| Assay Method Information | |
| | Inhibitory Activity Assay |
| Description: | In this experiment, an ADP-Glo kit was used to detect the inhibitory activity of the compounds against KIF18A. Each compound was prepared into a 10 mM stock solution with DMSO, which was then diluted at 1:5 to obtain 9 gradients with the initial final concentration set at 10 μM. Meanwhile, a control group and a blank group were also set up. The reaction buffer consisted of 15 mM Tris (pH 7.5), 10 mM MgCl2, and 0.01% Pluronic F-68. 4 nM KIF18A (1467) and the test compound were added to a 384-well plate and incubated at 37° C. for 20 mm. Then, 30 μM ATP was added to initiate the enzymatic reaction, and the plate was incubated at room temperature for 15 min. Finally, the ADP-Glo reagent was added to detect the luminescence intensity, where the luminescence intensity is directly proportional to the ADP content. Inhibition rate (%)=(1−Sample signal)/DMSO signal×100. Sample signal is the value of the compound group, and DMSO signal is the value of the DMSO control group. Then, the inhibition rate at each concentration was calculated, and the IC50 was determined by fitting using the GraphPadPrism 7.0 software. |
| Affinity data for this assay | |
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