Craig A. Belon

Optimization of Potent Hepatitis C Virus NS3 Helicase Inhibitors Isolated from the Yellow Dyes Thioflavine S and Primuline

A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2 ± 1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because P4 also interacted with DNA, more specific analogues were synthesized from the abundant dimeric component of primuline. Some of the 32 analogues prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6 ± 1 μM.

Monitoring Helicase Activity With Molecular Beacons

A high-throughput, fluorescence-based helicase assay using molecular beacons is described. The assay is tested using the NS3 helicase encoded by the hepatitis C virus (HCV) and is shown to accurately monitor helicase action on both DNA and RNA. In the assay, a ssDNA oligonucleotide molecular beacon, featuring a fluorescent moiety attached to one end and a quencher attached to the other, is annealed to a second longer DNA or RNA oligonucleotide. Upon strand separation by a helicase and ATP, the beacon strand forms an intramolecular hairpin that brings the tethered fluorescent and quencher molecules into juxtaposition, quenching fluorescence. Unlike currently available real-time helicase assays, the molecular beacon-based helicase assay is irreversible. As such, it does not require the addition of extra DNA strands to prevent products from re-annealing. Several variants of the new assay are described and experimentally verified using both Cy3 and Cy5 beacons, including one based on a sequence from the HCV genome. The HCV genome-based molecular beacon helicase assay is used to demonstrate how such an assay can be used in high-throughput screens and to analyze HCV helicase inhibitors.

Mechanism and specificity of a symmetrical benzimidazolephenylcarboxamide helicase inhibitor.

This study examines the effects of 1-N,4-N-bis[4-(1H-benzimidazol-2-yl)phenyl]benzene-1,4-dicarboxamide ((BIP)(2)B) on the NS3 helicase encoded by the hepatitis C virus (HCV). Molecular beacon-based helicase assays were used to show that (BIP)(2)B inhibits the ability of HCV helicase to separate a variety of RNA and DNA duplexes with half-maximal inhibitory concentrations ranging from 0.7 to 5 microM, depending on the nature of the substrate. In single turnover assays, (BIP)(2)B only inhibited unwinding reactions when it was preincubated with the helicase-nucleic acid complex. (BIP)(2)B quenched NS3 intrinsic protein fluorescence with an apparent dissociation constant of 5 microM, and in the presence of (BIP)(2)B, HCV helicase did not appear to interact with a fluorescent DNA oligonucleotide. In assays monitoring HCV helicase-catalyzed ATP hydrolysis, (BIP)(2)B only inhibited helicase-catalyzed ATP hydrolysis in the presence of intermediate concentrations of RNA, suggesting RNA and (BIP)(2)B compete for the same binding site. HCV helicases isolated from various HCV genotypes were similarly sensitive to (BIP)(2)B, with half-maximal inhibitory concentrations ranging from 0.7 to 2.4 microM. (BIP)(2)B also inhibited ATP hydrolysis catalyzed by related helicases from Dengue virus, Japanese encephalitis virus, and humans. (BIP)(2)B appeared to bind the HCV and human proteins with similar affinity (K(i) = 7 and 8 microM, respectively), but it bound the flavivirus proteins up to 270 times more tightly. Results are discussed in light of a molecular model of a (BIP)(2)B-HCV helicase complex, which is unable to bind nucleic acid, thus preventing the enzyme from separating double-stranded nucleic acid.

Ceestatin, a Novel Small Molecule Inhibitor of Hepatitis C Virus Replication, Inhibits 3-Hydroxy-3-Methylglutaryl-Coenzyme A Synthase

BACKGROUND Hepatitis C virus (HCV) chronically infects >170 million persons worldwide and is a leading cause of cirrhosis and hepatocellular carcinoma. The identification of more effective and better-tolerated agents for treating HCV is a high priority. We have reported elsewhere the discovery of the anti-HCV compound ceestatin using a high-throughput screen of a small molecule library. METHODS To identify host or viral protein targets in an unbiased fashion, we performed affinity chromatography, using tandem liquid chromatography/mass spectrometry to identify specific potential targets. RESULTS. Ceestatin binds to 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase and irreversibly inhibits HMG-CoA synthase in a dose-dependent manner. Ceestatins anti-HCV effects are reversed by addition of HMG-CoA, mevalonic acid, or geranylgeraniol. Treatment with small interfering RNA against HMG-CoA synthase led to a substantial reduction in HCV replication, further validating HMG-CoA synthase as an enzyme essential for HCV replication. CONCLUSIONS Ceestatin therefore exerts its anti-HCV effects through inhibition of HMG-CoA synthase. It may prove useful as an antiviral agent, as a probe to study HCV replication, and as a cholesterol-lowering agent. The logical stepwise process employed to discover the mechanism of action of ceestatin can serve as a general experimental strategy to uncover the targets on which novel uncharacterized anti-HCV compounds act.