Article

Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays

Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, University of Kansas Specialized Chemistry Center, University of Kansas, 2034 Becker Dr., Lawrence, KS 66047 and Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA.
Nucleic Acids Research (Impact Factor: 9.11). 06/2012; 40(17):8607-21. DOI: 10.1093/nar/gks623
Source: PubMed

ABSTRACT Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma's Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC(50) = 1.4 μM), suramin sodium salt (IC(50) = 3.6 μM), NF 023 hydrate (IC(50) = 6.2 μM) and tyrphostin AG 538 (IC(50) = 3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.

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