Promega Corporation , 2800 Woods Hollow Road , Madison , Wisconsin 53711 , United States.
Promega Biosciences Incorporated , 277 Granada Drive , San Luis Obispo , California 93401 , United States.
ACS Chem Biol. 2018 Sep 21;13(9):2758-2770. doi: 10.1021/acschembio.8b00692. Epub 2018 Aug 30.
A new generation of heterobifunctional small molecules, termed proteolysis targeting chimeras (PROTACs), targets proteins for degradation through recruitment to E3 ligases and holds significant therapeutic potential. Despite numerous successful examples, PROTAC small molecule development remains laborious and unpredictable, involving testing compounds for end-point degradation activity at fixed times and concentrations without resolving or optimizing for the important biological steps required for the process. Given the complexity of the ubiquitin proteasomal pathway, technologies that enable real-time characterization of PROTAC efficacy and mechanism of action are critical for accelerating compound development, profiling, and improving guidance of chemical structure-activity relationship. Here, we present an innovative, modular live-cell platform utilizing endogenous tagging technologies and apply it to monitoring PROTAC-mediated degradation of the bromodomain and extra-terminal family members. We show comprehensive real-time degradation and recovery profiles for each target, precisely quantifying degradation rates, maximal levels of degradation ( D), and time frame at D. These degradation metrics show specific PROTAC and family member-dependent responses that are closely associated with the key cellular protein interactions required for the process. Kinetic studies show cellular ternary complex stability influences potency and degradation efficacy. Meanwhile, the level of ubiquitination is highly correlated to degradation rate, indicating ubiquitination stemming from productive ternary complex formation is the main driver of the degradation rate. The approaches applied here highlight the steps at which the choice of E3 ligase handle can elicit different outcomes and discern individual parameters required for degradation, ultimately enabling chemical design strategies and rank ordering of potential therapeutic compounds.
新一代的杂双功能小分子，称为蛋白水解靶向嵌合体（PROTACs），通过募集到 E3 连接酶来靶向蛋白质降解，具有显著的治疗潜力。尽管有许多成功的例子，但 PROTAC 小分子的开发仍然是费力和不可预测的，需要在固定的时间和浓度下测试化合物的终点降解活性，而不解决或优化该过程所需的重要生物学步骤。鉴于泛素蛋白酶体途径的复杂性，能够实时表征 PROTAC 功效和作用机制的技术对于加速化合物的开发、分析和改善化学结构-活性关系的指导至关重要。在这里，我们提出了一种创新的、模块化的活细胞平台，利用内源性标记技术，并将其应用于监测溴结构域和额外末端家族成员的 PROTAC 介导的降解。我们展示了每个靶标的全面实时降解和恢复谱，精确地量化了降解率、最大降解水平（D）和 D 时间框架。这些降解指标显示出特定的 PROTAC 和家族成员依赖性反应，与该过程所需的关键细胞蛋白相互作用密切相关。动力学研究表明，细胞三元复合物的稳定性影响效力和降解效果。同时，泛素化水平与降解速率高度相关，表明源自有效三元复合物形成的泛素化是降解速率的主要驱动因素。这里应用的方法突出了选择 E3 连接酶处理的步骤可以产生不同的结果，并辨别降解所需的单个参数，最终使化学设计策略和潜在治疗化合物的排序成为可能。
