High throughput E3 ligase degron binding assays for novel PROTAC ligand discovery

The discovery of new small molecule ligands for E3 ligases will enable the creation of novel proteolysis targeting chimeras (PROTACs) and molecular glues to tackle traditionally undruggable proteins. Diversifying both the chemical matter for each E3 ligase and the type of ligases will be important to fully capture the potential of these targeted protein degradation modalities. A key step in this process is to establish an integrated screening platform for the rapid identification and optimization of small molecule ligands for E3 ligases. Here, we provide a method to evaluate E3 ligase ligands using AlphaScreen technology. AlphaScreen allows for the evaluation of a wide array of molecular interactions and is utilized extensively in small molecule screening campaigns. This bead-based proximity technology offers facile development for interactions across a wide range of affinities and can be adapted to interrogate E3 ligase-degron interactions. In this protocol, we demonstrate the development of AlphaScreen for E3 ligase ligand competition assays toward the discovery of new ligands for E3 ligases.