| Assay Method Information | |
| | Inhibitory Activity of Compounds Against EGFR Mutant Enzyme |
| Description: | The activity of the compounds of the present invention against EGFR mutant enzymes was measured using the HTRF system purchased from Cisbio as follows. For an EGFR mutant enzyme, the EGFR del19 enzyme was a recombinant protein purchased from Carna Biosciences, and the EGFR A763_Y764insFHEA enzyme was a recombinant protein purchased from SignalChem, respectively.The composition of the assay buffer used for activity measurement was 50 mM tris-HCl pH 7.5, 100 mM NaCl, 7.5 mM MgCl2, 3 mM KCl, 0.01% tween 20, 0.1% BSA, and 1 mM DTT. An enzymatic reaction was performed using a peptide substrate labeled with ATP at a concentration of 50 mM and biotin at a concentration of 0.5 mM. The analysis of the inhibitory effect of the compounds on EGFR activity was performed according to the following method.Component 1:4 μL of EGFR mutant enzymeComponent 2:2 μL of compound solutionComponent 3:4 μL of ATP and a biotin labeled peptideThe enzyme reaction begins by first mixing component 1 and component 2 and then adding component 3 thereto. After the reaction at 37° C. for 2 hours, 10 mL of a measurement solution consisting of streptavidin-XL665 and a europium-labeled anti-phosphotyrosine antibody purchased from Cisbio was added to the enzyme reaction solution and reacted at room temperature for 1 hour. Finally, the ratio of fluorescence values at 615 nm and 665 nm was measured using Perkin-Elmer's Envision instrument to calculate enzyme activity and confirm the inhibitory ability of the compounds. |
| Affinity data for this assay | |
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