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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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Early/Entry Inhibitors.A. The potencies (EC50) of early inhibitors (Inhs 1–5) against gt 1a/2a-Rluc virus (HCVcc) and HCV pseudo-particles (HCVpp) with the corresponding genotype 1 envelope glycoproteins were compared to identify virus entry inhibitors. Selectivity for HCVpp was confirmed using Vesicular stomatitis virus glycoprotein pseudo-particles (VSVpp) and cytotoxicity (CC50) using Cell-Titer Glo. A. Screen hits (Inhs 1–3) that demonstrated similar potency against HCVcc and HCVpp and exhibited selectivity relative to VSVpp and cytotoxicity (entry inhibitors). A control entry inhibitor (EI) was included to confirm the predicted outcome for a bonafide entry inhibitor. B. Screen hits (Inhs 4–5) that demonstrated reduced potency against HCVpp (HCVcc-specific early inhibitors). Genotype selectivity was assessed by comparing potency against HCVcc chimeras with genotype 1a, 1b or 2a structural proteins. C. Potency of the entry inhibitors (Inhs 1–3) and (D) HCVcc-specific early inhibitors (Inhs 4 & 5) against genotype 1a, 1b and 2a HCVcc and corresponding cytotoxicity.
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pone-0042609-g006: Early/Entry Inhibitors.A. The potencies (EC50) of early inhibitors (Inhs 1–5) against gt 1a/2a-Rluc virus (HCVcc) and HCV pseudo-particles (HCVpp) with the corresponding genotype 1 envelope glycoproteins were compared to identify virus entry inhibitors. Selectivity for HCVpp was confirmed using Vesicular stomatitis virus glycoprotein pseudo-particles (VSVpp) and cytotoxicity (CC50) using Cell-Titer Glo. A. Screen hits (Inhs 1–3) that demonstrated similar potency against HCVcc and HCVpp and exhibited selectivity relative to VSVpp and cytotoxicity (entry inhibitors). A control entry inhibitor (EI) was included to confirm the predicted outcome for a bonafide entry inhibitor. B. Screen hits (Inhs 4–5) that demonstrated reduced potency against HCVpp (HCVcc-specific early inhibitors). Genotype selectivity was assessed by comparing potency against HCVcc chimeras with genotype 1a, 1b or 2a structural proteins. C. Potency of the entry inhibitors (Inhs 1–3) and (D) HCVcc-specific early inhibitors (Inhs 4 & 5) against genotype 1a, 1b and 2a HCVcc and corresponding cytotoxicity.

Mentions: To identify entry-specific compounds, the 5 early-stage hits were tested against HCV pseudo-particles (HCVpp) harboring the HCV genotype 1a envelope glycoproteins matching those of the screening virus. Three of the 5 hits exhibited a similar profile as EI with similar potency against both HCVcc and HCVpp viruses (Fig. 6A). HCV selectivity was confirmed by counter-screening for activity against VSVpp and cytotoxicity (Fig. 6A). Together, these findings confirmed that Inh-1, 2 and 3 were HCV entry inhibitors. On the contrary, Inhs 4 and 5 showed no activity against HCVpp (Fig. 6B) suggesting that these hits could target an HCVcc-specific entry event or an early event not recapitulated by the pseudo-particle system (e.g., nucleocapsid uncoating). Next, genotype coverage of the inhibitors was determined using HCVcc chimeras harboring genotype 1a, 1b (432–4 isolate) or 2a (J6 isolate) structural proteins. Of the 3 entry inhibitors, Inh-1 was selective for genotype 1a, while Inhs 2 and 3 exhibited activity against genotype 1a and 1b but not 2a (Fig. 6C). A similar profile was observed using HCVpp harboring genotype 1a,1b or 2a envelopes (data not shown). For the HCVcc-specific inhibitors, both Inh-4 and Inh-5 exhibited similar potency against all 3 genotypes (Fig. 6D).


