| Assay Method Information | |
| | Cell-Free Kinase Inhibitory Activity |
| Description: | To a flat bottom 96 well white plate (Sumitomo Bakelite Co., Ltd., MS-8496W), 10 μl of FGFR1 protein (Carna Biosciences, Inc., 08-133) solution diluted to 1 μg/mL with an assay buffer (20 mM HEPES-NaOH, 0.01% Triton X-100, 2 mM DTT, and 5 mM MgCl2), 10 μL of an assay buffer solution containing CSK-tide substrate (Ana Spec Inc., 63843) in a final concentration of 1000 nM and ATP (Promega Corporation, V9102) in a final concentration of 58.3 μM, and 5 μl of a test substance diluted with the assay buffer were added, and the reaction was performed at room temperature for 1 hour. For measuring kinase activity, ADP-Glo Kinase Assay (Promega Corporation, V9102) was used. After the reaction, 25 μL of ADP-Glo reagent was added to each well of the plate, and the reaction was performed at room temperature for 40 minutes to stop the kinase reaction and to deplete the remaining ATP. The kinase detection reagent was further added, and the reaction was performed at room temperature for 40 minutes, so as to cause conversion from ADP to ATP, a luciferase/luciferin coupling reaction and a luminous reaction by ATP. To evaluate the enzyme activity, the amount of luminescence in each well was measured by Envision (PerkinElmer Co., Ltd.). The luminescence values of the wells containing the kinase protein without adding the test substance was defined as 100% and the luminescence values of the wells adding neither the test substance nor the kinase protein was defined as 0%. Then, a luminescence value ratio in the presence of the test substance was calculated. On the basis of this luminescence value ratio, the concentration of the test substance necessary for inhibiting the kinase activity by 50% (i.e., an IC50 value) was calculated.FGFR2 cell-free kinase inhibitory activity, FGFR3 cell-free kinase inhibitory activity, and FGFR4 cell-free kinase inhibitory activity were measured respectively by using FGFR2 protein (Carna Biosciences, Inc., 08-134), FGFR3 protein (Carna Biosciences, Inc., 08-135), or FGFR4 protein (Carna Biosciences, Inc., 08-136) in the same manner as the case of the aforementioned FGFR1 cell-free kinase inhibitory activity. However, with respect to a concentration of ATP, cell-free kinase inhibitory activity were evaluated in a final concentration of 35 μM for FGFR2, in a final concentration of 16.7 μM for FGFR3, and in a final concentration of 75 μM for FGFR4. For FGFR3 and FGFR4, the reaction with the test substance was performed at room temperature for 2 hours. |
| Affinity data for this assay | |
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