Characterization of Imidazo[4,5-d]Pyridazine Nucleosides as Modulators of Unwinding Reaction Mediated by West Nile Virus Nucleoside Triphosphatase/Helicase: Evidence for Activity on the Level of Substrate and/or Enzyme

The members of the family of Flaviviridae are small, enveloped RNA viruses with similar structures (31). The viral genome encodes a polyprotein of approximately 3,000 amino acids that is processed into three structural proteins (C, prM, and E) and at least seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (1, 6, 27). Among the nonstructural proteins, NS3 appears to be the most promising target for antiviral agents because of the multiple enzymatic activities associated with this protein. NS3 exhibits serine protease activity that resides in the NH2 terminus of the protein and nucleoside triphosphatase (NTPase) and RNA helicase activities located in the COOH terminus (10, 11, 12). NTPase/helicase, together with the NS5-associated RNA polymerase, is thought to be an essential component of the viral replicase complex (19). The NTPases/helicases are capable of enzymatically unwinding duplex RNA or DNA structures by disrupting the hydrogen bonds that keep the two strands together (17, 21). This is accomplished by a reaction that is coupled to the hydrolysis of a nucleoside triphosphate (NTP). Nonhydrolyzable ATP analogues did not substitute for ATP in the RNA-unwinding reaction, suggesting that ATP hydrolysis is required for this reaction (10). Although the helicase activity is dependent on the energy produced in the course of NTP hydrolysis, numerous observations show that the number of events of NTP hydrolysis per unwinding cycle is not a constant value (2, 5). Thus, potential specific inhibitors of the NTPases/helicases of members of the family Flaviviridae could act by any one or more of the following mechanisms: (i) inhibition of NTPase activity by interference with NTP binding (3), (ii) inhibition of NTPase activity by an allosteric mechanism (3), and (iii) inhibition of the coupling of NTP hydrolysis to the unwinding reaction (5). Other inhibitory mechanisms are also conceivable. These may involve modulation of interaction of the enzyme with its RNA or DNA substrate, for example, (iv) competitive inhibition of RNA binding (28) and (v) inhibition of the unwinding by steric blockage of translocation of the helicase along the polynucleotide chain (25). Because of the well-established antihelicase activities of numerous DNA-interacting agents, we were interested in developing NTPase/helicase inhibitors that act by interaction with a DNA substrate. In this report we describe the compounds 1-(2′-O-methyl-β-d-ribofuranosyl)imidazo[4,5-d]pyridazine-4,7(5H,6H)-dione (HMC-HO4), 1-(β-d-ribofuranosyl)imidazo[4,5-d]pyridazine-4,7(5H,6H)-dione (HMC-HO5), and 1-(2′-deoxy-α-d-ribofuranosyl)imidazo[4,5-d]pyridazine-4,7(5 H,6H)dione (HMC-HO1α). These compounds are analogues of purine nucleosides in which a pyridazine moiety replaces a pyrimidine fused to an imidazole ring. Our preliminary molecular modeling studies as well as the results of the experiments presented here suggest an interaction of these compounds with DNA. Surprisingly, the detailed kinetic analyses reported in this study revealed that this interaction results instead in an enhancement of the unwinding activities of the NTPases/helicases of the West Nile (WN) virus and of the related virus hepatitis C virus (HCV). On the other hand, the compounds were also discovered to interact directly with the enzymes investigated and uncouple their ATPase and helicase activities. In the case of HMC-HO4, this interaction resulted in a decrease in the level of the unwinding reaction mediated by the WN virus enzyme. These two very different effects occur at quite different concentrations of HMC-HO4. The HMC-HO4-mediated inhibition of the helicase activity correlated with the corresponding reduction in the level of WN virus replication in cell culture.