Bridging E3 Ligase Binding and Targeted Degradation Through Fluorescence Polarization.

Despite the rapid expansion of targeted protein degradation (TPD), early characterization of PROTACs and molecular glues still relies predominantly on cellular degradation assays, making it difficult to determine whether poor degradation results from suboptimal E3 ligase engagement or from subsequent steps in the degradation mechanism. Moreover, productive degradation depends on balanced binary interactions, as both insufficient and excessively strong binding to either the E3 ligase or the protein of interest (POI) can impair degrader activity. Here, we report robust fluorescence polarization (FP) assays for the quantitative determination of ligand binding to the two most widely exploited E3 ligases, cereblon (CRBN) and von Hippel-Lindau (VHL). Using proprietary fluorescent tracers, the assays were validated with chemically diverse molecular glues, clinically relevant PROTACs, and canonical ligands, yielding affinity values in excellent agreement with published data. Both assays were successfully miniaturized to 384-well format while maintaining analytical performance and subsequently implemented in standardized ready-to-use formats, facilitating reproducible affinity measurements across laboratories. These assays establish a rapid and accessible binding-first platform that complements cellular degradation studies; supports rational degrader discovery, profiling, and optimization; and enables the identification of structurally novel E3 ligase recruiters.