An In Vitro Pull-down Assay of the E3 Ligase:PROTAC:Substrate Ternary Complex to Identify Effective PROTACs.
An In Vitro Pull-down Assay of the E3 Ligase:PROTAC:Substrate Ternary Complex to Identify Effective PROTACs.
复制标题
E3 连接酶：PROTAC：底物三元复合物的体外下拉测定可识别有效的 PROTAC。
DOI：
10.1007/978-1-0716-1665-9_7
复制
发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Buhimschi,AlexandruD
中科院分区：
文献类型：
--
作者：
Bondeson,DanielP;Smith,BlakeE;Buhimschi,AlexandruD
评估 PROTAC 的特异性并确认其拟议的作用机制对于稳健的靶向蛋白质降解计划至关重要。由于其新颖的机制，需要新的测定方法来实现这些目标。我们和其他人已经证明，PROTAC 功效的常见解释是 PROTAC 在 E3 泛素连接酶和靶蛋白之间形成三元复合物的能力。在本章中，我们提供了一种简单的体外方法来快速且廉价地评估 PROTAC 分子的这一特性。我们提供了特定 E3 泛素连接酶 VHL 纯化的详细说明，以及可适用于任何 E3 连接酶和底物蛋白组合的通用方案。这种研究三元复合物的简便方法可以加强任何以 PROTAC 为重点的药物化学工作。
Assessing the specificity of PROTACs and confirming their proposed mechanism of action are critical for a robust targeted protein degradation program. Owing to their novel mechanism, new assays are needed to meet these goals. We and others have shown that a common explanation of PROTAC efficacy is the ability of the PROTAC to form a ternary complex between the E3 ubiquitin ligase and the target protein. In this chapter, we provide a simple in vitro method to quickly and inexpensively assess this property of PROTAC molecules. We provide detailed instructions for the purification of the specific E3 ubiquitin ligase VHL and then a generic protocol which can be adapted to any E3 ligase and substrate protein combination. This accessible method to study the ternary complex can strengthen any PROTAC-focused medicinal chemistry effort.