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)

Early/Entry Inhibitors.A. The potencies (EC50) of early inhibitors (Inhs 1–5) against gt 1a/2a-Rluc virus (HCVcc) and HCV pseudo-particles (HCVpp) with the corresponding genotype 1 envelope glycoproteins were compared to identify virus entry inhibitors. Selectivity for HCVpp was confirmed using Vesicular stomatitis virus glycoprotein pseudo-particles (VSVpp) and cytotoxicity (CC50) using Cell-Titer Glo. A. Screen hits (Inhs 1–3) that demonstrated similar potency against HCVcc and HCVpp and exhibited selectivity relative to VSVpp and cytotoxicity (entry inhibitors). A control entry inhibitor (EI) was included to confirm the predicted outcome for a bonafide entry inhibitor. B. Screen hits (Inhs 4–5) that demonstrated reduced potency against HCVpp (HCVcc-specific early inhibitors). Genotype selectivity was assessed by comparing potency against HCVcc chimeras with genotype 1a, 1b or 2a structural proteins. C. Potency of the entry inhibitors (Inhs 1–3) and (D) HCVcc-specific early inhibitors (Inhs 4 & 5) against genotype 1a, 1b and 2a HCVcc and corresponding cytotoxicity.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412796&req=5

pone-0042609-g006: Early/Entry Inhibitors.A. The potencies (EC50) of early inhibitors (Inhs 1–5) against gt 1a/2a-Rluc virus (HCVcc) and HCV pseudo-particles (HCVpp) with the corresponding genotype 1 envelope glycoproteins were compared to identify virus entry inhibitors. Selectivity for HCVpp was confirmed using Vesicular stomatitis virus glycoprotein pseudo-particles (VSVpp) and cytotoxicity (CC50) using Cell-Titer Glo. A. Screen hits (Inhs 1–3) that demonstrated similar potency against HCVcc and HCVpp and exhibited selectivity relative to VSVpp and cytotoxicity (entry inhibitors). A control entry inhibitor (EI) was included to confirm the predicted outcome for a bonafide entry inhibitor. B. Screen hits (Inhs 4–5) that demonstrated reduced potency against HCVpp (HCVcc-specific early inhibitors). Genotype selectivity was assessed by comparing potency against HCVcc chimeras with genotype 1a, 1b or 2a structural proteins. C. Potency of the entry inhibitors (Inhs 1–3) and (D) HCVcc-specific early inhibitors (Inhs 4 & 5) against genotype 1a, 1b and 2a HCVcc and corresponding cytotoxicity.
Mentions: To identify entry-specific compounds, the 5 early-stage hits were tested against HCV pseudo-particles (HCVpp) harboring the HCV genotype 1a envelope glycoproteins matching those of the screening virus. Three of the 5 hits exhibited a similar profile as EI with similar potency against both HCVcc and HCVpp viruses (Fig. 6A). HCV selectivity was confirmed by counter-screening for activity against VSVpp and cytotoxicity (Fig. 6A). Together, these findings confirmed that Inh-1, 2 and 3 were HCV entry inhibitors. On the contrary, Inhs 4 and 5 showed no activity against HCVpp (Fig. 6B) suggesting that these hits could target an HCVcc-specific entry event or an early event not recapitulated by the pseudo-particle system (e.g., nucleocapsid uncoating). Next, genotype coverage of the inhibitors was determined using HCVcc chimeras harboring genotype 1a, 1b (432–4 isolate) or 2a (J6 isolate) structural proteins. Of the 3 entry inhibitors, Inh-1 was selective for genotype 1a, while Inhs 2 and 3 exhibited activity against genotype 1a and 1b but not 2a (Fig. 6C). A similar profile was observed using HCVpp harboring genotype 1a,1b or 2a envelopes (data not shown). For the HCVcc-specific inhibitors, both Inh-4 and Inh-5 exhibited similar potency against all 3 genotypes (Fig. 6D).

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