SIN rather than AdoHcy should be considered as a good structural scaffold for future development of inhibitors for MTases from flavivirus families, or even more broadly for development of AdoMet-based inhibitors for any AdoMet-utilizing enzymes, as seen in a recent Nutlin-3 report. A viral titer reduction assay was used to determine the compounds effect on WNV. Approximately 2 x 105 human A549 cells in 1.0 ml of media were seeded into each well of a 24 well plate. At 24-30 hours after seeding, dilutions at 2X the 136553-81-6 biological activity desired concentration of the compound were made in 2 DMSO media and 50 ��l was added to wells in triplicate. Immediately following, 50 ��l of media containing WNV or DENV2 at a concentration to yield a multiplicity of infection 0.1 PFU/cell, was added to the wells. After one hour incubation at 37, 400 ��l of media containing the desired concentrations of the compound was added to the each well. After 42 hours incubation at 37, culture media was collected, and stored at -80 for later quantification using a plaque assay. For the plaque assay, Vero cell monolayers in 6-well plates were seeded 3-4 days prior to infection to achieve a confluent monolayer. Dilutions of the viral samples were made and 100��l of each dilution were inoculated into each of 2 wells, rocked gently to distribute virus, and incubated for 1 hour at 37. Cells are then overlaid with a nutrient medium containing 0.6 oxoid agar and incubated at37. After 2-5 days, depending on the virus a second overlay containing 2 neutral red is added to the cells and then incubated overnight. Plaques are counted daily for 1-2 days until no significant increase is seen. The Molecular Mechanics program CHARMM was used for the explicit solvent molecular dynamics simulations and their subsequent analysis. The CHARMM22 protein force field with the CMAP correction was used for the protein, the TIP3P model for the water, and Beglov and Roux parameters for the potassium and chloride ions. Parameters for AdoHcy were obtained from a previous study. These SIN parameters were adjusted to ensure transfer from appropriate chemical contexts. The final parameters used are provided in the supplementary material, where their origin is also annotated. The WNV MTase structure bound to SIN was used as the starting point for the
