NVS-PAK1-1

KD=7 nM. Caliper IC50 assay. The assay was performed using 384-well microtiter plates. Compounds were tested as 8-point dose responses. The assays were prepared by addition of 50 nL of compound solution in 90% DMSO directly into the empty plate. Subsequently, 4.5 μL of the enzyme solution (50 mM HEPES pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 10 mM beta-glycerolphosphate, 10 μM sodium orthovanadate, 3 mM MgCl2 and 0.6 nM PAK1 (249-545) wt nonphos, produced in-house by expression in E. coli cells and purified by affinity chromatography) was added to each well and the resulting solution was pre-incubated at 30°C for 60 min, followed by addition of 4.5 μL of the peptide/ATP solution (50 mM HEPES pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 10 mM beta-glycerolphosphate, 10 μM sodium orthovanadate, 3 mM MgCl2, 400 μM ATP and 4 μM peptide (5-Fluo-Ahx-AKRRRLSSLRA-COOH, Biosyntan GmbH). After 60 min incubation at 30°C, reactions were terminated by addition of 16 μL per well of the stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Product formation was measured in a microfluidic mobility shift assay (Caliper LC3000, PerkinElmer). IC50 values were derived from percent inhibition values at different compound concentrations by non-linear regression analysis.