Assay Method Information
Assay Name:  Determination of the Inhibitory Effect of the Compounds of the Present Invention on the Activity of EGFR del19/T790M/C797S and EGFR L858R/T790M/C797S Mutant Kinases
Description:  Experimental method: This experiment used Cisbio's HTRF kinase assay method (Cisbio #62TK0PEB), wherein a catalytic reaction occurred between the substrate polypeptide TK and ATP in the presence of the tyrosine kinase with EGFR del19/T790M/C797S or EGFR L858R/T790M/C797S mutation, the substance was phosphorylated, and the activity of the kinase was characterized by measuring the content of the phosphorylated substrate generated during the reaction, and the half inhibitory concentration IC50 of the compound on the activity of EGFR del19/T790M/C797S or EGFR L858R/T790M/C797S mutant kinase was obtained.Specific Experimental Operations were as Follows:the kinase reaction was carried out in a white 384-well plate (Perkin Elmer #6008280), and 1-5 μL of compounds of different concentrations which were diluted with 1% DMSO-containing ddH2O were added; to the positive control wells were added 1-5 μL of 1% DMSO-containing ddH2O, followed by 1-5 μL of 0.5-5 nM 4×EGFR del19/T790M/C797S or EGFR L858R/T790M/C797S mutant kinase solution which was diluted with Dilution buffer (5× kinase buffer, MgCl2 6.65 Mm, MnCl2 1.33 mM, DTT 1.33 mM); to the negative control wells were added 1-5 μL of Dilution buffer; to all the wells were added 1-5 μL of 4 μM 4× substrate TK solution which was prepared with 10× Dilution buffer, and finally added 1-5 μL of 24 μM 4×ATP solution which was diluted with Dilution buffer to start the reaction; after reacting at room temperature for 120 minutes, 10 μL of detection solution (TK antibody 16 nM, XL665 0.5 μM) was added to each well, same was reacted at room temperature in the dark for 20 minutes, and then the chemiluminescence value was detected using a BioTek Synergy H1 microplate reader.
Affinity data for this assay
 
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