| Assay Method Information | |
| | In-Vitro Inhibition of CDK Kinase Activity Assay |
| Description: | This experiment was used for determining the inhibition of the activities of CDK2, CDK7, CDK9 and CDK12 kinases by the compound. The kinase reaction carried out by the present invention was measured in a 384-well plate, the final measured volume was 16 μl, and the reaction temperature was 27° C. The concentrations of the kinases were determined by optimization experiment. The specific experimental process was as follows:1) preparation of kinase solutions:kinase solution (CDK2/Cyclin E1): the kinase was diluted in a assay buffer solution ((20 mM MES pH 6.75, 0.01% Tween 20, 0.05 mg/mL BSA, 2 mM MgCl2) to obtain an enzyme solution with the corresponding 2.4× concentration;kinase solution (CDK7/Cyclin H/MAT1): the kinase was diluted in the assay buffer solution (20 mM MES pH 6.75, 0.01% Tween 20, 0.05 mg/mL BSA, 6 mM MgCl2) to obtain an enzyme solution with the corresponding 2.4× concentration;kinase solution (CDK9/Cyclin T1): the kinase was diluted in the assay buffer solution (20 mM MES pH 6.75, 0.01% Tween 20, 0.05 mg/mL BSA, 10 mM MgCl2) to obtain an enzyme solution with the corresponding 2.4× concentration; andkinase solution (CDK12/Cyclin K): the kinase was diluted in a assay buffer solution (80 mM MES pH 6.5, 0.01% Tween 20, 0.05 mg/mL BSA, 10 mM MgCl2) to obtain an enzyme solution with the corresponding 2.4× concentration.2) Preparation of compound solutions: the compound was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM, the compound was diluted to 10 concentration gradients of 25 nM to 500 μM with DMSO during use, diluted in ultrapure water by 8.3 times respectively to obtain compound solutions with 6× concentration.3) Preparation of a polypeptide substrate and ATP solution: the polypeptide substrate and ATP were diluted in the assay buffer solution to obtain a polypeptide substrate and ATP mixed solution with 2.4× concentration.4) Kinase reaction process:2 ul of the test compound solution, 5 ul of the polypeptide substrate and ATP mixed solution and 5 ul of an enzyme solution were mixed and incubated at 27° C. (CDK2 was incubated for 60 min, CDK7 was incubated for 70 min, CDK9 was incubated for 70 min, and CDK12 was incubated for 280 min), and then 4 ul of EDTA with a concentration of 150 mM was added into each sample to terminate the reaction. The assay buffer solution containing 20 μM of staurosporine was used for replacing the compound solution as 100% inhibition, and DMSO was used for replacing the compound solution as 0% inhibition. Each test had at least two parallel controls. Final concentration of a reagent in CDK2 assay: ATP was 100 μM, the polypeptide substrate (5-FAM-YSPTSPSYSPTSPSYSPT SPSKKKK) was 2 μM, and CDK2/Cyclin E1 was 0.5 nM; final concentration of a reagent in CDK7 assay: ATP was 50 μM, the polypeptide substrate (5-FAM-YSPTSPSYSPTSPSYSPT SPSKKKK) was 2 μM, and CDK7/Cyclin H/MAT1 was 3 nM; final concentration of a reagent in CDK9 assay: ATP was 50 μM, the polypeptide substrate (FITC-Ahx-GSRTPMY-NH2) was 2 μM, and CDK9/Cyclin T1 was 8 nM; and final concentration of a reagent in CDK12 assay: ATP was 30 μM, the polypeptide substrate (FITC-Ahx-GSRTPMY-NH2) was 2 μM, and CDK12/Cyclin K was 50 nM.5) Data calculation and analysis: electrophoretic separation was carried out on a fluorescent substrate and a phosphorylated product on a Caliper EZ Reader II to analyze a reaction mixture. The data was calculated with GraphPad Prism version 9.0, and an IC50 value was obtained by adjusting a nonlinear regression model using a dose reaction curve. The calculation formula was Y=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((LogIC50−X)*HillSlope)). X represents a log value (log of dose or concentration); Y represents an inhibition rate (% inhibition, increasing as x increases) which was increased along with the increase of X; Top represents maximum response; Bottom represents baseline response; and HillSlope represents a curve slope. Due to the limitation of the detection lower limit of a CDK7 |
| Affinity data for this assay | |
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