| Assay Method Information | |
| | PD-1/PD-L1 Binding Assay |
| Description: | Binding assays were conducted in a low volume white 384-well polystyrene plate in a final volume of 20 μL. Compounds to be analyzed were first serially diluted in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1%. The assay was carried out at 25° C. in the PBS buffer (pH 7.4) with 0.05% Tween-20 and 0.1% BSA. Recombinant human PD-L1 protein (19-238) with a His-tag at the C-terminus was purchased from AcroBiosystems (PD1-H5229). Recombinant human PD-1 protein (25-167) with Fc tag at the C-terminus was also from AcroBiosystems (PD1-H5257). PD-L1 and PD-1 proteins were diluted in the assay buffer (final concentration −0.67 and 0.20 nM respectively) and 10 μL was added to the plate well. Plates were centrifuged and proteins were preincubated with compounds for 40 minutes. The incubation was followed by the addition of 10 μL of assay buffer supplemented with Alphascreen Ni chelate donor beads (PerkinElmer-AS101D) and Protein A Acceptor beads (PerkinElmer-6760137) at final concentration 2.5 μg/mL under reduced light. After plate sealing, the plate was incubated in the dark at room temperature for 120 minutes before reading on a PHERAstar FS plate reader (BMG Labtech). IC50 determination was performed by fitting the curve of percent control activity versus the log of the compound concentration using the GraphPad Prism 5.0 software. |
| Affinity data for this assay | |
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