Development of Non-conventional PROTACs

泛素(Ub)信号不仅调节蛋白酶体的蛋白质降解，而且还调节蛋白质的运输、受体- 介导的信号转导，以及有丝分裂吞噬和自噬的控制。Ub结合(E3连接酶)和 去结合(去泛素酶)酶和蛋白酶体一直是药物发现的目标，重点是 疾病相关蛋白的降解或保存，在蛋白酶体的批准方面取得了一些成功 靶向E3连接酶Cereblon的抑制剂和三种药物(沙利度胺、泊马度胺和来那度胺) 多发性骨髓瘤或套细胞淋巴瘤的治疗。Ub结合的非退化事件和 去卷曲疗法还没有被广泛用于治疗应用，尽管治疗假说 对这些功能的了解。目前，一种新的靶向和处理疾病相关蛋白的方法 正在引起人们的兴趣，并被称为靶向蛋白降解(TPD)。这项新兴技术使用双功能 被称为PROTAC(针对嵌合体的蛋白质分解)的分子，它结合到感兴趣的蛋白质上，而 同时通过E3Ub连接酶标记它进行降解。这项技术的独特之处在于， 靶蛋白不一定需要E3泛素连接酶的生理底物。这将打开 为“无法下药”的蛋白质组打开大门，包括信号转接子和转录因子。尽管 到目前为止，PROTACs的治疗应用一直被认为是蛋白质降解，涉及到化学 可能具有非降级应用，包括亚细胞定位或改变候选人的活动 以产生期望的药理效果的底物。这项提议的目标是开发一种新的应用程序 我们称之为“LOTAC”的PROTAC技术用于定位劫持E3Ub连接酶的嵌合体 TRAF6，已知催化K63连接的多泛素化。这一目标将通过实现 里程碑包括：1)从大肠杆菌中重组有活性的TRAF6并显示出与TRAF6特异的结合 化合物可以作为“LOTAC”配体；和2)证明TRAF6介导的多泛素化。 Foxp3使其细胞定位于胞浆和胞核。在第二阶段，TRAF6 LOTAC将 设计使用选择性、高亲和力的配体，与TRAF6结合并改变各种 通过K63连接的泛素化的底物。这项技术将帮助药物化学家开发LOTAC 通过改变治疗靶蛋白的亚细胞位置和/或活性而不是 通过蛋白酶体的降解来消除它们。这类LOTAC的成功开发将扩大其用途 这种新形式的基于泛素的疗法。

Ubiquitin (Ub) signaling regulates not only proteasomal protein degradation, but also protein trafficking, receptor- mediated signal transduction, and control of mitophagy and autophagy. Ub conjugating (E3 ligases) and deconjugating (deubiquitinases) enzymes and the proteasome have been targets of drug discovery focusing on degradation or preservation of disease-associated proteins, with some success in the approval of proteasome inhibitors and three drugs (thalidomide, pomalidomide, and lenalidomide) that target the E3 ligase Cereblon for the treatment of multiple myeloma or mantle cell lymphoma. The non-degradative events of Ub conjugation and deconjugation have not been widely exploited for therapeutic application, although therapeutic hypotheses lend themselves to these functions. Currently, a novel approach to target and dispose of disease-associated proteins is gaining interest and known as targeted protein degradation (TPD). This emerging technology uses bifunctional molecules known as PROTACs (Proteolysis targeting chimeras) that bind to a protein of interest while simultaneously tagging it for degradation via an E3 Ub ligase. What’s unique about this technology is that the target protein doesn’t necessarily need to by a physiological substrate for the E3 ubiquitin ligase. This opens the door for the “undruggable” proteome including signaling adaptors and transcription factors. Although the therapeutic application of PROTACs has heretofore been considered protein degradation, the chemistry involved could have non-degradative applications, including subcellular localization or altering the activity of candidate substrates to generate a desired pharmacologic effect. The goal of this proposal is to develop a new application of PROTAC technology which we term “LOTAC” for localization targeting chimeras that hijacks the E3 Ub ligase TRAF6, which is known to catalyze K63-linked polyubiquitination. This goal will be addressed by achieving milestones including: 1) reconstituting active TRAF6 from E. coli and showing that specific TRAF6-binding compounds can function as “LOTAC” ligands; and 2) demonstrating that TRAF6 mediated polyubiquitination of FOXP3 causes its cellular localization from the cytosol to the nucleus. In Phase II, TRAF6 LOTACs will be designed using selective, high affinity ligands that bind to TRAF6 and alter the subcellular location of various substrates via K63-linked ubiquitination. This technology will assist medicinal chemists in developing LOTAC molecules that work by changing the subcellular location and/or activity of therapeutic target proteins rather than by eliminating them by proteasomal degradation. Successful development of such LOTACs will expand the utility of this novel form of ubiquitin-based therapy.