Figure 3.

Differential inhibition of WT and PA I38T FluB-ht by BXA.A, 165 nM of WT or PA I38T was mixed with increasing concentrations of BXA and inhibition quantified. The dotted line represents the half-maximal inhibitory concentration (IC₅₀) of BXA. Error bars represent the standard deviation of at least three independent experiments. B, dependence of the IC₅₀ value on FluB-ht concentration. Plots of IC₅₀ versus enzyme concentration for both WT and PA I38T yield a linear relationship with a slope of approximately 0.5 (WT = 0.56, PA I38T = 0.58.) (C) Close-up of the Y-axis from panel B emphasizing the apparent inhibition constant (Ki^app) of BXA for WT and PA I38T FluB-ht. The dotted line indicates the threshold below which robust IC₅₀ determination was not possible due to insufficient endonuclease activity. Percent error for WT IC₅₀ measurements was 27% while percent error for PA I38T was 36%. All experiments were performed under the following final conditions: 100 nM 20-nt substrate, 30 mM Tris-pH 7.5, 5% DMSO, 25 mM NaCl, BXA as specified, FluB-ht as indicated, 1.7 μM vRNA, and 5 mM MgCl₂.