关键词: HiBiT NanoBiT PROTAC Targeted Protein Degradation WDR5 degradation kinetics degrader kinetics live cells screening

Mesh : Proteolysis Proteins / metabolism Ubiquitin-Protein Ligases / metabolism

来  源:   DOI:10.1016/j.chembiol.2023.06.002

Abstract:
The multi-step degradation process of PROteolysis TArgeting Chimeras (PROTACs) poses a challenge for their rational development, as the rate-limiting steps that determine PROTACs efficiency remain largely unknown. Moreover, the slow throughput of currently used endpoint assays does not allow the comprehensive analysis of larger series of PROTACs. Here, we developed cell-based assays using the NanoLuciferase and HaloTag that allow measuring PROTAC-induced degradation and ternary complex formation kinetics and stability in cells. Using PROTACs developed for the degradation of WD40 repeat domain protein 5 (WDR5), the characterization of the mode of action of these PROTACs in the early degradation cascade revealed a key role of ternary complex formation and stability. Comparing a series of ternary complex crystal structures highlighted the importance of an efficient E3-target interface for ternary complex stability. The developed assays outline a strategy for the rational optimization of PROTACs using a series of live cell assays monitoring key steps of the early PROTAC-induced degradation pathway.
摘要:
多步降解过程的ProteesolutionTogeting嵌合体(PROTACs)对其合理发展提出了挑战,因为决定PROTACs效率的限速步骤在很大程度上仍然未知。此外,当前使用的终点测定的通量缓慢,不允许对更大系列的PROTACs进行全面分析.这里,我们使用NanoLuciferase和HaloTag开发了基于细胞的测定法,该测定法允许测量PROTAC诱导的降解和三元复合物形成动力学以及细胞中的稳定性。使用开发用于降解WD40重复结构域蛋白5(WDR5)的PROTACs,这些PROTACs在早期降解级联中的作用模式的表征揭示了三元复合物形成和稳定性的关键作用。比较一系列三元络合物晶体结构强调了有效的E3-靶界面对于三元络合物稳定性的重要性。开发的测定法概述了使用一系列活细胞测定法监测早期PROTAC诱导的降解途径的关键步骤的合理优化PROTAC的策略。
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