Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays |
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Authors: | Mukherjee Sourav Hanson Alicia M Shadrick William R Ndjomou Jean Sweeney Noreena L Hernandez John J Bartczak Diana Li Kelin Frankowski Kevin J Heck Julie A Arnold Leggy A Schoenen Frank J Frick David N |
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Affiliation: | 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. |
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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) (IC50 = 1.4 μM), suramin sodium salt (IC50 = 3.6 μM), NF 023 hydrate (IC50 = 6.2 μM) and tyrphostin AG 538 (IC50 = 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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