, 1997; Wu et al., 2005; Xu et al., 2005; Yamashita et al., 2008). The amino acid sequences of the NS3 helicase domain of JEV exhibited 65%, 44% and 23% homology to those of DEN, YFV and HCV, respectively (Yamashita et al., 2008). The crystal structures of the NS3 helicases of DEN (Xu et al., 2005) and YFV (Wu et al., 2005) are similar to that of JEV, but slightly different from HCV (Yao et al., 1997). Yamashita et al. (2008) emphasized that the distance between domains 1 and 2 of HCV helicase is longer than

that in most flavivirus NS3 helicases. This leads to the conclusion that MLN8237 chemical structure the HCV helicase has a larger ATP-binding pocket than other flaviviruses, and that the folding of domain 3 of the HCV helicase is unique, whereas the folding of JEV is very similar to those of other flaviviruses, including DEN and YFV (Yamashita et al., 2008). Superposition of JEV, DEN, YFV and HCV helicases further clarified that the HCV helicase has a unique conformation in the NTPase-binding region and domain 3 in comparison with JEV, DEN and YFV helicases (Yamashita et al., 2008). In particular, the conformation of motifs I and II of HCV helicase was different from Doxorubicin nmr that of JEV, DEN and YFV helicases. The distance between motifs I and II

of Cα of HCV and the other flaviviruses was 6.7 and 3.5 Å, respectively (Yamashita et al., 2008). There was also a 4.7 Å difference in the distance of Nz of Lys200 in the motif I between JEV and HCV, suggesting that HCV helicase has a wider ATP-binding pocket than other flaviviruses (Yamashita et al., 2008). In contrast to the structure of motifs I and II, that of motif VI was well conserved among the flavivirus helicases, including Rucaparib purchase HCV. Although a subtle difference is observed, the ATP-binding residues in JEV, DEN, YFV, and HCV helicases are well conserved, suggesting that flavivirus

helicases possess similar mechanisms of ATP hydrolysis, which reflects the lack of specificity of compounds 1 and 2. The virtual screening performed allowed the noncompetitive mode of action of 3 and 4 to be confirmed, as they were not identified as hits for the ATP-binding site. Although the antiviral activity of the identified hits needs to be confirmed in experimental studies, the reliability of the computational results obtained is enhanced by several factors. As mentioned, the refined crystal structure of the catalytic domain of JEV NS3 helicase/NTPase was utilized to construct the pharmacophore model. Moreover, the residues constituting the ATP-binding site were identified in the mutational analysis. Finally, the application of consensus screening procedure improved the hit ranking list. The consensus scoring procedure has been demonstrated to improve virtual screening results significantly (Feher, 2006). It was reported that consensus scoring usually substantially enhances virtual screening performance, contributing to better enrichments.