CHEMBL5738469

Inhibitory Activity Assay: The tested compounds and the reference compounds are dissolved in DMSO to prepare a high concentration of storage solution. The stock solution of reference compound was diluted with DMSO to prepare a 100× solution. In the first column of the working plate, 8 μL above tested compounds and 8 μL 100× reference compound were respectively added as the highest concentration, and then the highest concentrations were subjected to three times dilution to obtain 11 concentrations and prepare 100× solution. 0.5 μL solution was transferred from the above plate to the detection plate. To each well was added 0.5 μL 100× compound solution. For HPE and ZPE control wells, 0.5 μL 100% DMSO was added.25 μL 2×IDO1 (His-tag) enzyme solution (containing L-ascorbic acid, methylene blue, and catalase) was added to each well. 25 μL reaction solution without IDO1 (His-tag) enzyme was added into HPE control well. The test plate was centrifugated at 1000 rpm for 1 minute to mix well. Then, the test plate was incubated at room temperature for 30 minutes. 25 μL above 2× substrate (L-tryptophan) solution was added to each well. The test plate was centrifugated at 1000 rpm for 1 minute to mix well. The detection plate was placed on ELISA (SpectraMax M5e), the temperature was set at 25° C., and the absorbance (OD value) was measured at 320 nm every 10 minutes till 60 minutes.