Sweet and Blind Spots in E3 Ligase Ligand Space Revealed by a Thermophoresis-Based Assay

Repurposing E3 ubiquitin ligases for targeted protein degradation via customized Molecular Glues or proteolysis-targeting chimeras (PROTACs) is an increasingly important therapeutic modality. Currently, a major limitation in the design of suitable Molecular Glues and PROTACs is our fragmentary understanding of E3 Ligases and their ligand space. We here describe a quantitative assay for the discovery and characterization of E3 Ligase ligands that is based on the thermophoretic behavior of a custom reporter ligand. Thereby, it is orthogonal to commonly employed fluorescence-based assays and less affected by the optical properties of test compounds. It can be employed for the high-throughput screening of compound libraries for a given Ligase but also for hit validation, which we demonstrate with the identification of unexpected well-binders and non-binders, yielding new insights into the ligand space of Cereblon (CRBN).