Chemical Proteomics Approach to Discover Novel Small Molecule E3 Ligase recruiters for Targeted Protein degradation (TPD)

项目摘要 靶向蛋白降解（TPD）是一种新兴的治疗方式，有可能克服 传统药理学抑制方法的局限性。TPD使用小分子（降解剂）劫持 通过募集E3泛素连接酶到感兴趣的蛋白质（POI）， 否则不被认为是基底。尽管TPD方法前景广阔，但主要瓶颈 仍然存在，包括：1）针对不可用药致病蛋白的配体发现。2)缺乏可用的 E3连接酶募集者可以用于TPD应用，因为E3连接酶的数量有限， 尽管存在>600种E3连接酶，但大多数已发表的降解剂使用cereblon和VHL的配体 在人类基因组中。因此，发现新的E3连接酶配体， 扩大新疗法的范围。最近，几个研究小组发现半胱氨酸反应性共价键 针对先前未靶向的E3连接酶的招募人员已经通过使用化学试剂成功地用于TPD中， 蛋白质组学方法虽然靶向半胱氨酸作为一个有用的起点，有必要考虑 其他类型的化学来产生共价E3募集物，用于靶向新的E3连接酶，以扩大 波士顿警局一种这样的可能性是靶向E3上潜在的亲核残基，例如赖氨酸、酪氨酸和丝氨酸 连接酶。然而，靶向赖氨酸、酪氨酸和丝氨酸的特异性生物相容性亲电体是不稳定的。 对于TPD应用来说，很大程度上是未开发的。本提案的最终目标是设计、综合和验证 新的E3连接酶配体，能够开发新的降解剂。为此，我们将使用 化学蛋白质组学策略，利用广泛的反应性，赖氨酸，酪氨酸和丝氨酸定向亲电 弹头偶联到细胞内蛋白的选择性配体（BRD 4的JQ 1或FKBP 12的SLF），以筛选 通过E3连接酶的共价加合作用的异双功能降解剂化合物。为此我们 已经成功开发了一系列化合物来促进配体诱导的蛋白质降解。这些 概念验证降解剂导致靶蛋白的诱导泛素化和蛋白酶体降解。我们 将进行进一步的化学蛋白质组学研究，以确定这些降解剂的E3连接酶靶点， 降解活性。通过我们的新方法，我们希望看到E3连接酶的全部功能被招募， 靶向蛋白质降解进一步富集，为下一代组织和疾病铺平道路， 特定的分子降解剂。

Project Summary Targeted protein degradation (TPD) is an emerging therapeutic modality with the potential to overcome limitations of traditional pharmacological inhibition approaches. TPD uses small molecules (degraders) to hijack the cellular degradation machinery by recruiting E3 ubiquitin ligases to proteins of interest (POI), which would not otherwise be recognized as substrate. Despite the tremendous promise of TPD approach, major bottlenecks still exist, including: 1) ligand discovery against undruggable disease-causing proteins. 2) a dearth of available E3 ligase recruiters that can be exploited for TPD applications, as there are limited number of E3 ligases, with most of the published degraders using ligands for cereblon and VHL, despite the existence of >600 E3 ligases in the human genome. Therefore, it is a great deal of interest in the discovering of new E3 ligase ligands to expand the scope of new therapeutics. Recently, several research groups discovered cysteine-reactive covalent recruiters against previously untargeted E3 ligases that have been successfully used in TPD by using chemical proteomic approaches. While targeting cysteines serves as a useful starting point, there is a need to consider other types of chemistry to generate covalent E3 recruiters for targeting new E3 ligases to expand the scope of TPD. One such possibility is to target potential nucleophilic residue, such as lysine, tyrosine, and serine on E3 ligases, respectively. However, specific biocompatible electrophiles targeting lysines, tyrosine, and serine are largely unexplored for TPD application. The ultimate goal of this proposal is to design, synthesize and validate new E3 ligases ligands that enable the development of novel degraders. To achieve this, we will use a chemoproteomic strategy that leverages broadly reactive, lysine, tyrosine, and serine-directed electrophilic warheads coupled to selective ligands for intracellular proteins (JQ1 for BRD4 or SLF for FKBP12) to screen for heterobifunctional degrader compounds that operate by covalent adduction of E3 ligases. To this end, we have already succeeded in developing a series of compounds to promote ligand-induced protein degradation. These proof-of-concept degraders led to the induced ubiquitination and proteasomal degradation of target proteins. We will perform further chemoproteomic studies to identify the E3 ligase targets of these degraders responsible for the degradation activity. With our novel approaches, we expect to see the repertoire of E3 ligases recruited for targeted protein degradation to be further enriched, paving the way for the next generation of tissue- and disease- specific molecular degraders.