Identification and Analysis of Inhibitors Targeting the Hepatitis C Virus NS3 Helicase

Abstract

This chapter describes two types of FRET-based fluorescence assays that can be used to identify and analyze compounds that inhibit the helicase encoded by the hepatitis C virus (HCV). Both assays use a fluorescently labeled DNA or RNA oligonucleotide to monitor helicase-catalyzed strand separation, and they differ from other real-time helicase assays in that they do not require the presence of other nucleic acids to trap the reaction products. The first assay is a molecular beacon-based helicase assay (MBHA) that monitors helicase-catalyzed displacement of a hairpin-forming oligonucleotide with a fluorescent moiety on one end and a quencher on the other. DNA-based MBHAs have been used extensively for high-throughput screening (HTS), but RNA-based MBHAs are typically less useful because of poor signal to background ratios. In the second assay discussed, the fluorophore and quencher are split between two hairpin-forming oligonucleotides annealed in tandem to a third oligonucleotide. This split beacon helicase assay can be used for HTS with either DNA or RNA oligonucleotides. These assays should be useful to the many labs searching for HCV helicase inhibitors in order to develop new HCV therapies that are still desperately needed.

Primuline Derivatives That Mimic RNA to Stimulate Hepatitis C Virus NS3 Helicase-catalyzed ATP Hydrolysis

“…HCV helicase is needed for HCV replication (31), the protein was first crystallized over 15 years ago, and numerous high throughput assays are available that can be used to identify inhibitors (32)(33)(34). However, relatively few HCV helicase inhibitors have been reported and the mechanism of action of most known HCV helicase inhibitors is still not clear.…”

Bicyclic octahydrocyclohepta[ b ]pyrrol-4(1 H )one derivatives as novel selective anti-hepatitis C virus agents

“…Reduction of 6 led exclusively to one stereoisomer (38). The configuration of the newly formed stereocenter was elucidated using X-Ray analysis of derivative 39 ( All the synthesized compounds were then tested in HCV replicon assays using the same protocol described above for the initial set of 21 compounds ( Table 2).…”

“…Finally, compounds derived from functionalization (34)(35)(36)(37) or reduction (38) of the keto group of compound 6 were also synthesized. Hydrazide 34, semicarbazide 35 and two oximes (36,37) were prepared by refluxing compound 6 in ethanol with appropriate nucleophile (Scheme 3).…”

“…Molecular beacon-based NS3 helicase assays were performed as described by Hanson et al [38]. • Compound 34 was the most potent derivative against both genotypes 1b and 2a.…”

Discovering New Medicines Targeting Helicases: Challenges and Recent Progress

“…11 Second, compounds that bind the helicase substrate can be detected because they decrease substrate fluorescence in the absence of the helicase (or ATP). 24,142 As discussed below, DNA binding compounds are frequent, nonspecific hits in helicase assays, and identifying them early is critical for efficient helicase inhibitor development.…”