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bioRxiv · 10.1101/2025.08.20.671298

Single-Assay Characterization of Ternary Complex Assembly and Activity in Targeted Protein Degradation

Yu, C. H.·Dougherty, V.·Lv, D.·Ebadi, P.·Dubey, S.·Nayak, D.·Nayak, A.·Zhou, D.·Olsen, S. K.·Ivanov, D. N.
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Abstract

Targeted protein degradation (TPD) is a rapidly advancing therapeutic strategy that selectively eliminates disease-associated proteins by co-opting the cells protein degradation machinery. Covalent modification of proteins with ubiquitin is a critical event in TPD, yet the analytical tools for quantifying the ubiquitination kinetics have been limited. Here, we present a real-time, high-throughput fluorescent 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 monitored reaction enables rigorous and quantitative analysis of ubiquitination kinetics. We show that this assay can be used to measure key degrader characteristics such as 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.

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Yu, C. H., Dougherty, V., Lv, D., Ebadi, P., Dubey, S., Nayak, D., Nayak, A., Zhou, D., Olsen, S. K., Ivanov, D. N.. 2025-08-22. Single-Assay Characterization of Ternary Complex Assembly and Activity in Targeted Protein Degradation. https://doi.org/10.1101/2025.08.20.671298

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