Inhibition of PD-1/PD-L1 interaction was tested for 4 high and low potent predicted compounds using the PD1/PD-L1 HTRF assay kit from Cisbio US, Inc. The assay protocol was used as mentioned in the kit for each predicted compound (4b, 4c, 4d and 4e) and the BMS control compound (4a). Briefly, 2 µL of the compound, 4 µL from a 25 nM Tag1-PD-L1 protein solution and 4 µL from a 250 nM Tag2-PD1 protein were added into a Cisbio's HTRF 96-well low volume white plate. Then, the plate was incubated for 15 minutes at room temperature. Next, 10 µL from pre-mixed anti-tag detection reagents (5 µL from 1X anti-Tag1-Eu 3+ and 5 µL from 1X anti-Tag2-XL665) were added and the sealed plate was incubated for 2 hours at room temperature. Finally, the plate sealer was removed, and measurements were taken using a HTRF ® compatible reader.