Development of Non-conventional PROTACs

泛素（Ub）信号不仅调节蛋白酶体蛋白的降解，而且还调节蛋白质运输、受体介导的蛋白质转运和蛋白质转运。 介导的信号转导以及控制线粒体自噬和自噬。Ub缀合（E3连接酶）和 去结合（去泛素化酶）酶和蛋白酶体已经成为药物发现的靶点， 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 抑制剂和三种药物（沙利度胺，泊马度胺和来那度胺），靶向E3连接酶Cereblon， 多发性骨髓瘤或套细胞淋巴瘤的治疗。Ub结合的非降解事件和 去缀合尚未被广泛用于治疗应用，尽管治疗假设提供了 自己这些功能。目前，一种靶向和处理疾病相关蛋白的新方法 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（蛋白水解靶向嵌合体）的分子，其结合到感兴趣的蛋白质， 同时通过E3 Ub连接酶标记其降解。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 这是一扇“不可药物化”的蛋白质组的大门，包括信号衔接子和转录因子。虽然 迄今为止，PROTAC的治疗应用被认为是蛋白质降解，所涉及的化学过程 可以具有非降解应用，包括亚细胞定位或改变候选物的活性。 底物以产生所需的药理学作用。本提案的目标是开发一种新的应用程序 我们称之为“LOTAC”，用于定位靶向劫持E3 Ub连接酶的嵌合体 TRAF 6，已知其催化K63连接的多聚泛素化。这一目标将通过实现 里程碑包括：1）从E.显示特异性TRAF 6结合 化合物可以作为“LOTAC”配体起作用;和2）证明TRAF 6介导的LOTAC的多聚泛素化可以作为“LOTAC”配体起作用;和 F0 XP 3导致其从胞质溶胶到细胞核的细胞定位。在第二阶段，TRAF 6 LOTAC将 设计使用选择性的，高亲和力的配体，结合TRAF 6，并改变各种亚细胞位置， 通过K63连接的泛素化的底物。这项技术将有助于药物化学家开发LOTAC 这些分子通过改变治疗靶蛋白的亚细胞位置和/或活性而起作用， 通过蛋白酶体降解来消除它们。这种LOTAC的成功开发将扩大效用 这种新型的基于泛素的治疗方法。

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.