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Author Spotlight: Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes
Published on: January 12, 2024
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Single-Assay Characterization of Ternary Complex Assembly and Activity in Targeted Protein Degradation.
Corey H Yu1, Vi Dougherty1, Dongwen Lv2,3
1Department of Biochemistry and Structural Biology, The University of Texas Health at San Antonio, San Antonio, TX 78229, USA.
Biorxiv : the Preprint Server for Biology
|September 2, 2025
Summary
A new fluorescent assay enables real-time monitoring of ubiquitination kinetics for targeted protein degradation (TPD). This tool accelerates the discovery and optimization of novel therapeutic degraders and molecular glues.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Targeted protein degradation (TPD) is a key therapeutic strategy leveraging cellular machinery to eliminate disease proteins.
- Quantifying ubiquitination kinetics, crucial for TPD, has been limited by available analytical tools.
Purpose of the Study:
- To develop a real-time, high-throughput fluorescent assay for monitoring ubiquitination kinetics.
- To enable quantitative analysis of crucial degrader characteristics and accelerate drug discovery.
Main Methods:
- Utilized purified, FRET-active E2-Ub conjugates for a real-time fluorescent assay.
- Monitored ubiquitin transfer in a single-step, single-turnover reaction.
- Applied the assay to measure degrader affinity, ternary complex assembly, and catalytic efficiency.
Main Results:
- The assay requires no target protein or ligase engineering, minimizing artifacts.
- Demonstrated accurate measurement of key parameters for heterobifunctional degraders and molecular glues.
- Achieved high sensitivity and accuracy in characterizing the ternary complex.
Conclusions:
- The developed assay provides a versatile and quantitative method for analyzing ubiquitination kinetics.
- This tool significantly enhances the discovery and optimization of targeted protein degraders.
- Empowers researchers with a comprehensive approach to characterizing molecular glue and PROTAC-like degraders.
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