0 (compare Table 2). The screening and docking results Pifithrin-�� chemical structure were combined in the consensus scoring procedure to give the final ranking list of 15 hits. Docking of the most potent hit 8 (ZINC07570349) (Fig. 7a) reveals that the main interactions of this ligand involve the network of hydrogen bonds between Lys200, Glu286 and one of the NH hydrogen atoms of the thiourea moiety of the ligand as well as its hydroxylic group. The phenyl ring of 8 is placed in the hydrophobic cavity formed by Val227, Val228, Phe289 and Ile411.

Docking of the next hit, 9 (ZINC05339577), also revealed engagement of the crucial residues of the JEV NS3 helicase/NTPase with the potential inhibitor (Fig. 7b). In this case two hydroxylic groups of the ligand form hydrogen bonds with Glu286. Additionally, the side chain of Arg202 is engaged in the hydrogen bond with the oxirane moiety of 9, similarly as in the case of ring-expanded nucleoside 2. The ketone and hydroxylic groups of 9 interact with the NH hydrogen atoms of the main chains of Thr201 and Lys202. In the case of 10 (ZINC01590677), which was the first hit in the Screen Library procedure, apart from the already mentioned Arg202 (which forms a bond with the oxygen atom of the ligand) and Thr201 (interacting with the one

of NH hydrogen atoms), Glu231 also seems to be engaged, as it forms a hydrogen bond with the other NH hydrogen atoms (Fig. 7c). The fourth hit, 11 (ZINC11756980) (Fig. 7d), interacts with both Arg202 and Arg464 (through Clomifene its diazole nitrogen atom and the carbonyl group, respectively). Moreover, its

amino group interacts with Asn417 and, through water Atezolizumab in vitro molecule, with Arg461. In the case of 12 (ZINC10674215), similarly to 10 and 11, the side chains of Arg202, Glu231 and Arg464 are engaged in the hydrogen bonds with the ligand hydroxylic and carbonyl group, whereas the next compound identified, 13 (ZINC06668757), interacts through water molecules with the side chain of Arg464 and with the main chains of Gly199 and Lys200. The compound, 14 (ZINC04887000), is also worth mentioning because it possesses a pentose moiety and in this regard is similar to nucleosides. It forms hydrogen bonds with the side chains of Arg202 and Glu286. The other eight potential inhibitors 15–22 identified interact with the binding pocket of JEV NS3 helicase/NTPase in a similar way to 8–14. However, they are characterized by significantly lower scores, which indicates a worse fit to the binding site. It is worth emphasizing that among 15 identified potential inhibitors only one of them, 14, exhibits partial similarity to the natural ligand, ATP. The others constitute novel chemotypes of JEV NS3 helicase/NTPase inhibitors. Additionally, lipophilicity and the ability to cross the blood–brain barrier for identified hits were calculated with Preadmet server (preadmet.bmdrc.org). The results are presented in Table 3.