Figure 4. MEKK2 inhibitory activity of a panel of known kinase inhibitors and IC₅₀ determination for screening hits.

(A) A panel of marketed and clinical trial kinase inhibitors was screened at 1 μM compound concentration using the MEKK2 activity assay. The compounds were screened in duplicate wells in three independent experiments and the normalized data from all experiments was aggregated by averaging percent inhibition values obtained from all experiments and plotting them as shown. Error bars represent standard deviation of the three experiments. (B) Concentration response data for the hit compound from the large kinase-focused library screen and a structural analog was normalized to controls with and without MEKK2 and plotted as percent specific activity. One hit (compound 1, ●) confirmed activity with an average IC₅₀ of 299 ± 71 nM and a close structural analog (compound 2, ■) resulted in an IC₅₀ of >30 μM. IC₅₀ data are representative of at least three independent experiments. (C) The compounds resulting in ≥50% inhibition from the known kinase inhibitor panel were tested in concentration response experiments, except for sunitinib which was used as a control. IC₅₀ determinations for bosutinib (■), crizotinib (●), and pazopanib (▲) resulted in IC₅₀ values (nM) and SD of 59 ± 34, 75 ± 35, 698 ± 163, respectively. IC₅₀ data are representative of three independent experiments.