| Assay Method Information | |
| | IC50 Test of Compound YJZ5118 on CDK12, CDK13, and CDK9 Kinases |
| Description: | The inhibitory activities of the test compound on the kinases were evaluated by LANCE Ultra assay, which was used for detecting ATP-dependent phosphorylation of a ULight-4E-BP1 (Thr37/Thr46) substrate peptide (150 nM). In short, enzymatic reactions were performed in a reaction buffer (25 mM HEPES (pH 7.5), 10 mM MgCl2, 0.01% BSA, 0.01% Tween 20, and 1 mM DTT). The assay was performed in a 384-well plate (6 μL). A final concentration of an ATP substrate was 10 M, and a final concentration of the ULight-4E-BP1 (Thr37/Thr46) substrate peptide was 150 nM. A final concentration of CDK9, CDK12, or CDK13 was 0.3, 30, or 30 nM, respectively. Pre-incubation of the compound and the enzyme was performed at room temperature for 60 min. The reaction was stopped by adding 10 mM EDTA and 0.15 nM Eu-labeled anti-phospho-eIF4E-binding protein 1 (Thr37/46) antibody in LANCE detection buffer after incubation at room temperature for 15, 60, or 90 min. Time-resolved fluorescence (excitation, 320 nm; emission donor, 615 nm; emission acceptor, 665 nm) was monitored using an EnVision spectrophotometer (PerkinElmer). Readings were computed as (acceptor count/donor count)×1000. IC50 values were derived by fitting a sigmoidal dose-response curve to a plot of assay readouts of inhibitor concentrations. |
| Affinity data for this assay | |
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