title: A time-resolved fluorescence-resonance energy transfer assay for identifying inhibitors of hepatitis C virus core dimerization.
abstract: Binding of hepatitis C virus (HCV) RNA to core, the capsid protein, results in the formation of the nucleocapsid, the first step in the assembly of the viral particle. A novel assay was developed to discover small molecule inhibitors of core dimerization. This assay is based on time-resolved fluorescence resonance energy transfer (TR-FRET) between anti-tag antibodies labeled with either europium cryptate (Eu) or allophycocyanin (XL-665). The N-terminal 106-residue portion of core protein (core106) was tagged with either glutathione-S-transferase (GST) or a Flag peptide. Tag-free core106 was selected as the reference inhibitor. The assay was used to screen the library of pharmacologically active compounds (LOPAC) consisting of 1,280 compounds and a 2,240-compound library from the Center for Chemical Methodology and Library Development at Boston University (CMLD-BU). Ten of the 28 hits from the primary TR-FRET run were confirmed in a secondary amplified luminescent proximity homogeneous assay (ALPHA screen). One hit was further characterized by dose-response analysis yielding an IC(50) of 9.3 microM. This 513 Da compound was shown to inhibit HCV production in cultured hepatoma cells.
authors.affiliation: Department of Infectology, The Scripps Research Institute-Scripps Florida, Jupiter, Florida 33458, USA.
authors.forename: Smitha
authors.initials: S
authors.lastname: Kota
authors.forename: Louis
authors.initials: L
authors.lastname: Scampavia
authors.forename: Timothy
authors.initials: T
authors.lastname: Spicer
authors.forename: Aaron B
authors.initials: AB
authors.lastname: Beeler
authors.forename: Virginia
authors.initials: V
authors.lastname: Takahashi
authors.forename: John K
authors.initials: JK
authors.lastname: Snyder
authors.forename: John A
authors.initials: JA
authors.lastname: Porco
authors.forename: Peter
authors.initials: P
authors.lastname: Hodder
authors.forename: Arthur Donny
authors.initials: AD
authors.lastname: Strosberg
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D016174:Hepacivirus / Q000187:drug effects*; D055503:Protein Multimerization / Q000187:drug effects*; D014758:Viral Core Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry
chemical_list: D000998:Antiviral Agents; D014758:Viral Core Proteins