PROTACS: new computational methods and targets

PROtealysis TArgeting Chimeras，也被称为PROTACs，是由两个蛋白质配体通过连接体连接而成的双功能分子。一侧的配体与e3泛素连接酶结合，该酶以蛋白质降解为目标，而另一侧的配体与需要从细胞中移除的不需要的蛋白质结合。自2001年首次描述PROTACs以来，该技术已被应用于越来越多的目标，首批癌症治疗药物目前正在临床试验中。所有PROTACs都是基于已知的e3连接酶的配体，在许多情况下，Von Hippel-Lindau肿瘤抑制因子（VHL）连接到已知或新的靶蛋白配体。然而，直到最近，人们才清楚地认识到，连接剂的长度和化学性质，在大多数情况下由聚乙二醇、烷基、炔或三唑组成，在PROTAC化合物的生物活性中起着重要作用。由于潜在连接器的数量几乎是无限的，因此需要新的计算方法来指导和支持新的protac的开发。在与剑桥晶体学数据中心（CCDC）的合作中，我们将开发用于连接器设计的分子建模工具，该工具使用VHL连接酶和目标蛋白的结构，并结合从成功的连接器中获得的经验规则。这些工具最初将使用一个完善的PROTACs模型系统进行测试和验证，VHL结合人类溴结构域4 (Brd4)，这是癌症治疗的主要靶点，其中已经确定了三元VHL- protac -Brd4复合物的晶体结构。与此同时，我们将靶向Eppstein-Barr病毒核抗原1 (EBNA1)，这是在所有与Eppstein-Barr病毒感染相关的人类肿瘤中一致表达的关键蛋白。一些VHL和Brd4配体已经在文献中描述过，并且可以在市场上买到。含有新型连接剂的新型PROTAC化合物将与该部门的有机化学小组合作合成。结合VHL和目标蛋白，将使用Nanotemper Dianthus筛选中实现的新型光谱移位技术进行测试。该技术的高灵敏度使得它在384孔板高通量格式的三元体系中研究连续结合事件特别有用。最有希望的化合物将被转发到我们的结构生物学平台，包括结晶和晶体结构测定以及冷冻电子显微镜。

PROtealysis TArgeting Chimeras, also designated PROTACs, are bifunctional molecules composed of two protein ligands connected by a linker. The ligand on one side binds to the E3-ubiquitin ligase, which targets a protein for degradation, while the other ligand binds the unwanted proteins that needs to be removed from the cell. Since PROTACs had first been described in 2001, the technology has been applied to an increasing range of targets, with the first cancer therapeutics now in clinical trials. All PROTACs are based on known ligands for an E3-ligase on one side, in many cases, the Von Hippel-Lindau tumor suppressor (VHL) connected to a known or novel ligand for the target protein. However, only recently, it has become clear that the length and chemistry of the linker, in most cases composed of polyethylene glycols, or alkyls, alkynes or triazoles, plays a major role in the biological activity of the PROTAC compound. With a virtually unlimited number of potential linkers, new computational methods are needed to guide and support the development of new PROTACs. In this collaboration with the Cambridge Crystallographic Data Centre (CCDC) we will develop molecular modelling tools for linker design that use the structures of the VHL ligase and the target protein in combination with empirical rules derived from successful linkers. These tools will initially be tested and validated using one well-established PROTACs model system, VHL bound to the human bromodomain 4 (Brd4), a major target for cancer therapies, where crystals structures of the ternary VHL-PROTAC-Brd4 complex have been determined. In parallel we will target the Eppstein-Barr virus nuclear antigen 1 (EBNA1), the key protein, consistently expressed in all human tumours that are linked to Eppstein-Barr viral infections. Several VHL and Brd4 ligands have been described in the literature and are commercially available. New PROTAC compound containing novel linkers will be synthesized in collaboration with the organic chemistry groups in the department. Binding to both, the VHL as well as the target protein, will be tested using the novel spectral-shift technology implemented in Nanotemper Dianthus screening. The high sensitivity of this techniques makes it particularly useful to investigate consecutive binding events in ternary systems in 384-well plate high-throughput format. The most promising compounds will be forwarded to our structural biology platform including crystallisation and crystal structure determination as well as cryo Electron Microscopy.