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.

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 μM ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 μM, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure–activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines.

The flavivirus nonstructural protein 3 (NS3) is a protease and helicase, and on the basis of its similarity to its homologue encoded by the hepatitis C virus (HCV), the flavivirus NS3 might be a promising drug target. Few flavivirus helicase inhibitors have been reported, in part, because few specific inhibitors have been identified when nucleic acid unwinding assays have been used to screen for helicase inhibitors. To explore the possibility that compounds inhibiting NS3-catalyzed ATP hydrolysis might function as antivirals even if they do not inhibit RNA unwinding in vitro, we designed a robust dengue virus (DENV) NS3 ATPase assay suitable for high-throughput screening. Members of two classes of inhibitory compounds were further tested in DENV helicase-catalyzed RNA unwinding assays, assays monitoring HCV helicase action, subgenomic DENV replicon assays, and cell viability assays and for their ability to inhibit West Nile virus (Kunjin subtype) replication in cells. The first class contained analogues of NIH molecular probe ML283, a benzothiazole oligomer derived from the dye primuline, and they also inhibited HCV helicase and DENV NS3-catalyzed RNA unwinding. The most intriguing ML283 analogue inhibited DENV NS3 with an IC50 value of 500 nM and was active against the DENV replicon. The second class contained specific DENV ATPase inhibitors that did not inhibit DENV RNA unwinding or reactions catalyzed by HCV helicase. Members of this class contained a 4-hydroxy-3-(5-methylfuran-2-carbonyl)-2H-pyrrol-5-one scaffold, and about 20 μM of the most potent pyrrolone inhibited both DENV replicons and West Nile virus replication in cells by 50%.