title: Novel cell-based hepatitis C virus infection assay for quantitative high-throughput screening of anti-hepatitis C virus compounds.
abstract: Therapy for hepatitis C virus (HCV) infection has advanced with the recent approval of direct-acting antivirals in combination with peginterferon and ribavirin. New antivirals with novel targets are still needed to further improve the treatment of hepatitis C. Previously reported screening methods for HCV inhibitors either are limited to a virus-specific function or apply a screening method at a single dose, which usually leads to high false-positive or -negative rates. We developed a quantitative high-throughput screening (qHTS) assay platform with a cell-based HCV infection system. This highly sensitive assay can be miniaturized to a 1,536-well format for screening of large chemical libraries. All candidates are screened over a 7-concentration dose range to give EC50s (compound concentrations at 50% efficacy) and dose-response curves. Using this assay format, we screened a library of pharmacologically active compounds (LOPAC). Based on the profile of dose-dependent curves of HCV inhibition and cytotoxicity, 22 compounds with adequate curves and EC50s of <10 μM were selected for validation. In two additional independent assays, 17 of them demonstrated specific inhibition of HCV infection. Ten potential candidates with efficacies of >70% and CC50s (compound concentrations at 50% cytotoxicity) of <30 μM from these validated hits were characterized for their target stages in the HCV replication cycle. In this screen, we identified both known and novel hits with diverse structural and functional features targeting various stages of the HCV replication cycle. The pilot screen demonstrates that this assay system is highly robust and effective in identifying novel HCV inhibitors and that it can be readily applied to large-scale screening of small-molecule libraries.
authors.affiliation: Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Zongyi
authors.initials: Z
authors.lastname: Hu
authors.forename: Keng-Hsin
authors.initials: KH
authors.lastname: Lan
authors.forename: Shanshan
authors.initials: S
authors.lastname: He
authors.forename: Manju
authors.initials: M
authors.lastname: Swaroop
authors.forename: Xin
authors.initials: X
authors.lastname: Hu
authors.forename: Noel
authors.initials: N
authors.lastname: Southall
authors.forename: Wei
authors.initials: W
authors.lastname: Zheng
authors.forename: T Jake
authors.initials: TJ
authors.lastname: Liang
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D016174:Hepacivirus; D006526:Hepatitis C; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000821:virology; D057166:High-Throughput Screening Assays; D006801:Humans; D020128:Inhibitory Concentration 50; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D008156:Luciferases; D054852:Small Molecule Libraries; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase