3.2. In Vitro Kinase Inhibition Assays

To verify the Syk inhibitory activity of the test compounds, a luminescent kinase assay was performed to measure the ADP produced in a kinase reaction utilizing an ADP-Glo^TM kinase assay kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. In this assay, the ADP produced by the Syk activity was converted to ATP, which is the substrate of luciferase, consequently leading to the production of light. Therefore, the luminescent signal directly correlates with the kinase inhibitory activity of the test compounds. Staurosporine (CAS No. 62996-74-1) was used as a positive control for the Syk activity assay. A kinase buffer containing 5% DMSO was used as a negative control. All test compounds were purchased from the National Institutes for Food and Drug Control (Beijing, China), and the purity of each compound was greater than 98% based on HPLC analysis.

All reactions were performed in white, non-binding 384-well plates with flat bottoms (Corning #3824, Corning Glass Works, Corning, NY, USA). The kinase reaction was performed in Tris buffer (40 mM, pH 7.5) containing 20 mM MgCl₂, 0.1 mg/mL bovine serum albumin and 50.0 μM DTT. Four microliters of Syk (ab60886) solution (1 ng/μL in kinase buffer) and 2.0 μL of test compound solution (150 μM in kinase buffer containing 5% DMSO) were incubated for 15 min at 27 °C. Then, 4.0 μL of substrate solution (0.2 μg/μL polypeptide (4:1 Glu, Tyr) (Abcam catalogue no. ab204877, Cambridge, UK) in a kinase buffer containing 10.0 μM ATP) was added to activate the kinase reaction for 1 hour at 27 °C. The kinase reaction was terminated by adding 5 μL ADP-Glo™ reagent into 5.0 μL Syk reaction solution. The mixture was incubated for 40 min to deplete the unconsumed ATP. Finally, 10.0 μL of kinase detection reagent was added, and the mixture was incubated for 1 h to convert ADP to ATP; then, luciferase and luciferin were added to detect ATP. The luminescence was determined using an Envision 2104 Multilabel Reader (Perkin-Elmer, Eden Prairie, MN, USA). To avoid false negative results caused by the self-luminescence of compounds, the luminescence values of 300 compounds were detected. The fluorescence values were quantified in relative fluorescence units (RFU).