Targeted protein stabilization using Protein Rescue Targeting Chimeras (PRESTACs)

靶向泛素途径是治疗多种毁灭性疾病的潜在选择，包括 癌症和神经退化最近的焦点是异型双功能蛋白水解靶向嵌合体， 将E3连接酶募集到感兴趣的靶标以用于靶标的泛素化和降解的PROTAC是一种新的靶向蛋白酶。 很有前途的治疗方法已经开发了PROTAC来降解广泛的蛋白质家族，并且 新的目标不断被研究。在细胞中，连接酶的泛素结合通过去- 缀合蛋白酶（DUBs），并且这两种酶类别的动力学调节蛋白质周转， 分割和其他过程。一些细胞病变是由一个关键的过度降解驱动的 蛋白质，例如肿瘤抑制因子，并且病理状态可以通过减弱降解来校正， 从而节省关键蛋白质。形式上，DUB可以被异双功能分子招募， 去泛素化靶蛋白并防止其降解，因此假设， 称为蛋白质拯救靶向嵌合体（PRESTAC）的异型双功能分子可以通过以下方式产生： 将DUB配体与靶配体化学连接，并评估它们的生理功能。Progenra拥有 鉴定并表征了结合USP 7并激活其DUB功能的新型小分子USP 7配体。 这些分子，沿着与目标靶点的配体，将用于设计和合成PRESTAC， 这将代表DUB活性与疾病相关靶点的首次联系，并提供了一种新的工具， 扩展基于UPS的治疗。作为概念的证明，将合成双功能分子以招募 DUB USP 7稳定核受体PPAR γ，已知有几种激动剂。噻唑烷二酮类 靶向PPAR β的TZDs通过诱导降低胰岛素抵抗的基因来治疗2型糖尿病。配体结合 TZDs对PPAR β的转录调控作用，同时也导致了PPAR β的泛素化和蛋白酶体的降解。 降解因此，稳定PPAR γ将延长其活化并增强这些药物的作用。 PRESTAC将使用Progenra的USP 7激活剂和罗格列酮设计，罗格列酮是一种TZD， 对过氧化物酶体增殖物激活受体的影响也得到了很好的表征。PRESTAC候选人将进行生物化学评估， 在NIH 3 T3细胞中，它们形成三元复合物（USP 7-PRESTAC-PPAR γ）的能力， 稳定PPARm，并影响PPARm活化的下游靶标（例如，PPARm报告基因测定， 脂联素水平）。

Targeting the ubiquitin pathway is a potential option for treating a variety of devastating diseases, including cancers and neurodegeneration. Recent focus on heterobifunctional proteolysis targeting chimeras, (PROTACs), which recruit E3 ligases to targets of interest for ubiquitination and degradation of the target, is a promising therapeutic avenue. PROTACs have been developed to degrade a wide range of protein families, and new targets are continually being investigated. In cells, ubiquitin conjugation by ligases is reversed by de- conjugating proteases (DUBs), and the dynamics of these two enzyme classes regulates protein turnover, compartmentation, and other processes. Some cell pathologies are driven by excessive degradation of a key protein, for example, a tumor suppressor, and the pathological state can be corrected by attenuating degradation, thereby sparing the critical protein. Formally, DUBs could be recruited by a heterobifunctional molecule to deubiquitinate target proteins and prevent their degradation, and it is therefore hypothesized that heterobifunctional molecules called Protein Rescue Targeting Chimeras (PRESTACs) can be generated by chemically linking a DUB ligand to a target ligand and evaluated for their physiological function. Progenra has identified and characterized novel small molecule USP7 ligands that bind USP7 and activate its DUB function. These molecules, along with ligands for targets of interest, will be used to design and synthesize PRESTACs, which will represent the first tethering of DUB activity to disease-relevant targets and provide a novel tool for expansion of UPS-based therapies. As proof of concept, bifunctional molecules will be synthesized to recruit the DUB USP7 to stabilize the nuclear receptor PPAR, for which several agonists are known. Thiazolidinediones (TZDs) targeting PPAR treat type 2 diabetes by inducing genes that decrease insulin resistance. Ligand binding of TZDs to PPAR not only regulates transcription, but also leads to PPAR ubiquitination and proteasomal degradation. Thus, stabilizing PPAR will prolong its activation and augment the effects of these drugs. PRESTACs will be designed using Progenra’s USP7 activators and rosiglitazone, a TZD whose interaction with and effects on PPAR are well-characterized. PRESTAC candidates will be evaluated biochemically, biophysically, and in NIH 3T3 cells for their ability to form ternary complexes (USP7-PRESTAC-PPAR), to stabilize PPAR, and to affect downstream targets of PPAR activation (for example, PPAR reporter assays, levels of adiponectin).