| Assay Method Information | |
| | Enzyme Activity Test |
| Description: | The activity of the compounds prepared in Examples against wild-type RET kinase was screened using the Kinase activity Assay method at ATP Km concentration, and staurosporine was used as a reference substance. The biological activity screening of the compounds will be determined repeatedly at 10 concentrations.1. Sample to be TestedEach sample was prepared into a solution with a concentration of 10 mM.2. Experimental MethodI. Preparing Basic Buffer Solution and Quenching Buffer Solution for Experimental Kinase20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO.II. Preparing Compounds for Experimental KinaseThe test compound was dissolved in 100% dimethyl sulfoxide to a specific concentration. Integra Viaflo Assist was used to assist DMSO for (continuous) dilution. III. Reaction StepsKinase was added to the newly prepared basic reaction bufferAdding any desired cofactors to the substrate solution.Adding wild-type RET kinase into the substrate solution and gently mixing;Acoustic technology (Echo 550; Nanoliter range) was used to feed the compounds in 100% of dimethyl sulfoxide into the kinase reaction mixture and incubated at room temperature for 20 minutes.33P-ATP (specific activity 10 Ci/l) was added to the reaction mixture to start the reaction.Incubated at room temperature for 2 hoursRadioactivity was detected by filter-binding method.Kinase activity data was expressed as the percentage of remaining kinase activity in the test sample compared to the vehicle (dimethyl sulfoxide) reaction. Prism (GRAPHPAD software) was used to obtain IC50 value and curve-fitting. |
| Affinity data for this assay | |
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