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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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Related in: MedlinePlus

Outline of primary screen, re-test and hit triage strategy.Potent and selective hits identified from a primary screen were subjected to a myriad of life cycle stage assays, genotype coverage and resistance studies to group hits according to target.
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pone-0042609-g003: Outline of primary screen, re-test and hit triage strategy.Potent and selective hits identified from a primary screen were subjected to a myriad of life cycle stage assays, genotype coverage and resistance studies to group hits according to target.

Mentions: Figure 3 outlines the strategy to triage potent and selective leads and rapidly categorize screen hits. This included removing known HCV actives, and selections based on potency and lack of cytotoxicity, as well as confirming activity. These steps identified actives for which further assay results were less likely to be confused with subtle cytotoxicity. Then secondary assays such as compound chemotype clustering and assays that distinguish compounds based on the mechanism of activity were used. The results of the screen are presented in Table 2. Primary screening (1,175,504 compounds) and re-tests identified 9,025 compounds (0.77% hit rate) with ≥70% inhibition of HCV at 6 µM and ≤30% cytotoxicity as measured by CellTiter-Glo (Table 2). To expeditiously identify the more potent leads (e.g., EC50≤1 µM), hits were re-tested at 0.6 µM (0.1× initial screening concentration) and 632 compounds were selected based on ≥30% inhibition. Dose-response curves were used to determine EC50, as well as the 50% cytotoxicity concentration (CC50). There were 386 compounds with an EC50≤1 µM and 307 compounds with a therapeutic index (TI) ≥10 (CC50/EC50). To ensure none of the screen hits directly affected Renilla luciferase, the compounds were tested against Vesicular stomatitis virus (VSV) pseudo-typed retroviral particles harboring either a firefly or Renilla luciferase reporter. Seventy five compounds were eliminated and the remaining 232 screen hits were clustered based on similarity using atom pair descriptors and in-house software. Seventeen compounds, each representing a unique chemical chemotype, were advanced for further analysis. Therapeutic indices for these hits ranged from 22 to >6,000 (Fig. 4A) and each demonstrated similar potency using either the Renilla luciferase or HCV Core Cellomics readouts confirming their specificity for HCV (Fig. 4B).


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)

Outline of primary screen, re-test and hit triage strategy.Potent and selective hits identified from a primary screen were subjected to a myriad of life cycle stage assays, genotype coverage and resistance studies to group hits according to target.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042609-g003: Outline of primary screen, re-test and hit triage strategy.Potent and selective hits identified from a primary screen were subjected to a myriad of life cycle stage assays, genotype coverage and resistance studies to group hits according to target.
Mentions: Figure 3 outlines the strategy to triage potent and selective leads and rapidly categorize screen hits. This included removing known HCV actives, and selections based on potency and lack of cytotoxicity, as well as confirming activity. These steps identified actives for which further assay results were less likely to be confused with subtle cytotoxicity. Then secondary assays such as compound chemotype clustering and assays that distinguish compounds based on the mechanism of activity were used. The results of the screen are presented in Table 2. Primary screening (1,175,504 compounds) and re-tests identified 9,025 compounds (0.77% hit rate) with ≥70% inhibition of HCV at 6 µM and ≤30% cytotoxicity as measured by CellTiter-Glo (Table 2). To expeditiously identify the more potent leads (e.g., EC50≤1 µM), hits were re-tested at 0.6 µM (0.1× initial screening concentration) and 632 compounds were selected based on ≥30% inhibition. Dose-response curves were used to determine EC50, as well as the 50% cytotoxicity concentration (CC50). There were 386 compounds with an EC50≤1 µM and 307 compounds with a therapeutic index (TI) ≥10 (CC50/EC50). To ensure none of the screen hits directly affected Renilla luciferase, the compounds were tested against Vesicular stomatitis virus (VSV) pseudo-typed retroviral particles harboring either a firefly or Renilla luciferase reporter. Seventy five compounds were eliminated and the remaining 232 screen hits were clustered based on similarity using atom pair descriptors and in-house software. Seventeen compounds, each representing a unique chemical chemotype, were advanced for further analysis. Therapeutic indices for these hits ranged from 22 to >6,000 (Fig. 4A) and each demonstrated similar potency using either the Renilla luciferase or HCV Core Cellomics readouts confirming their specificity for HCV (Fig. 4B).

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