A series of ring-expanded ("fat") nucleoside analogues (RENs) containing the 6-aminoimidazo[4,5-e][1,3]diazepine-4,8-dione ring system have been synthesized and screened for inhibition of NTPase/helicase of the West Nile Virus (WNV). To assess the selectivity of RENs against the viral enzymes, a truncated form of human enzyme Suv3((Delta)(1)(-)(159)) was also included in the study. Ring-expanded nucleosides 16, 17, and 19, which possess the long C(12), C(14), and C(18) side-chains, respectively, at position 6, as well as the ring-expanded heterocycle 39, which contains aralkyl substitution at position 1, were all found to have excellent profiles of activity and selectivity toward the viral versus human enzymes against the West Nile Virus (IC(50) ranging 1-10 muM). Compound 30, while being an equally potent inhibitor of WNV, was found to be somewhat less selective, whereas compound 36, which is an alpha-anomeric counterpart of 30, exhibited potent and selective inhibition of WNV (IC(50) 1-3 muM). The same compounds that showed potent inhibition of viral helicase activity completely failed to show any activity against the viral NTPase reaction even up to 500 muM. However, at concentrations >500 muM of RENs and the ATP concentrations >10 times the K(m) value of the enzyme, a significant activation of NTPase activity was observed. This activating effect underwent further dramatic enhancement (>1000%) by further increases in ATP concentration in the reaction mixture, suggesting that the viral helicase and NTPase reactions are not coupled. A tentative mechanistic model has been proposed to explain the observed results.