title: High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon.
abstract: We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5'-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3'-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.
authors.affiliation: Department of Life Science and Medical Bio-Science, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Tani
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Akimitsu
authors.forename: Osamu
authors.initials: O
authors.lastname: Fujita
authors.forename: Yasuyoshi
authors.initials: Y
authors.lastname: Matsuda
authors.forename: Ryo
authors.initials: R
authors.lastname: Miyata
authors.forename: Satoshi
authors.initials: S
authors.lastname: Tsuneda
authors.forename: Masayuki
authors.initials: M
authors.lastname: Igarashi
authors.forename: Yuji
authors.initials: Y
authors.lastname: Sekiguchi
authors.forename: Naohiro
authors.initials: N
authors.lastname: Noda
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D004265:DNA Helicases / Q000037:antagonists & inhibitors*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D004265:DNA Helicases; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases