Figure 1.

Virtual screening and experimental verification: (A) comparison of experimentally determined and docked conformations of SIN in the SAM-binding pocket of the WNV MTase; (B) inhibition of the N7 and 2′-O methylation activities of the WNV MTase by 17 top ranking compounds at 150 µM concentration, analyzed on TLC plates. The N7 methylation was measured by conversion of G*pppA-RNA→m⁷G*pppA-RNA; the 2′-O methylation was measured by conversion of m⁷G*pppA-RNA→m⁷G*pppAm-RNA (the asterisk indicates that the following phosphate is ³²P labeled; the RNA represents the first 90 nucleotides of the WNV genome). The spots representing different cap structures on TLC plates were quantified by a PhosphorImager. The specific activity (%) was defined as intensity (m⁷G*pppA)/(intensity (G*pppA) + intensity (m⁷G*pppA)) × 100) for N7 methylation and as intensity (m⁷G*pppAm)/(intensity (m⁷G*pppA) + intensity (m⁷G*pppAm)) × 100 for 2′-O methylation. The methylation activity without compounds was set at 100, and the relative methylation activity with a particular compound was defined as specific activity (compound)/specific activity (no compound) × 100.