Two identified MTase RNA binding sites identified 431898-65-6 compounds with potency, but not specificity. A potential third route of flavivirus MTase inhibition is to target the GTP binding site using nucleoside analogs to prevent the binding of the capped portion of the viral RNA and its subsequentmethylation. Ribavirin, a nucleoside analog used clinically to treat various RNA virus infections, has been shown to bind to the DENVMTase GTP binding site and inhibit RNA cap methylation in vitro. Interestingly, we have identified nucleoside analogs that appear to bind to both the GTP binding site as well as the SAMbinding pocket, inhibitingMTase activity in vitro and viral replication. These compounds, along with those identified in this study, give us further insight into the chemical scaffolds most likely to inhibit flavivirusMTase proteins. The remaining compounds were docked into the binding site. Finally, the predicted binding modes were post-filtered and promising compounds were short-listed for purchase. To derive a compound set for virtual screening, an in-house virtual library containing 4,177,660 commercially available compounds was filtered by the following selection criteria: at least one but not more than five hydrogen-bond donors, at least one but not more than ten hydrogen-bond acceptors, at least nine but not more than 23 heavy atoms and a clogP between 21 and 4. In addition, the number of rotatable bonds was restricted to less than seven, the total DEL-22379 charge between 21 and 1, and at least one but not more than two ring systems were allowed. Compounds containing unwanted functionalities were excluded. Only compounds that fulfilled all requirements were taken to the next step. The selected subset was further filtered using a protein-based pharmacophore. When deriving the pharmacophore we aimed to strike a reasonable balance between a complex query which potentially retrieves very potent compounds but has only a very low hit rate and a relaxed query retrieving many compounds which prove not be active. To not be over descriptive we decided to only include interactions to His25 which is essential for recognition of the cytosine moiety of the substrate. In all structures containing ligands interacting with this residue, ND presumably carries a hydrogen atom to hydrogen bond with the cytidine moiety of the ligands. However, in the crystal structures His25 NE is solvent exposed and not involved in a hydrogen-bonding network. Accordingly, it is possible that not ND but NE carries a hydrogen atom when challenged with ligands presenting a hydrogen-bond donor funct
