title: A fluorescence-based screening assay for identification of hepatitis C virus NS3 helicase inhibitors and characterization of their inhibitory mechanism.
abstract: Hepatitis C virus (HCV) can establish a chronic infection in the majority of individuals infected, resulting in liver cirrhosis and hepatocellular carcinoma. Because the current standard treatment for HCV infection has limitations in terms of severe side effects, the emergence of drug resistance, and drug-drug interactions, it is desirable to develop novel antivirals that target viral proteins involved in viral replication. HCV nonstructural protein 3 (NS3) helicase, which unwinds double-stranded nucleic acids to yield single-stranded nucleic acids, is one possible target for new drug development, because it plays an essential role in viral replication. In this chapter, we describe a helicase assay based on fluorescence resonance energy transfer (FRET) that can be used for high-throughput screening of HCV NS3 helicase inhibitors. The assay uses a double-stranded RNA (dsRNA) substrate with a fluorophore-labeled strand hybridized to a quencher-labeled strand and monitors the increase in fluorescence intensity resulting from helicase-catalyzed unwinding of the dsRNA substrate. We further describe radioactive assays to directly visualize RNA strands unwound by helicase and to evaluate the ATPase and RNA-binding activities of NS3, which are linked to helicase activity, for characterization of the inhibitory mechanism.
authors.affiliation: Department of Life Science and Medical Bioscience, Waseda University, Tokyo, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Furuta
authors.forename: Kazi Abdus
authors.initials: KA
authors.lastname: Salam
authors.forename: Hidenori
authors.initials: H
authors.lastname: Tani
authors.forename: Satoshi
authors.initials: S
authors.lastname: Tsuneda
authors.forename: Yuji
authors.initials: Y
authors.lastname: Sekiguchi
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Akimitsu
authors.forename: Naohiro
authors.initials: N
authors.lastname: Noda
mesh_terms: D001681:Biological Assay / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D016174:Hepacivirus / Q000201:enzymology*; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases