Kinase assays

Recombinant DYRK1B (2.5 ng per reaction; catalog no. D09-10BG-10; SignalChem, Richmond, BC, Canada) phosphorylation of DyrkTide (40 µM;, RRRFRPASPLRGPPK; catalog no. D96-58; SignalChem) was measured in a reaction buffer containing 10 mM MgCl₂, 20 mM Tris pH 7.5, and 10 μM adenosine triphosphate. Drug candidates (CC-401 and STF-785) were assayed at fourfold dilutions starting at 20 μM; final 2% dimethyl sulfoxide (DMSO). Reactions were terminated after 90 minutes at 30°C and kinase activity was measured using ADP-Glo Kinase Assay (V6930; Promega, Madison, WI). An analogous experiment was conducted with p27 (25 ng per reaction; catalog no. 56279; Abcam) as the reaction substrate instead of DyrkTide, with other reaction components unchanged. Reactions were conducted in the presence and absence of 1 μM CC-401 or 1% DMSO. Control reactions lacking DYRK1A (2.5 ng per reaction, SignalChem D09-10G-10) or adenosine triphosphate were also conducted. After 60 minutes at 30°C, reactions were quenched and run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions. Total p27 and phospho-p27 were quantified by western blot. Kinome screening and kinase inhibition assays were performed by DiscoveRx (San Diego, CA) (18).