Mukherjee, SouravHanson, Alicia M.Shadrick, William R.Ndjomou, JeanSweeney, Noreena L.Hernadez, John J.Bartczak, DianaLi, KelinFrankowski, Kevin J.Heck, Julie A.Arnold, Leggy A.Schoenen, Frank J.Frick, David N.2014-04-112014-04-112012-06-27Mukherjee, Sourav, Alicia M Hanson, William R Shadrick, Jean Ndjomou, Noreena L Sweeney, John J Hernandez, Diana Bartczak, et al. 2012. “Identification and Analysis of Hepatitis C Virus NS3 Helicase Inhibitors Using Nucleic Acid Binding Assays.” Nucleic Acids Research 40 (17): 8607–21. http://dx.doi.org/10.1093/nar/gks623.https://hdl.handle.net/1808/13459Typical 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.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.http://creativecommons.org/licenses/by-nc/3.0Article10.1093/nar/gks623openAccess