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.

A series of ring-expanded ("fat") heterocycles, nucleoside and nucleotide analogues (RENs) containing the imidazo[4,5-e][1,3]diazepine ring system (9, 14, 15, 18, 24-26, 28, 31, and 33) and imidazo[4,5-e][1,2,4]triazepine ring systems (30b, 30c, 32, and 34), have been synthesized as potential inhibitors of NTPases/helicases of Flaviviridae, including the West Nile virus (WNV), hepatitis C virus (HCV), and Japanese encephalitis virus (JEV). An amino-terminal truncated form of human enzyme Suv3(delta1-159) was also included in the study so as to assess the selectivity of RENs against the viral enzymes. The analogues of RENs included structural variations at position 1 of the heterocyclic base and contained changes in both the type of sugar moieties (ribo, 2'-deoxyribo, and acyclic sugars) and the mode of attachment (alpha versus beta anomeric configuration) of those sugars to the heterocyclic base. The target RENs were biochemically screened separately against the helicase and ATPase activities of the viral NTPases/helicases. A number of RENs inhibited the viral helicase activity with IC50 values that ranged in micromolar concentrations and exhibited differential selectivity between the viral enzymes. In view of the observed tight complex between some nucleosides and RNA and/or DNA substrates of a helicase, the mechanism of action of RENs might involve their interaction with the appropriate substrate through binding to the major or minor groove of the double helix. The REN-5'-triphosphates, on the other hand, did not influence the above unwinding reaction, but instead exerted the inhibitory effect on the ATPase activity of the enzymes. The activity was found to be highly dependent upon the low concentration levels of the substrate ATP. At concentrations >500 microM of RENs and the ATP concentrations >10 times the Km 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. A tentative mechanistic model has been proposed to explain the observed results, which includes an additional allosteric binding site on the viral NTPases/helicases that can be occupied by nucleoside/nucleotide-type molecules such as RENs.

Ubiquitous protein kinase CK2 participates in a variety of key cellular functions. We have explored CK2 involvement in angiogenesis. As shown previously, CK2 inhibition reduced endothelial cell proliferation, survival and migration, tube formation, and secondary sprouting on Matrigel. Intraperitoneally administered CK2 inhibitors significantly reduced preretinal neovascularization in a mouse model of proliferative retinopathy. In this model, CK2 inhibitors had an additive effect with somatostatin analog, octreotide, resulting in marked dose reduction for the drug to achieve the same effect. CK2 inhibitors may thus emerge as potent future drugs aimed at inhibiting pathological angiogenesis. Immunostaining of the retina revealed predominant CK2 expression in astrocytes. In human diabetic retinas, mRNA levels of all CK2 subunits decreased, consistent with increased apoptosis. Importantly, a specific CK2 inhibitor prevented recruitment of bone marrow-derived hematopoietic stem cells to areas of retinal neovascularization. This may provide a novel mechanism of action of CK2 inhibitors on newly forming vessels.

Maf1 protein is a global negative regulator of RNA polymerase (Pol) III transcription conserved from yeast to man. We report that phosphorylation of Maf1 by casein kinase II (CK2), a highly evolutionarily conserved eukaryotic kinase, is required for efficient Pol III transcription. Both recombinant human and yeast CK2 were able to phosphorylate purified human or yeast Maf1, indicating that Maf1 can be a direct substrate of CK2. Upon transfer of Saccharomyces cerevisiae from repressive to favorable growth conditions, CK2 activity is required for the release of Maf1 from Pol III bound to a tRNA gene and for subsequent activation of tRNA transcription. In a yeast strain lacking Maf1, CK2 inhibition showed no effect on tRNA synthesis, confirming that CK2 activates Pol III via Maf1. Additionally, CK2 was found to associate with tRNA genes, and this association is enhanced in absence of Maf1, especially under repressive conditions. These results corroborate the previously reported TFIIIB-CK2 interaction and indicate an important role of CK2-mediated Maf1 phosphorylation in triggering Pol III activation.RNA polymerase III regulation | transfer RNA | casein kinase II regulation R NA polymerase (Pol) III is responsible for the transcription of some 300 different genes in yeast (class III genes), mostly tRNA genes (1). In-depth analyses of the yeast Pol III transcription system have revealed a cascade of protein-DNA and protein-protein interactions leading to the recruitment of Pol III to its target tRNA genes: binding of the six-subunit TFIIIC factor to the intragenic promoter, TFIIIC-directed recruitment and assembly of the three subunits of TFIIIB (TBP, Brf1, and Bdp1) and subsequent recruitment of the 17-subunit Pol III enzyme (2). High rate of tRNA transcription is achieved through many rounds of reinitiation by Pol III on stable DNA-bound complexes of the initiation factor TFIIIB (3, 4).Pol III is under control of the general negative regulator Maf1 (5, 6), which binds to Pol III clamp and rearranges specific subcomplex C82/34/31, which is required for transcription initiation (7). In the repressive complex, Maf1 impairs recruitment of Pol III to a complex of promoter DNA with the initiation factors TFIIB and thus prevents closed-complex formation (4, 7). Maf1 is essential for repressing Pol III transcription in yeast and mediates several signaling pathways (8). In addition to the down-regulation that occurs normally in the stationary phase, Pol III repression accompanying starvation, respiratory growth, as well as oxidative and replication stress, also requires Maf1 (9-11). Maf1 inhibits Pol III transcription via a mechanism that depends on the dephosphorylation and nuclear accumulation of Maf1 followed by its physical association with Pol III at Pol III-transcribed genes genomewide (6, 12). In contrast Maf1 phosphorylation occurs in favorable growth conditions and is linked to cytoplasmic localization of Maf1 (6, 13).Maf1 was recently found to be phosphorylated by protein kinases PKA (14, 15), Sch9 (16-18), and TO...

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