Discovery and analysis of potential antivirals targeting the nonstructural 3 helicase of hepatitis C and dengue viruses

The non-structural protein 3 (NS3) of the hepatitis C virus (HCV) and dengue virus (DENV) is an important component of the viral replicase, and harbors several enzymatic activities. It contains a serine protease at the N-terminus and a helicase at the C-terminus. The serine protease has been an effective antiviral target, but to date, there are not any approved antiviral drugs targeting the helicase protein. The primary hypothesis that my thesis addresses is that the viral helicase is an attractive drug target because it is needed for the survival of the virus, and contains ATP and RNA binding sites that can be targets for inhibition. The focus of this investigation is to apply the mechanism-based approach to discover inhibitors of the NS3 helicase (NS3h). In vitro high-throughput screening (HTS) assays were developed and automated to target the three different ways in which the helicase's activity and degree of inhibition can be measured, i.e., 1) its ability to unwind duplex DNA/RNA 2) its ability to bind nucleic acids and 3) its ability to hydrolyze ATP. All three HTS assays developed were very good having a Z' greater than 0.5. The Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) binding HTS assay cost 14¢/well and had a Z' of 0.59. The TR-FRET based unwinding HTS assay cost 22¢/well and had a Z' of 0.74. A Biomol-Green based ATPase assay cost 2.5¢/well and was the most robust having a z' of 0.79. A biochemical characterization to study the mechanism of action of a terminal pyridine-containing benzothiazole that stimulates the helicase's ability to hydrolyze ATP is also reported. Molecular modeling and site-directed mutagenesis studies suggest that the stimulatory compound binds in the RNA-binding cleft near key residues Arg-393, Glu-493 and Ser-231. The model also shows the pyridine moiety of the stimulatory compound contacting a helix lining the ATP binding site. Gu and Rice (2010) postulated that this "spring helix" contacts bound nucleic acid and helps couple ATP hydrolysis to helicase translocation along nucleic acids. Based on this model, we hypothesized that this favorable interaction with the "spring helix" allows this pyridine-terminated benzothiazole to mimic RNA and thus stimulate the helicase-catalyzed ATP hydrolysis. Upon investigating the kinetics of helicase-catalyzed ATP hydrolysis, the stimulatory compound binds the enzyme tightly with a Ki of 4 +/- 1.2 microM.;The Biomol Green-based ATPase assay was used to find inhibitors of Dengue NS3 helicase. It was designed to detect compounds that hinder ATP binding, ATP hydrolysis, or RNA binding. Analysis of validation experiments show that the assay is statistically robust with a Z' factor of 0.8. A pilot screen of different compound libraries comprised of 5,880 compounds identified 47 inhibitors that cause at least 50% inhibition of ATP hydrolysis by the helicase. To verify whether some of the identified flavivirus inhibitors can be used as molecular probes in cells, replicon assays were run that measured the ability of some of the target hits in the collection to inhibit Renilla luciferase, after cells harboring replicons were exposed to compounds for 72 hours. The effect of the same compound concentration on BHK cell viability was also monitored. Two classes of inhibitors were discovered to have access to be antivirals. The first class consisted of benzothiazoles derived from NIH molecular probe ML283, which was developed to inhibit HCV. A benzothiazole derivative inhibited DENV NS3 with an IC50 value of 0.5 +/- 0.1 microM and was active against DENV replicon. The second class comprised of pyrrolone inhibitors. The most potent pyrrolone derivative that was a specific DENV inhibitor inhibited both DENV replicon and West Nile virus replication in cells by 50%.;To assist in the characterization of inhibitors, x-ray crystallography was investigated to delineate a possible binding pose of the compound with helicase. Purified DENV helicase yielded acceptable crystals that diffracted on average to 2.7A. Whisker-seeding optimization experiments yielded bigger, better quality crystals that diffracted to 2.3A. Co-crystallization and soaking experiments with two of the most potent inhibitors: aurintricarboxylic acid (ATA) and suramin did not show the small molecules bound to the protein. Structure was solved by molecular replacement using PDB file 2BMF as the search model. Our robust high-throughput assay and efforts in x-ray crystallography provide a solid starting point for identifying potential inhibitors and understanding their interactions with the helicase.