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Identification and Analysis of Hepatitis C Virus NS3 Helicase Inhibitors Using Nucleic Acid Binding Assays

Identification and Analysis of Hepatitis C Virus NS3 Helicase Inhibitors Using Nucleic Acid Binding Assays

Sourav Mukherjee, Alicia M Hanson, William R Shadrick, Jean Ndjomou, Noreena L Sweeney, John J Hernandez, Diana Bartczak, Kelin Li, Kevin J Frankowski, Julie A Heck, Leggy A Arnold, Frank J Schoenen, David N Frick

Nucleic Acids Res. 2012 Sep 1;40(17):8607-21.

PMID: 22740655

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
ALP104869310	NF 023 hydrate		104869-31-0 (anhydrous)	Price
AP129464	Suramin sodium salt		129-46-4	Price

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