| Assay Method Information | |
| | Measurement of Inhibitory Ability of the Compounds Represented by Formula 1 According to the Present Invention Against EGFR Ex20 Insertion Mutant Enzyme |
| Description: | An experiment to measure the activity of the compounds of the present invention against EGFR mutant enzymes was performed as follows using the HTRF system sold by Cisbio. For the EGFR mutant enzyme, EGFR A763_Y764insFHEA enzyme, a recombinant protein provided by SignalChem was purchased and used.The composition of the assay buffer used for measuring the activity 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. Here, an enzyme reaction was performed using ATP at a concentration of 50 mM and a peptide substrate labeled with biotin at a concentration of 0.5 mM. Analysis of the inhibitory effect of the compounds against the EGFR activity was conducted according to the following analysis reaction recipe.Component 1: 4 μL of an EGFR mutant enzymeComponent 2: 2 μL of a compound solutionComponent 3: 4 μL of ATP and a peptide labeled with biotinThe enzyme reaction was started by first mixing Component 1 and Component 2 and then adding Component 3. After reaction at 37° C. for 2 hours, 10 mL of measurement solution consisting of streptavidin-XL665 and europium-labeled anti-phosphotyrosine antibody provided by 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 obtained using the Envision equipment from Perkin-Elmer to quantitatively measure the enzyme activity and confirmed the inhibitory ability of the compounds. |
| Affinity data for this assay | |
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