West Nile virus (WN virus), a member of the family of Flaviviridae, is a small enveloped single-stranded RNA positivestrand virus. The viral genome encodes a monocistronic polyprotein of 3,430 amino acids that is processed into three structural proteins, protein M, capsid protein C, and glycoprotein E, and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (10, 11, 52). The processing of the polyprotein is carried out by the host signal peptidase associated with the endoplasmic reticulum and viral proteases. The polyprotein of WN virus and its processing are similar to those of the pestivirus-and hepatitis C virus (HCV)-related viruses (36,44,55). Sequence analysis of the nonstructural region of WN virus polyprotein revealed numerous conserved motifs specific for serine proteases, RNA helicase with intrinsic RNA-stimulated nucleoside triphosphatase (NTPase) localized in the NS3 protein, and RNA-directed RNA polymerase associated with the NS5 protein (3, 16, 17). These predictions were partially confirmed by verifying the enzymatic properties of a COOH-terminal segment of NS3 released from a membrane fraction of infected cells by subtilisin (54). Further information about the interactions and functions of the viral proteins was obtained by using synthesized recombined proteins of Flaviviridae or HCV-related viruses (19,23,47,49,50).Due to multiple enzymatic and biological activities associated with NS3, this protein appears to be the most promising target for antiviral agents. The protease activity of NS3 is the subject of numerous studies and has been well characterized previously (24, 31). However, despite the importance of enzymes modulating RNA structures in diverse metabolic processes and their critical role in the life cycles of viruses whose genomes are composed of RNA, only limited information on the viral helicases or helicase-like enzymes is available.Helicases are capable of enzymatically unwinding duplex DNA or RNA structures by disrupting the hydrogen bonds that keep the two strands together (18,21). The unwinding reaction is accomplished by the hydrolysis of ␥-phosphate of nucleotide triphosphate (NTP). Based on sequence comparisons, the viral helicases have been divided into three superfamilies. The WN virus helicase is a member of superfamily II (SFII), which includes helicases from bymovirus, potyvirus, pestivirus, herpesvirus, poxvirus, HCV, and other Flaviviridae (22). All of the helicases contain seven highly conserved amino acid sequences (motifs I to VII) that are located on the surfaces of domains 1 and 2 of the three-domain enzymes. The involvement of the motifs in NTP binding, NTP hydrolysis, and the binding of polynucleotide(s) was well explained by resolving the crystal structures of several enzymes (25,57). However, these structures did not elucidate the mechanisms coupling ATP hydrolysis to the unwinding reaction. Although numerous studies about the quantification of the interaction of SFII helicases with NTP and polynucleotides were performed, uniform resul...

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.

N4-Hydroxy-dCMP (N4-OH-dCMP), N4-methoxy-dCMP (N4-OMe-dCMP), and their 5-fluoro congeners (syntheses of which are described) were all slow-binding inhibitors of Ehrlich carcinoma thymidylate synthase (TS), competitive with respect to dUMP, and had differing kinetic constants describing interactions with the two TS binding sites. N4-OH-dCMP was not a substrate (no dihydrofolate produced; no tritium released with 5-3H-labeled molecule), and its inactivation of TS was methylenetetrahydrofolate-dependent, hence mechanism-based, with arrest of a step posterior to addition of cofactor and blocking abstraction of the C(5) hydrogen. Ki values for N4-OH-dCMP and its 5-fluoro analogue were in the range 10(-7) - 10(-8) M, 2-3 orders of magnitude higher for the corresponding N4-OMe analogues. The 5-methyl analogue of N4-OH-dCMP was 10(4)-fold less potent, pointing to the anti rotamer of the imino form of exocyclic N4-OH, relative to the ring N(3), as the active species. This is consistent with weaker slow-binding inhibition of the altered enzyme from 5-FdUrd-resistant, relative to parent, L1210 cells by both FdUMP and N4-OH-dCMP, suggesting interaction of both N4-OH and C(5)-F groups with the same region of the active center. Kinetic studies with purified enzyme from five sources, viz., Ehrlich carcinoma, L1210 parental, and 5-FdUrd-resistant cells, regenerating rat liver, and the tapeworm Hymenolepis diminuta, demonstrated that addition of a 5-fluoro substituent to N4-OH-dCMP increased its affinity from 2- to 20-fold for the enzyme from different sources.(ABSTRACT TRUNCATED AT 250 WORDS)

To improve anti-helical activity of analogues of 1H-benzotriazole and 1H-benzimidazole their N-alkyl derivatives were synthesized and tested for antihelicase activity against enzymes of selected Flaviviridae including hepatitis C virus (HCV), West Nile virus (WNV), Dengue virus (DENV) and Japanese encephalitis virus (JEV). 1- and 2-alkyl derivatives of 4,5,6,7-tetrabromo-1H-benzotriazole were obtained by direct alkylation of 4,5,6,7-tetrabromo-1H-benzotriazole with the use of respective alkyl halides in the presence of KOH in methanol, to give a mixture of 1- and 2- isomers, which was separated by flash column chromatography in good yield. The proportion of isomers strongly depended on the reaction time and temperature. 1- and 2-hydroxyethyl and 1- and 2-chloroethyl derivatives of the tetrabromobenzo-triazole were synthesized with the use of 2-bromoethanol and 1-bromo-2-chloroethane respectively as alkylating agents. N-alkylation of this benzotriazole compound enhanced inhibitory activity and selectivity towards the helicase activity of HCV NTPase/helicase. The most active were the 2-methyl, 2-ethyl and 2-propyl derivatives (IC50 approximately 6.5 microM in the presence of DNA as a substrate). Derivatives of the benzotriazole in which hydroxyethyl or chloroethyl replaced the alkyl substituents lost their inhibitory activity. Brominated or methylated benzotriazole N(1) ribosides also did not exert helicase inhibitory activity. Although a number of N(1) and N(2) alkyl derivatives exerted good HCV and WNV helicase inhibitory activity when DNA was used as substrate, the activity was strongly decreased or even disappeared when RNA was used as substrate. The cytotoxicity tests in Vero and HeLa Tat cells showed a substantial decrease of cytotoxicity of N-alkyl derivatives as compared to the parent benzotriazole.