HCV-selective genome replication inhibitors. All of the hits exhibited similar potency against HCVcc and a genotype 2a replicon (Fig. 7A), confirming that these compounds block replication of the viral genome. Of the 8 inhibitors, 6 (Inhs 6–11) exhibited reduced potency against both genotype 1a HCV and BVDV replicons which was indistinguishable from cytotoxicity (Fig. 7B) showing that these agents were selective for genotype 2a HCV. On the contrary, Inhs 12 and 13 exhibited similar activity against genotype 2a and 1a HCV as well as BVDV replicons (Fig. 7C) and these activities were separable from cytotoxicity. Taken together, these results demonstrated that these inhibitors were not selective for HCV but rather blocked the replication of both viruses.


High-throughput screening and rapid inhibitor triage using an infectious chimeric Hepatitis C virus.

Wichroski MJ, Fang J, Eggers BJ, Rose RE, Mazzucco CE, Pokornowski KA, Baldick CJ, Anthony MN, Dowling CJ, Barber LE, Leet JE, Beno BR, Gerritz SW, Agler ML, Cockett MI, Tenney DJ - PLoS ONE (2012)

HCV Genome Replication Inhibitors.A. The genome replication inhibitors (Inhs 6–13) were confirmed by comparing potency (EC50) against the 1a/2a-Rluc virus and a corresponding JFH1 replicon. B & C. Genotype 2a and HCV selectivity were assessed by comparing inhibitor potency against genotype 2a, genotype 1a and BVDV replicons and compounds were subsequently grouped into genotype 2a-selective (B) and non-selective (C).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412796&req=5

pone-0042609-g007: HCV Genome Replication Inhibitors.A. The genome replication inhibitors (Inhs 6–13) were confirmed by comparing potency (EC50) against the 1a/2a-Rluc virus and a corresponding JFH1 replicon. B & C. Genotype 2a and HCV selectivity were assessed by comparing inhibitor potency against genotype 2a, genotype 1a and BVDV replicons and compounds were subsequently grouped into genotype 2a-selective (B) and non-selective (C).
Mentions: HCV and Bovine viral diarrhea virus (BVDV) replicons were used to determine if Inhs 7–13 were HCV-selective genome replication inhibitors. All of the hits exhibited similar potency against HCVcc and a genotype 2a replicon (Fig. 7A), confirming that these compounds block replication of the viral genome. Of the 8 inhibitors, 6 (Inhs 6–11) exhibited reduced potency against both genotype 1a HCV and BVDV replicons which was indistinguishable from cytotoxicity (Fig. 7B) showing that these agents were selective for genotype 2a HCV. On the contrary, Inhs 12 and 13 exhibited similar activity against genotype 2a and 1a HCV as well as BVDV replicons (Fig. 7C) and these activities were separable from cytotoxicity. Taken together, these results demonstrated that these inhibitors were not selective for HCV but rather blocked the replication of both viruses.

Bottom Line: The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors.Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism.Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.

View Article: PubMed Central - PubMed

Affiliation: Bristol-Myers Squibb Research and Development, Wallingford, Connecticut, United States of America.

ABSTRACT
The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening). The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors. Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism. Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.

Show MeSH
Related in: MedlinePlus