| Assay Method Information | |
| | Autotaxin (ATX) Enzyme Activity Inhibition Assay |
| Description: | The inhibitory activities of the compounds on the autotaxin enzyme were detected using the Autotaxin Inhibitor Screening Assay Kit (Cayman, 700580). First, the test compound was prepared as a 10 mM stock solution in DMSO solvent, and then the stock solution was diluted with DMSO to 8 gradient concentrations. Subsequently, the 8 concentrations were diluted with Autotaxin Assay buffer (1×) provided in the kit into 19× compound working solutions (DMSO content was 1.9%). The Autotaxin Assay Reagent (10×) was taken out and diluted by 10 times with Autotaxin Assay Buffer (1×). The Autotaxin Substrate was taken out, dissolved by adding 1.2 mL of Autotaxin Assay Buffer (1×), mixed evenly and stood at room temperature. In a 96-well plate, 150 μL of Autotaxin Assay Buffer (1×), 10 μL of the prepared and diluted 19× compound working solutions, 10 μL of Autotaxin Assay Reagent (1×), and 20 μL of the dissolved Autotaxin Substrate were added to each of the wells for each concentration, and homogenously mixed. The 96-well plate was shaken in a constant temperature shaker at 37° C. and incubated in the dark for 30 min, and then the plate was taken out and placed on a microplate reader to read OD405. The experimental results were input into GraphPad Prism software, and the IC50 of each compound was calculated by fitting. |
| Affinity data for this assay | |
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