Yu Corey H, Dougherty Vi, Lv Dongwen, Ebadi Pirouz, Dubey Shikha, Nayak Digant, Nayak Anindita, Zhou Daohong, Olsen Shaun K, Ivanov Dmitri N
Department of Biochemistry and Structural Biology, The University of Texas Health at San Antonio, San Antonio, TX 78229, USA.
Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, Augusta, Georgia, 30912, USA.
bioRxiv. 2025 Aug 22:2025.08.20.671298. doi: 10.1101/2025.08.20.671298.
Targeted protein degradation (TPD) is a rapidly advancing therapeutic strategy that selectively eliminates disease-associated proteins by co-opting the cell's 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.
靶向蛋白质降解（TPD）是一种快速发展的治疗策略，它通过利用细胞的蛋白质降解机制来选择性地清除与疾病相关的蛋白质。蛋白质与泛素的共价修饰是TPD中的关键事件，但用于量化泛素化动力学的分析工具一直很有限。在此，我们展示了一种实时、高通量荧光测定法，该方法利用纯化的、具有荧光共振能量转移（FRET）活性的E2-泛素缀合物来监测泛素转移。该测定法具有高度通用性，无需对靶蛋白或连接酶进行工程改造，从而加速测定法的开发并将产生假象的风险降至最低。所监测反应的单步、单周转性质能够对泛素化动力学进行严格且定量的分析。我们表明，该测定法可用于测量关键降解剂特性，如降解剂对靶蛋白的亲和力、降解剂对连接酶的亲和力、三元复合物组装的亲和力以及三元复合物的催化效率。这种用于三元复合物表征的全面、单测定法的高灵敏度和准确性将助力异双功能降解剂和分子胶的发现与优化。
