Beyond Binding Affinity: Detailed Profiling of Protein–Ligand Interactions with Time-Resolved FRET

Discovering new E3 ligase ligands and accurate characterizations of the critical binary interactions between these ligands and the ligases are fundamental to the rational design of new E3 ligase ligands and the expansion of proteolysis-targeting chimeras. Herein, we developed a time-resolved Förster resonance energy transfer (TR-FRET) platform for multiparameter profiling of protein-ligand interactions to access previously obscured insights from conventional affinity-based assays. This platform was employed to investigate interactions ranging from model systems (HaloTag and VHL) to novel E3 ligase CDC20 with Apcin. From the multiexponential fitting of photoluminescence decays, key parameters (distinct FRET subpopulations, their amplitudes, efficiencies, and distances) directly report the protein binding site occupancy (or fraction of bound ligand) as well as potential alterations in binding geometry or molecular orientation. The heatmap visualization further converts complex binding data into an intuitive format, providing medicinal chemists with a practical and expandable tool to guide ligand optimization across diverse target proteins and E3 ligases.