| Assay Method Information | |
| | Inhibitory Effect of the Compounds of the Examples of the Present Invention on PI3Kα/β/γ/δ Kinase Activity |
| Description: | Experimental instruments: Centrifuge (5810R, purchased from Eppendorf), pipette (purchased from Eppendorf or Rainin), and microplate reader (Model: SynergyH1 full-function microplate reader, purchased from BioTek, USA).Experimental method: Promega's ADP-Glo lipid kinase assay method (Promega #V9102) was applied in this experiment. Lipid kinase PI3Kα/β/γ/δ catalyzed the generation of ADP from ATP in the presence of the substrate PIP2: 3PS and ATP. The lipid kinase activity was characterized by measuring the ADP content in the reaction, and the half inhibitory concentration IC50 of the compound on inhibiting PI3Kα/β/γ/δ kinase activity was thus obtained.The specific experimental process is as follows:The kinase reaction was carried out in a white 384-well plate (Perkin Elmer #6007299). 2 μL of the compound solution of different concentrations diluted with ddH2O containing 1% DMSO was added to each well. 2 μL of ddH2O containing 1% DMSO was added to the positive control well. 2 μL of 0.1˜2 nM PI3K kinase solution diluted with 5× kinase buffer (HEPES 250 mM, MgCl2 15 mM, NaCl 250 mM, BSA 0.05%) was added to each well. 2 μL of 5× kinase buffer was added to the negative control well. 4 μL of 50 μM substrate PIP2: 3PS (Promega #V1701) formulated with 10×Dilution buffer and ddH2O was added to all wells. Finally, 2 μL of 50˜100 μM ATP solution diluted with water was added to start the reaction. After the reaction was carried out at room temperature for 90 to 120 minutes, 10 μL of ADP-Glo Reagent (containing 10 mM MgCl2) was added to each well, and reacted at room temperature for 60 minutes to remove excess ATP in the reaction. 20 μL of Kinase Detection Reagent was added to each well, and reacted at room temperature in the dark for 20 minutes. The chemiluminescence value was measured by the BioTek Synergy H1 microplate reader |
| Affinity data for this assay | |
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