| Assay Method Information | |
| | Biochemical Kinase Assay |
| Description: | Reagents and materials: ABL (ThermoFisher, Cat. No. PV3585), ABL T315I(Thermo Fisher, Cat. No. PR7429B), Ret (Carna, Cat. No. 08-159-10 ug), RET<sup>V804M, Active (Signalchem, Cat. No. R02-12GG), ATP (Sigma, Cat. No. A7699-1G), DMSO (Sigma, Cat. No. D2650), 96-well plate (Corning, Cat. No. 3365), 384-well plate (Greiner, Cat. No. 784076), buffer solution (Thermo Fisher, Cat. No. PR4876B). Specific Assay Method: Compound formulation: The test compounds were dissolved in DMSO to prepare a 20 mM stock solution. Before use, the compounds were diluted in DMSO to 0.1 mM, and diluted into 11 concentrations with a 3-fold gradient. When addition, dilutions of 4-fold final concentration were then made with buffer solution. Kinase assay method: After preparing the buffer, the enzyme was mixed with pre-formulated and diluted compounds of different concentrations in duplicate for each concentration, and placed at room temperature for 30 minutes. The corresponding substrate and ATP were added thereto,and reacted at room temperature for 60 minutes (both a negative and a positive control were set). After the reaction, antibody was added for detection, Envision detection was carried out after 60 minutes incubation at room temperature, and data was collected. Data analysis and fitting were made according to XLfit5 software. Compounds of the present disclosure were tested in the above kinase inhibition assay, and it is found that the compounds of the present disclosure have potent activity against ABL, ABLT315I, Ret and RETV804M. |
| Affinity data for this assay | |
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