ADP-Glo biochemical assay

The ADP-Glo™ kinase assay (Promega, Madison, WI) was used to screen A674563 for its FLT3 inhibition effects. The kinase reaction system contains 4.95 μL FLT3-WT (40 ng/μL) or FLT3-ITD (40 ng/μL), 0.55 μL of serially diluted A674563, and 5.5 μL FLT3 substrate Poly (4:1 Glu, Tyr) peptide (0.2 μg/μL) (Promega, Madison, WI) with 100 μM ATP (final concentration: 50 μM) (Promega, Madison, WI). The reaction in each tube was started immediately by adding ATP and kept going for an hour under 37°C. After the tube cooled for 5 minutes at room temperature, 5 μL solvent reactions were carried out in a 384-well plate. Then 5 μL of ADP-Glo™ reagent was added into each well to stop the reaction and consume the remaining ADP within 40 minutes. At the end, 10 μL of kinase detection reagent was added into the well and incubated for 30 minutes to produce a luminescence signal. Luminescence signal was measured with an automated plate reader (Perkin-Elmer Envision) and each measurement was performed in triplicate.