| Assay Method Information | |
| | PD-1/PD-L1 Binding Assay (Alphascreen) |
| Description: | The assays were conducted in a standard black 384-well polystyrene plate with a final volume of 20 μL. Inhibitors were first serially diluted in DMSO and then added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1%. Add 100 nL/well of compound to the 384 reaction plate (6008280, PerkinElmer) with Echo and centrifuge at 1000 rpm for 1 minute. Add 5 μL/well 4×PD-L1 solutions to the 384 reaction plate, centrifuge at 1000 rpm for 1 minute, and add 5 μL/well 4×PD-1 solutions, centrifuge at 1000 rpm for 1 minute, and incubate at 25° C. for 15 minutes. The concentrations of the compounds were 300, 100, 33.33, 11.11, 3.70, 1.23, 0.41, 0.137, 0.046, 0.015, 0 nM, respectively. Add 10 μL/well 2× Anti-6×His AlphaLISA Acceptor beads and Streptavidin Donor beads solution (PerkinElmer-AL356F) to the above 384 reaction plate, centrifuge at 1000 rpm for 1 minute, and incubate at 25° C. in the dark for 120 minutes. Read the AlphaLISA signal value using the Envision Reader. IC50 determination was performed by fitting the curve of percent control activity versus the log of the inhibitor concentration using the GraphPad Prism 8.0 software. |
| Affinity data for this assay | |
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