A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease
Fig 5
Suppression of NPGB-induced SLC enhancement by candidate allosteric inhibitors.
(A) Active-site inhibitor NPGB can induce enhanced SLC which can be suppressed by allosteric inhibitors NSC135618, 260594, and 146771 at high concentration. The N2CN3N was at 150 nM. The compounds were at 40 μM. NSC135618 or NPGB was first incubated with the SLC protease construct for 30 min. in different orders, prior to addition of the luciferin substrate, followed by luminescence detection. N = 3. ***, P < 0.01, by one-way ANOVA. (B) The candidate compounds do not significantly affect the luciferase activity of FLuc (150 nM). The experimental procedure was the same as above. (C) Inhibition of the NPGB-induced SLC enhancement by a concentration series of candidate inhibitors, as indicated. In all panels, DMSO only control was set as 100% relative luminescence (RLU). Luciferase activities of the N2CN3N protease construct with NPGB and/or various compounds were set as percentage of the DMSO control. N = 3. ***, P < 0.01, by one-way ANOVA.