A search has been initiated for lead inhibitors of the nonstructural protein 3 (NS3)-associated NTPase/helicase activities of hepatitis C virus, the related West Nile virus, Japanese encephalitis virus and the human mitochondrial Suv3 enzyme. Random screening of a broad range of unrelated low-molecular mass compounds, employing both RNA and DNA substrates, revealed that 4,5,6,7-tetrabromobenzotriazole (TBBT) hitherto known as a potent highly selective inhibitor of protein kinase 2, is a good inhibitor of the helicase, but not NTPase, activity of hepatitis C virus NTPase/helicase. The IC 50 is approximately 20 lM with a DNA substrate, but only 60 lM with an RNA substrate. Several related analogues of TBBT were enzymeand/or substrate-specific inhibitors. For example, 5,6-dichloro-1-(b-D-ribofuranosyl)benzotriazole (DRBT) was a good, and selective, inhibitor of the West Nile virus enzyme with an RNA substrate (IC 50 0.3 lM), but much weaker with a DNA substrate (IC 50 3 lM). Preincubation of the enzymes, but not substrates, with DRBT enhanced inhibitory potency, e.g. the IC 50 vs the hepatitis C virus helicase activity was reduced from 1.5 to 0.1 lM. No effect of preincubation was noted with TBBT, suggesting a different mode of interaction with the enzyme. The tetrachloro congener of TBBT, 4,5,6,7,-tetrachlorobenzotriazole (TCBT; a much weaker inhibitor of casein kinase 2) is also a much weaker inhibitor than TBBT of all four helicases. Kinetic studies, supplemented by comparison of ATP-binding sites, indicated that, unlike the case with casein kinase 2, the mode of action of the inhibitors vs the helicases is not by interaction with the catalytic ATP-binding site, but rather by occupation of an allosteric nucleoside/nucleotide binding site. The halogeno benzimidazoles and benzotriazoles included in this study are excellent lead compounds for the development of more potent inhibitors of hepatitis C virus and other viral NTPase/helicases.

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Hepatitis C virus (HCV), West Nile virus (WNV), Japanese encephalitis virus ( JEV), and dengue virus (DENV) are members of Flaviviridae. They are deadly human pathogens and new and better therapeutic strategies are desperately needed. However, there are considerable barriers to the development of anti-Flaviviridae therapeutics, including the persistence of the virus, its genetic diversity during replication in the host and, for example, in the case of HCV, the lack of reproducible infectious culture systems. Similarities in the key Flaviviridae enzymes (with respect to conserved subdomain organization and arrangement of structural motifs) may indicate the potential usefulness of ribonucleoside analogues in the treatment of these viral infections. Over the last few years our understanding of the Flaviviridae replication cycle and the key enzymes involved in it has increased, giving hope for development of new antiviral agents.The Flaviviridae genome is a positive-stranded RNA, with a single open reading frame that translates into a single polyprotein. This polyprotein is co-and post-translationally processed by cellular and viral proteases into structural and non-structural (NS) proteins (Rosenberg, 2001). Among the NS proteins, NS3 and NS5 -the major components of the so called 'replication complex' -appear to be the most promising targets for antiviral therapy because of their well-characterized enzymatic activities (Tan et al.,

Synthesis and interactions of guanosine, inosine and ribavirin 5'-fluorosulfonylbenzoyl esters with hepatitis C virus (HCV) and Flaviviruses NTPase/helicase and polymerase are described.

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