DENV2 and DENV3. The IC50 values for inhibition of the WNV and DENV3 MTase activities by AdoHcy were estimated to be in low micromolar or even nanomolar range. In contrast, our results showed that AdoHcy and its derivatives do not significantly inhibit both the N-7 and 2′-O activities for MTases from four different viruses, even at very high concentrations. Although there are some differences in assay conditions such as buffers, pH, substrates, and constructs of enzymes used, it is hard to believe that they will account for the large discrepancies. As shown in Figure 3, in the absence of the positive control SIN inhibitor, all enzymes could efficiently carry out the N-7 and 2′-O MTase reactions in our experiments. Under the same conditions as for AdoHcy, SIN could efficiently abolish the MTase activities of all MTases from the four viruses, which is consistent with our previous results showing that SIN inhibited both MTase activities of the WNV MTase with IC50 about 14 ��M using the TLC method. In contrast, AdoHcy under the same conditions failed to inhibit the enzymatic activities. The discrepancies may more reasonably be attributed to the different methods used to monitor the reactions. We monitored the reaction product, m7G*pppA and double methylated m7G*pppAm, using the TLC method. Although this method is low throughput, its advantage is the ability to directly “visualize” and quantify the reaction product. Alternative higher throughput monitoring methods could possibly quantify non-specific binding of radiolabeled materials and/or signals arising from incorporation of radio-labeled materials to other positions of RNA. Previous studies employed the SPA-based scintillation assay in which -AdoMet was used as a co-factor and activity was monitored by scintillation counting of the transfer of -labeled methyl group to the viral RNA. Nonspecific binding of radio-labeled materials or incorporation of radio-labeled materials to positions other than N-7 and 2′-O of the RNA could affect the activity reported by this assay. It was reported that N-7 and 2′-O reactions might only account for one-third of the total signals and that a large fraction of signals were unresolved when using the SPA method. In particular, the flavivirus MTase was reported to also carry out 2′-O methylation of internal adenosines in the viral RNA. The unresolved signals therefore could be from methylations of internal adenosines of the RNA. The Fulvestrant Estrogen Receptor inhibitor presence of these unresolved signals may thus affect how the results from inhibition studies using the SPA method were interpreted. It is possible that AdoHcy might mainly inhibit the internal methylation activity of flavivirus MTase, for which the hypothesis requires further investigation. The weak inhibition of the N-7 and 2′-O activities of flavivirus by AdoHcy are consistent with functional analysis indicating that it does not suppress viral growth till a high concentration is reached. In contrast, SIN inhibits both N-7 and 2′-O activities of the WNV MTase with IC50 of 14 ��M in vitro, and can also efficiently inhibit the growth of WNV with an EC50 of 27 ��M. The ineffectiveness of AdoHcy in virus growth inhibition is also consistent with results from a number of studies showing that the circulating blood levels of AdoHcy are as high as 0.77 ��M, and the levels of AdoMet are as high as 2.6 ��M. The binding affinity of AdoHcy for the DENV3 MTase was also shown to be much lower than those of AdoMet and SIN. The low affinity of AdoHcy for the MTase may facilitate the by-product release from the MTase and replenishment with a fresh AdoMet for a new cycle of methylation reaction. Structural comparison also supports the results. Superposition of the crystal structures of the WNV MTase-SIN and MTase-AdoHcy complexes reveals that SIN binds to the AdoMet pocket of the MTase in a conformation similar to that of AdoHcy in the MTase-AdoHcy complex. However, the free amine NE of the C-NH2 group of SIN, i.e., the group that replaces the SCH3 group of AdoMet, makes at least five additional contacts with the MTase, which include a pair of potential hydrogen bonds between the NE atom of SIN and the OD1 and O atoms of the MTase catalytically Reversine essential residue D146. The structural results correlate very well with MM-PBSA analysis of binding of SIN and AdoHcy to the WNV MTase, which showed that SIN binds the WNV MTase more favorably than AdoHcy by 6.8 kcal/mol, and that the NH2 group of SIN alone makes the largest contribution.