| Assay Method Information | |
| | Inhibitory Activity of Compounds against RIPK1 Enzyme |
| Description: | The compound to be tested was dissolved in DMSO to prepare a 10 mM stock solution, diluted 3.16 times with DMSO into a series of concentration gradients, and then MOPS pH 7.0 buffer solution was used to dilute 50 times to prepare a working solution, and mixed well with 36 nM RIPK1 (final concentration), 0.33 mg/ml substrate MBP. Afterwards, 10 mM magnesium ions and 155 μM phosphorus 33 isotope-labeled ATP were added to react, The final concentration of DMSO was 2%. After the reaction was proceeded at room temperature for 2 hours, phosphoric acid was added to terminate. The final reaction system was processed and detected using a liquid scintillation counter. After the test results are subtracted from the blank control and compared with the reading value of the control group to convert to the activity percentage, which was plotted as a curve with the final concentration of the corresponding compound, and four-parameter fitting was used to obtain the IC50 of the compound that inhibits RIPK1 enzyme activity. It can be seen from the experimental results that the exemplary compounds of the present invention possessed high inhibitory activity against RIPK1, with an IC50 value of less than 200 nM (for example, 0.1 nM to 200 nM); IC50 values of some compounds were even less than 100 nM (for example. 0.1 nM to 100 nM) or less than 50 nM (for example, 0.1 nM to 50 nM). |
| Affinity data for this assay | |
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