Institut für Pharmazeutische Chemie, Goethe-University Frankfurt, Biozentrum, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany; Structural Genomics Consortium, Goethe-University Frankfurt, Buchmann Institute for Life Sciences, Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany.
Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences, Oncological Sciences and Neuroscience, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
Cell Chem Biol. 2023 Jul 20;30(7):753-765.e8. doi: 10.1016/j.chembiol.2023.06.002. Epub 2023 Jun 23.
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.
蛋白水解靶向嵌合体（PROTACs）的多步降解过程对其合理开发构成了挑战，因为决定 PROTACs 效率的限速步骤在很大程度上仍然未知。此外，目前使用的终点测定法的缓慢通量不允许对更大系列的 PROTACs 进行全面分析。在这里，我们使用 NanoLuciferase 和 HaloTag 开发了基于细胞的测定法，这些测定法可用于测量 PROTAC 诱导的降解以及三元复合物形成的动力学和稳定性。使用针对 WD40 重复结构域蛋白 5（WDR5）降解开发的 PROTACs，对这些 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。比较一系列三元复合物晶体结构突出了有效 E3-靶接口对于三元复合物稳定性的重要性。所开发的测定法概述了一种使用一系列监测早期 PROTAC 诱导降解途径关键步骤的活细胞测定法来合理优化 PROTACs 的策略。
