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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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Life cycle stage triage.A. Performance (EC50) of control entry (EI), genome replication (BMS-339) and late-stage (LY411575) inhibitors in single- vs. multi-cycle replication assay formats. B. Screen hits (Inhs 1–13) that demonstrated similar potency in single- and multi-cycle replication formats (single- & multi-cycle inhibitors). C. Screen hits (Inhs 14–17) that demonstrated greater potency in multi-cycle replication assay format (multi-cycle specific inhibitors). D. Performance of control entry (EI) and genome replication (BMS-339) inhibitors following infection with gt 1a/2a-Rluc virus or direct transfection of the corresponding virus genomic RNA. E. Screen hits (Inhs 1–5) that demonstrated reduced potency following direct transfection of virus genomic RNA (early inhibitors). F. Screen hits (Inhs 6–13) that demonstrated similar potency in infection and transfection assays (genome replication inhibitors).
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pone-0042609-g005: Life cycle stage triage.A. Performance (EC50) of control entry (EI), genome replication (BMS-339) and late-stage (LY411575) inhibitors in single- vs. multi-cycle replication assay formats. B. Screen hits (Inhs 1–13) that demonstrated similar potency in single- and multi-cycle replication formats (single- & multi-cycle inhibitors). C. Screen hits (Inhs 14–17) that demonstrated greater potency in multi-cycle replication assay format (multi-cycle specific inhibitors). D. Performance of control entry (EI) and genome replication (BMS-339) inhibitors following infection with gt 1a/2a-Rluc virus or direct transfection of the corresponding virus genomic RNA. E. Screen hits (Inhs 1–5) that demonstrated reduced potency following direct transfection of virus genomic RNA (early inhibitors). F. Screen hits (Inhs 6–13) that demonstrated similar potency in infection and transfection assays (genome replication inhibitors).

Mentions: Table 3 outlines the assays used to deconvolute screen hits according to virus life cycle stage target and predicts the expected profile for inhibitors targeting early (e.g., entry and nucleocapsid uncoating), genome replication or late (e.g., virus assembly and release) events in the virus life cycle. As a first step in elucidating the stage at which the hits were active, the potencies of the compounds were compared using single and multi-cycle HCVcc infectivity assays. As previously determined (Fig. 1), if the assay was constrained to 48 hours, luciferase activity largely represented the first replication cycle only. In contrast, >90% of the signal after 96 h pi was attributed to subsequent rounds of replication. As expected, control entry (EI) and genome replication (BMS-339) inhibitors exhibited similar potency at 48 and 96 h pi, while the late-stage inhibitor (LY411575) was significantly more potent (>200 fold) at 96 h (Fig. 5A). Of the 17 hits selected for further analysis, 13 showed similar potencies (<3-fold difference) at both 48 and 96 h pi (Fig. 5B) while 4 hits (Inhs 14, 15, 16 & 17) showed a marked increase in potency (32–145 fold) at 96 h (Fig. 5C). These results suggested those 13 inhibitors with similar potencies at 48 and 96 h act coincident with early or genome replication inhibition, while the 4 with increased potency at 96 h with late-stage inhibition.


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)

Life cycle stage triage.A. Performance (EC50) of control entry (EI), genome replication (BMS-339) and late-stage (LY411575) inhibitors in single- vs. multi-cycle replication assay formats. B. Screen hits (Inhs 1–13) that demonstrated similar potency in single- and multi-cycle replication formats (single- & multi-cycle inhibitors). C. Screen hits (Inhs 14–17) that demonstrated greater potency in multi-cycle replication assay format (multi-cycle specific inhibitors). D. Performance of control entry (EI) and genome replication (BMS-339) inhibitors following infection with gt 1a/2a-Rluc virus or direct transfection of the corresponding virus genomic RNA. E. Screen hits (Inhs 1–5) that demonstrated reduced potency following direct transfection of virus genomic RNA (early inhibitors). F. Screen hits (Inhs 6–13) that demonstrated similar potency in infection and transfection assays (genome replication inhibitors).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412796&req=5

pone-0042609-g005: Life cycle stage triage.A. Performance (EC50) of control entry (EI), genome replication (BMS-339) and late-stage (LY411575) inhibitors in single- vs. multi-cycle replication assay formats. B. Screen hits (Inhs 1–13) that demonstrated similar potency in single- and multi-cycle replication formats (single- & multi-cycle inhibitors). C. Screen hits (Inhs 14–17) that demonstrated greater potency in multi-cycle replication assay format (multi-cycle specific inhibitors). D. Performance of control entry (EI) and genome replication (BMS-339) inhibitors following infection with gt 1a/2a-Rluc virus or direct transfection of the corresponding virus genomic RNA. E. Screen hits (Inhs 1–5) that demonstrated reduced potency following direct transfection of virus genomic RNA (early inhibitors). F. Screen hits (Inhs 6–13) that demonstrated similar potency in infection and transfection assays (genome replication inhibitors).
Mentions: Table 3 outlines the assays used to deconvolute screen hits according to virus life cycle stage target and predicts the expected profile for inhibitors targeting early (e.g., entry and nucleocapsid uncoating), genome replication or late (e.g., virus assembly and release) events in the virus life cycle. As a first step in elucidating the stage at which the hits were active, the potencies of the compounds were compared using single and multi-cycle HCVcc infectivity assays. As previously determined (Fig. 1), if the assay was constrained to 48 hours, luciferase activity largely represented the first replication cycle only. In contrast, >90% of the signal after 96 h pi was attributed to subsequent rounds of replication. As expected, control entry (EI) and genome replication (BMS-339) inhibitors exhibited similar potency at 48 and 96 h pi, while the late-stage inhibitor (LY411575) was significantly more potent (>200 fold) at 96 h (Fig. 5A). Of the 17 hits selected for further analysis, 13 showed similar potencies (<3-fold difference) at both 48 and 96 h pi (Fig. 5B) while 4 hits (Inhs 14, 15, 16 & 17) showed a marked increase in potency (32–145 fold) at 96 h (Fig. 5C). These results suggested those 13 inhibitors with similar potencies at 48 and 96 h act coincident with early or genome replication inhibition, while the 4 with increased potency at 96 h with late-stage inhibition.

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