Novel Approaches for PROTAC Drug Discovery

PROTAC是一种新的治疗类别，由小分子结合蛋白组成。 靶蛋白和泛素（Ub）E3连接酶，使得能够选择性靶降解。PROTAC的优势 包括精确选择性、对弱结合剂的耐受性以及在有限靶结合的情况下的最大降解。 第一个PROTAC采用E3连接酶组分pVHL来降解靶蛋白。大约在同一时间， 沙利度胺和相关类似物（IMiDs）被成功地重新用作抗癌药物，随后 当沙利度胺被证明能与 cereblon（CRBN），Cullin 4型泛素连接酶的pVHL样亚基。IMiD促进E3的相互作用 控制T细胞免疫的连接酶转录因子。在细胞中，Ub介导的信号调节蛋白质含量， 位置和活性，主要通过蛋白质降解，以及泛素连接酶的失调， 许多毁灭性的疾病。因此，连接酶是有前途的药物靶标和PROTAC的载体。约700人 Ub连接酶，只有CRBN和pVHL已被用于PROTAC开发，这一过程受到几个因素的阻碍。 问题.化学家快速合成新的PROTAC和时间之间存在脱节- 消耗，人工制品敏感的免疫印迹细胞测定用于评估它们。此外，PROTAC的绑定 由于空间位阻，在错误的位点或在靶点的泛素化， 错误的链构型（K63 vs K48），或细胞内代谢或进入细胞的能力差。因此难以 PROTAC化学家开发有意义的结构活性关系，这对于临床前研究至关重要。 发展本文提出开发用于PROTAC的连接酶选择性高通量细胞测定。 介导的靶蛋白泛素化。在第一阶段，生命传感器将采用独特的亲和矩阵，称为 TUBE（串联泛素结合实体）以高通量模式分析泛素化模式， 细胞内的内源性蛋白质这种方法提供了加速发现新PROTAC的潜力， 在泛素化和降解之间建立关系，消除低通量、耗时 蛋白质印迹分析，并导致及时识别和开发新的PROTAC药物， 药物化学家将能够有效和合理地设计PROTAC，最终包括两个 降解性和非降解性泛素化。第一阶段将通过建立明确的关系来完成 细胞中CRBN和HDM2连接酶靶蛋白的PROTAC介导的泛素化和降解之间的关系 并调整LifeSensors的TUBE技术，以监测PROTAC的泛素化和降解， 在细胞中的吞吐量方式。在第二阶段，该技术将扩展到整个连接酶家族。

PROteolysis TArgeting Chimeras (PROTACs) is a new therapeutic class comprised of small molecules binding a target protein and a ubiquitin (Ub) E3 ligase, enabling selective target degradation. PROTACs’ advantages include exquisite selectivity, tolerance of weak binders, and maximal degradation with limited target engagement. The first PROTACs employed the E3 ligase component pVHL to degrade target proteins. Around the same time, thalidomide and related analogs (IMiDs) were successfully repurposed as anti-cancer agents, and subsequently their ubiquitin ligase-associated molecular mechanism was discovered, when thalidomide was shown to bind to cereblon (CRBN), a pVHL-like subunit of a Cullin 4-type ubiquitin ligase. IMiDs promote interaction of this E3 ligase transcription factors that control T cell immunity. In cells, Ub-mediated signaling regulates protein content, location, and activity, primarily through protein degradation, and dysregulation of ubiquitin ligases is linked to numerous devastating diseases. Thus, ligases are promising drug targets and vehicles for PROTACs. Of ~700 Ub ligases, only CRBN and pVHL have been exploited for PROTAC development, a process hindered by several issues. There is a disconnect between the rapid synthesis of new PROTACs by chemists and the time- consuming, artifact-susceptible immunoblot cell assays used to evaluate them. Moreover, binding of a PROTAC to its target does not ensure degradation, owing to steric hindrance, ubiquitylation at the wrong site or in the wrong chain configuration (K63 vs K48), or metabolism inside or poor penetrance into cells. Thus, it is difficult for PROTAC chemists to develop meaningful structure activity relationships, which are essential for preclinical development. It is proposed here to develop ligase-selective, high-throughput cellular assays for PROTAC- mediated ubiquitylation of target proteins. In phase I, LifeSensors will employ unique affinity matrices called TUBEs (Tandem Ubiquitin Binding Entities) in a high throughput mode to analyze ubiquitylation patterns of endogenous proteins in cells. This approach offers the potential to expedite discovery of novel PROTACs, establish a relationship between ubiquitylation and degradation, eliminate low throughput, time-consuming western blot analysis, and lead to the timely identification and development of novel PROTAC drugs, as medicinal chemists will be able to design PROTACs efficiently and rationally, eventually encompassing both degradative and non-degradative ubiquitylation. Phase I will be accomplished by establishing a clear relationship between PROTAC-mediated ubiquitylation and degradation for CRBN and HDM2 ligase target proteins in cells and adapting LifeSensors’s TUBEs technology to monitor PROTAC ubiquitylation and degradation in a high throughput fashion in cells. In Phase II the technology will be expanded to entire ligase families.