Fluorescent primuline derivatives inhibit hepatitis C virus NS3-catalyzed RNA unwinding, peptide hydrolysis and viral replicase formation.

The hepatitis C virus (HCV) multifunctional nonstructural protein 3 (NS3) is a protease that cleaves viral and host proteins and a helicase that separates DNA and RNA structures in reactions fueled by ATP hydrolysis. Li et al. (2012) recently synthesized a series of new NS3 helicase inhibitors from the benzothiazole dimer component of the fluorescent yellow dye primuline. This study further characterizes a subset of these primuline derivatives with respect to their specificity, mechanism of action, and effect on cells harboring HCV subgenomic replicons. All compounds inhibited DNA and RNA unwinding catalyzed by NS3 from different HCV genotypes, but only some inhibited the NS3 protease function, and few had any effect on HCV NS3 catalyzed ATP hydrolysis. A different subset contained potent inhibitors of RNA stimulated ATP hydrolysis catalyzed by the related NS3 protein from Dengue virus. In assays monitoring intrinsic protein fluorescence in the absence of nucleic acids, the compounds cooperatively bound NS3 with K(d)s that reflect their potency in assays. The fluorescent properties of the primuline derivatives both in vitro and in cells are also described. The primuline derivative that was the most active against subgenomic replicons in cells caused a 14-fold drop in HCV RNA levels (IC(50)=5±2µM). In cells, the most effective primuline derivative did not inhibit the cellular activity of NS3 protease but disrupted HCV replicase structures.

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

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.