Covalent Ligand Screening Uncovers a RNF4 E3 Ligase Recruiter for Targeted Protein Degradation Applications
Covalent Ligand Screening Uncovers a RNF4 E3 Ligase Recruiter for Targeted Protein Degradation Applications
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DOI：
10.1021/acschembio.8b01083
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发表时间：
2019-11-01
影响因子：
4
通讯作者：
Nomura, Daniel K.
中科院分区：
文献类型：
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作者：
Ward, Carl C.;Kleinman, Jordan I.;Nomura, Daniel K.
靶向蛋白质降解已经成为一种强大的药物发现策略，可以将不可药物的蛋白质作为蛋白质酶体降解的靶点。这种方法通常使用异双功能降解物，包括连接到E3连接酶招募者的蛋白质靶向配体，以泛素化和标记蛋白酶体降解感兴趣的蛋白质。然而，这种方法的一个挑战是，尽管人类基因组中有数百种已知的E3连接酶，但目前只有几个E3连接酶招募者存在于有针对性的蛋白质降解应用中。在这里，我们利用基于活性的蛋白质图谱(ABPP)的共价配体筛选方法来识别与E3泛素连接酶RNF4反应的半胱氨酸反应性小分子，并为基于RNF4的降解物的设计提供化学起点。此筛选中的HIT共价配体与环区的两个锌配位半胱氨酸C132和C135中的任何一个反应，对RNF4的活性没有影响。我们进一步优化了这种HIT的效力，并将这个潜在的RNF4招募者整合到与BET溴域蛋白家族的抑制剂JQ1相连的双功能降解物中。我们证明了得到的化合物CCW 28-3能够以蛋白酶体和RNF4依赖的方式降解BRD4。在这项研究中，我们已经证明了使用化学蛋白质组学启用的共价配体筛选平台来扩大E3连接酶招募者的范围的可行性，这些募集者可以被用于靶向的蛋白质降解应用。
Targeted protein degradation has arisen as a powerful strategy for drug discovery allowing the targeting of undruggable proteins for proteasomal degradation. This approach most often employs heterobifunctional degraders consisting of a protein-targeting ligand linked to an E3 ligase recruiter to ubiquitinate and mark proteins of interest for proteasomal degradation. One challenge with this approach, however, is that only a few E3 ligase recruiters currently exist for targeted protein degradation applications, despite the hundreds of known E3 ligases in the human genome. Here, we utilized activity-based protein profiling (ABPP)-based covalent ligand screening approaches to identify cysteine-reactive small-molecules that react with the E3 ubiquitin ligase RNF4 and provide chemical starting points for the design of RNF4-based degraders. The hit covalent ligand from this screen reacted with either of two zinc-coordinating cysteines in the RING domain, C132 and C135, with no effect on RNF4 activity. We further optimized the potency of this hit and incorporated this potential RNF4 recruiter into a bifunctional degrader linked to JQ1, an inhibitor of the BET family of bromodomain proteins. We demonstrate that the resulting compound CCW 28-3 is capable of degrading BRD4 in a proteasome- and RNF4-dependent manner. In this study, we have shown the feasibility of using chemoproteomics-enabled covalent ligand screening platforms to expand the scope of E3 ligase recruiters that can be exploited for targeted protein degradation applications.