| Assay Method Information | |
| | Inhibitory Activity of Compounds Against EGFR Mutant Enzyme |
| Description: | The activity of the compounds of the present invention against HER mutant enzymes was measured using the HTRF system purchased by Cisbio as follows. As a HER2 mutant enzyme, the HER2 A775_G776insYVMA mutant enzyme was used by purchasing a recombinant protein provided by Carna Biosciences.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. In addition thereto, the 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 activity inhibitory effect of the compounds on HER2 A775_G776insYVMA mutation was performed according to the following method.Component 1:4 μL of HER2 A775_G776insYVMA 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 equipment to calculate enzyme activity and confirm the inhibitory ability of the compounds. |
| Affinity data for this assay | |
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