NS3 helicase

The NTPase/ helicase domain of dengue NS3 protein is located within the C-terminal end and contains seven conserved motifs associated with SF2 class of RNA NTPases and helicases (Gorbalenya et al 1989, 1993). NS3 helicase activity is essential for unwinding dsRNA, an intermediate step during viral RNA synthesis. The unwinding mechanism is an energy-dependent reaction driven by ATP

FIG. 3. Homology model of Dengue 2 CF40-Gly-NS3pro185 in complex with Ac-Lys-Arg-Arg-H.

hydrolysis. Mutational analysis of dengue 2 NS3 protein within and outside the helicase motifs have shown that absence of helicase activity correlates directly with lack of viral replication (Matusan et al 2001). NS3 helicase activity is potentially an interesting antiviral target, however so far these domains have proven to be challenging targets for modern drug discovery.

It is important to consider that helicases are involved in a variety of cellular processes involving nucleic acid metabolism, e.g. replication, transcription, translation, DNA recombination and repair. Because of the conserved nature of helicase motor function, inhibitors targeting helicases can be potent inhibitors of host protein. To effectively target dengue viral helicase, it is very important to select an inhibitor which specifically inhibits dengue viral helicase activity. In this direction, we are pursuing both a structure based approach as elaborated by the previous presentation, as well as a screening based approach. In the latter, we have developed a FRET (fluorescence resonance energy transfer)-based helicase unwinding assay (Fig. 4) in high-throughput format which will be used to evaluate compound libraries. Since helicase unwinding activity is driven by ATP hydrolysis, the FRET-based assay can select inhibitors which affect both the ATPase activity and RNA unwinding activity. We have also developed a secondary assay which detects ATPase activity which will help determine mode of action of inhibitors. In addition to HTS, the recently resolved crystal structure of the catalytically active helicase domain (Xu et al 2005) will permit fragment based screening (Rees et al 2004) as well as in silico high-throughput docking to identify potential helicase inhibitors.

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