| Assay Method Information | |
| | RET Kinase Assay |
| Description: | The experimental steps are as follows:(1) The test compound (the compound of the present disclosure, and compound 164 in WO2018017983A1 as a control) was dissolved in 100% DMSO to a final concentration of 10 mM.(2) 4 μL of the test compound solution prepared in step (1) was dissolved with 46 μL of 100% DMSO, and the solution obtained in this step was numbered as No. 2.(3) No. 2 solution was subjected to subsequent gradient dilution with a dilution factor of 5 times (i.e. 20 μL of 100% DMSO was added to 5 μL of the compound), a total of 9 gradients, numbered 3 to 11.Note: No. 2 was not used for the dilution in step (4).(Unless otherwise specified, the following steps need to be carried out on ice)(4) The buffer provided in the kit (Cisbio, Cat. No. 62TK0PEB) was used to continuously serially dilute the solutions numbered from 3 to 11 with a dilution factor of 20 times (that is, adding 19 μL of buffer to the solutions numbered from 3 to 11). At this time, the final concentration range of the test compound in the system No. 3 to 11 was 3200 nM˜0.008 nM (9 gradients), and the final concentration of DMSO was 2%.(5) 9 compound solutions of gradient concentration in step (4) were added into a 384-well plate in order according to their concentration at 4 μL per well, and two duplicate wells were set.(6) 2 μL of human RET protein was added to each well and incubated on ice for 10 minutes.(7) 2 μL of ATP (Sigma #A7699) and 2 μL of biotinylated polypeptide substrate (Cisbio, Cat. No. 62TK0PEB) were added to each well to start the phosphorylation reaction, and incubated at 37° C. for half an hour.(8) 5 μL of anti-phosphotyrosine antibody coupled with europium compound (provided in the kit, Cat. No. 62TK0PEB) and 5 μL of streptavidin coupled with modified allophycocyanin XL665 (Cisbio, Cat. No. 62TK0PEB) were added to each well.(9) The plate was continued to incubate for 1 hour at room temperature. After the incubation, the TF-FRET mode of the microplate reader (BMG Labtech, model: FLUOStar Omega) was adopted to measure the fluorescence intensity at an excitation wavelength of 304 nM and emission wavelengths of 615 nM and 665 nM in each well. The ratio would be calculated automatically.(10) By comparing the fluorescence intensity ratio in the control group, the inhibition rate of the compound at each concentration was calculated, and GraphPad Prism 5 was used to perform curve fitting with logarithmic concentration-inhibition rate to calculate the IC50 value of the compound. |
| Affinity data for this assay | |
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