CHEMBL5738546

PD1-PDL1 HTRF Binding Activity Test: The effect of compounds and positive compounds of the examples of the present invention on the interaction between PD-1 and PD-L1 was determined by the PD-1/PD-L1 binding assay kit from Cisbio (#64ICP01PEG or 64ICP01PEH). The detailed experimental process was as follows:1. Pre-diluted compound solution, 4 μL of Tag1-PD-L1 and 4 μL of Tag2-PD1 were added to each well of a 384-well plate;2. After the mixture was incubated at room temperature for 15 min, 5 μL of anti-Tag1-Eu3+ antibody and 5 μL of anti-Tag2-XL665 antibody were then added;3. After incubated for 2 hrs at room temperature or overnight at 4° C., plates were read on Envision of Pelkin Elmer; and readings at 665 nm and 620 nm were recorded, and the ratio of the two readings was taken as a reading for each well;4. The reading of each well after compound treatment was compared with the reading of DMSO treated wells to obtain the percent inhibition of the compound; and5. IC50 values of compounds and positive compounds of the examples of the present invention were determined by non-linear regression analysis of percent inhibition at different compound concentrations.