Kinase Assay to Determine the IC50 Values

The IC₅₀ values against ALK5, TGFBR2, casein kinase 1 delta (CK1δ), and epsilon (CK1ε) of selected compounds were determined using LanthaScreen Eu Kinase binding assay (Life Technologies, Rockville, MD). The assays were performed following the manufacturers’ protocols in white 384‐well plates (Cat. No. 3572; Corning Life Sciences, Acton, MA). SB203580 and TA‐01 were tested as controls in all assays. The compounds were dissolved in DMSO (Hybri‐Max Cat. No. D2650; Sigma‐Aldrich, St. Louis, MO, 5 mM stocks) and diluted to a final concentration of 1% (vol/vol) DMSO for all assays. Each data point was carried out in triplicate. The assay was run according to the protocol developed by Invitrogen (Carlsbad, CA). Tecan Infinite M1000 Pro microplate reader was used for fluorescence measurements (LanthaScreen Eu kinase binding assay; excitation λ = 317 nm, emission λ = 665, 620 nm). Inhibition curves and IC₅₀ values were generated by nonlinear regression analysis and data represent mean ± SEM. Results are included in Supporting information Table S1.