A real-time FRET ubiquitin transfer assay for quantitative characterization of ternary complexes in targeted protein...

Inhibiting or redirecting E3 ubiquitin ligase activity with drug-like small molecules is of growing therapeutic interest, yet broadly applicable and robust assays for quantifying E3-catalyzed ubiquitination remain limited. Here, we present a real-time, high-throughput fluorescence assay utilizing purified, FRET-active E2-Ub conjugates to monitor ubiquitin transfer. This assay is highly versatile, requiring no engineering of the target protein or ligase, thereby accelerating assay development and minimizing the risk of artifacts. The single-step, single-turnover nature of the reaction enables rigorous and quantitative analysis of ubiquitination kinetics. We show that this assay can be used to measure key degrader characteristics, including degrader affinity for the target protein, degrader affinity for the ligase, affinity of ternary complex assembly, and catalytic efficiency of the ternary complex. The high sensitivity and accuracy of this comprehensive, single-assay approach to ternary complex characterization will empower the discovery and optimization of heterobifunctional degraders and molecular glues.