Identification of false positives in a HTRF screen for inhibitors of programmed cell death protein 1 and its ligand

Abstract: The programmed cell death protein 1 (PD-1) receptor (CD279), expressed on the surface of activated T cells, and its ligand PD-L1 (CD274), expressed on the surface of dendritic cells or macrophages, are important targets in multiple cancers. The PD-1 and PD-L1 interaction functions as an immune checkpoint that helps maintain immune homeostasis and inhibits autoimmunity. In cancer, immune checkpoint mechanisms often suppress the nascent anti-tumor immune response. Blocking the PD-1 - PD-L1 interaction is promising for cancer immunotherapies. Monoclonal antibodies (e. g. rituximab, nivolumab) have been approved for treatment of various cancers. However, to date there have been no approved small molecule PD-1/PD-L1 inhibitors. Small molecules have advantages over monoclonal antibodies, including simpler manufacturing, more stability and reduced likelihood of immunogenicity. A TR-FRET PD-1/PD-L1 interaction assay was developed to screen small molecules from the BioAscent diversity library for inhibitors that are able to block the PD-1/PD-L1 interaction and serve as suitable starting points for drug development. False positives were identified based on 2 facts: 1. Compounds binding to the PD-1/PD-L1 interaction surface should take some time to reach a new equilibrium. 2. Genuine positives should only affect FRET values. Identification of false positives allowed the over 5000 library compounds to selected down to 5 candidates for further evaluation saving time and money that might have been expended characterizing false positives.