A Time-Resolved Fluorescence-Resonance Energy Transfer Assay for Identifying Inhibitors of Hepatitis C Virus Core Dimerization

作者：Kota Smitha; Scampavia Louis; Spicer Timothy; Beeler Aaron B; Takahashi Virginia; Snyder John K; Porco John A Jr; Hodder Peter; Stro**erg Arthur Donny^*

来源：Assay and Drug Development Technologies, 2010, 8(1): 96-105.

DOI：10.1089/adt.2009.0217

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 mu M. This 513 Da compound was shown to inhibit HCV production in cultured hepatoma cells.

• 出版日期2010-2