Figure 1. High throughput screen identifies small molecule activators and inhibitors of Ras GTPase superfamily.

Figure 1High throughput screen identifies small molecule activators and inhibitors of Ras GTPase superfamily

(A) Left panel shows a plot of forward scatter versus a logarithm of side scatter. Singlet beads are selected by drawing a gate as shown. Middle panel shows a plot of the logarithm of green fluorescence versus counts. Right panel shows a plot of the logarithm of red fluorescence versus logarithm of side scatter. The seven different bead sets are obvious and gates were drawn about each set. (B) GST-GTPases were conjugated to fluorescent GSH-beads and assays were initiated with the addition of BODIPY-GTP and library compounds in 384 well plates. HyperCyt time-resolved data (time versus logarithm of green fluorescence) collected in the FL8 red fluorescence channel are shown for one 384-well plate. Each block represents a group of 20 samples, separated by blank wells. Each sample contains data for 6 targets and a GSH-GFP control (Y axis: fluorescence intensity, X axis: time). Arrow denotes positive well containing MLS000088004, inset 2x magnification of positive ‘hit’ in primary screen. (C) Graph depicting the MLSCN screen of 194,738 compounds’ effect on Cdc42WT (black), Rac1WT (red) and Rac1ACT (green). Percent response was calculated by the following equation, % Response = 100*(TestSample − Blocked Control)/(Unblocked Control − Blocked Control), which lead to 100 % response corresponding to compounds with no effect. Compounds designated as potential hits were those with % Response beyond the cutoff: % Response > 120% for activators and % Response < 80 % for inhibitors, see dotted line in graph. Encircled with a black rectangle are the calculated response values corresponding to MLS000088004; 209 % response for Cdc42, 174 % response for Rac1, and 152 % response for Rac1 activated mutant. D) Dose dependent activation of GTPases by MLS000088004 with corresponding table of reported results. n=3.

From: Three small molecule pan activator families of Ras-related GTPases

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Probe Reports from the NIH Molecular Libraries Program [Internet].

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