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

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Abstract
Compounds that interact with DNA or RNA generally act as inhibitors of enzymes that unwind DNA or RNA. In the present study we describe the synthesis and properties of some nucleoside analogues that interact with double-stranded DNA but that, in contrast, facilitate the unwinding reaction mediated by West Nile (WN) virus nucleoside triphosphatase (NTPase)/helicase. The nucleoside analogues described, 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, and 1-(2′-deoxy-α-d-ribofuranosyl)imidazo[4,5-d]pyridazine-4,7(5H,6H)dione, all contain the imidazo[4,5-d]pyridazine ring system. The extent of the enhancing effect on helicase activity was found to be dependent on the time of exposure of the DNA substrate to the compounds and their concentrations. The nucleoside analogues were nevertheless found to be capable of uncoupling the ATPase and helicase activities of the enzyme by a mechanism operating on the level of the enzyme. Thus, in the case of HMC-HO4, the direct interaction with the enzyme caused inhibition of its helicase activity, with a half-maximal inhibitory concentration of 30 μM. The similar potency of the compound against replication of WN virus in cell culture suggests that inhibition of the helicase activity of the viral enzyme is responsible for the observed antiviral activity of HMC-HO4 and may indeed represent an important mode of action of antiviral drugs in general. Comparative studies performed with the related NTPase/helicase from hepatitis C virus revealed that the extent of the effects mediated by imidazo[4,5-d]pyridazine nucleosides is enzyme specific. The substances described may represent a starting point for the development of a new class of helicase-specific antivirals.