title: A cell-based, microplate colorimetric screen identifies 7,8-benzoflavone and green tea gallate catechins as inhibitors of the hepatitis C virus.
abstract: We describe a cell-based, microplate colorimetric screen for anti-hepatitis C virus (HCV) drugs that exploits the HCV-JFH1 viral culture system. Antiviral activity was assessed by measuring protection against the HCV-JFH1-induced cytopathic effect (CPE) in Huh7.5.1 cells using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) viability assay. The use of serum-free medium substantially sensitized Huh7.5.1 cells to HCV-induced CPE, causing sufficient cell death to perform colorimetric assays for anti-HCV activity in 96-well plates. As a proof of concept, we carried out a pilot screen of an inhibitor library and identified cyclosporin A and tamoxifen, two compounds with reported anti-HCV activity. Using the assay, we discovered the anti-HCV properties of the plant flavonoids epigallocatechin gallate (EGCG) and 7,8-benzoflavone (α-naphthoflavone). Other gallate-type catechins and flavones also displayed anti-HCV activity, but 5,6-benzoflavone (β-naphthoflavone), flavanone, and non-gallate catechins were inactive. EGCG apparently acted mainly on HCV entry, although it may also block other steps. In contrast, 7,8-benzoflavone was presumed to inhibit later stages of the HCV life cycle. This assay is simple, reliable and cost-effective; does not require any specially engineered cell lines or viruses; and should be useful in the identification of compounds with anti-HCV activity.
authors.affiliation: Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo, Japan. fukazawa@nih.go.jp
authors.forename: Hidesuke
authors.initials: H
authors.lastname: Fukazawa
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Suzuki
authors.forename: Takaji
authors.initials: T
authors.lastname: Wakita
authors.forename: Yuko
authors.initials: Y
authors.lastname: Murakami
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology / Q000627:therapeutic use*; D001571:Benzoflavones / Q000494:pharmacology / Q000627:therapeutic use*; D028241:Camellia sinensis / Q000737:chemistry*; D002392:Catechin / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D045744:Cell Line, Tumor; D016572:Cyclosporine / Q000494:pharmacology / Q000627:therapeutic use; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D047309:Flavones / Q000494:pharmacology; D016174:Hepacivirus / Q000187:drug effects*; D006526:Hepatitis C / Q000188:drug therapy* / Q000821:virology; D006801:Humans; D008517:Phytotherapy; D015203:Reproducibility of Results; D013629:Tamoxifen / Q000494:pharmacology / Q000627:therapeutic use
chemical_list: D000998:Antiviral Agents; D001571:Benzoflavones; D047309:Flavones; D013629:Tamoxifen; C011512:alpha-naphthoflavone; D016572:Cyclosporine; D002392:Catechin; C045651:epigallocatechin gallate