Development of native ion mobility mass spectrometry methods to study PROTAC systems

传统上，开发小分子抑制剂是为了与特定的蛋白质结合，从而阻止它在细胞中发挥其功能。在最近的生物技术方法中，已经创造出了降解目标蛋白质的分子，而不是抑制它。与抑制剂相比，蛋白质降解剂的主要优势是降解剂的效果更持久，实现效果所需的浓度更低。此外，降解剂适用于更广泛的蛋白质谱，因为结合不限于特定的活性部位。到目前为止，最流行的降解物类型被称为蛋白质水解靶向嵌合体(PROTAC)，它是同时与目标蛋白质和E3连接酶结合的双功能配体，将蛋白质带入复合体，从而使E3连接酶标记目标蛋白质以供细胞降解。PROTAC已经针对多种医学相关蛋白进行了开发，如临床试验中探索的致癌雄激素受体和雌激素受体。目前开发新型PROTAC的一个限制是无法快速有效地直接测量三组分络合物的形成。天然质谱学(NMS)是分析E3连接酶、PROTACs和底物蛋白等复杂混合物中物种的有效方法。这是因为它能够报告动态蛋白质混合物中存在的多种结合化学计量学，包括种群数量较少的物种。当结合离子迁移率时，这是一种互补的气相技术，也可以分离不同构象的蛋白质或蛋白质复合体。该博士项目的目的是进一步开发用于分析PROTACs和其他蛋白质降解物的天然离子迁移质谱学方法，这些蛋白质降解物具有很强的治疗癌症和其他疾病的潜力。

Small molecule inhibitors have traditionally been developed to bind to a specific protein, thereby preventing it from carrying out its function in the cell. In recent biotechnological approaches, molecules have been created that degrade a target protein, rather than inhibit it. Major advantages of protein degraders over inhibitors are the longer-lasting effects of degraders and the lower concentrations required to achieve efficacy. Moreover, degraders are applicable to a wider spectrum of proteins since binding is not limited to a specific active site. The most popular type of degraders to date are called proteolysis targeting chimeras (PROTACs), which are bifunctional ligands that bind simultaneously to the targeted protein and an E3 ligase, bringing the proteins into a complex, so that the E3 ligase labels the targeted protein for degradation by the cell. PROTACs have been developed against a variety of medically relevant proteins, such as the tumorigenic Androgen Receptor and Estrogen Receptor, as explored in clinical trials. A current limitation in the development of novel PROTACs is the ability to directly measure the formation of three-component complexes in a fast and efficient manner. Native mass spectrometry (nMS) is an efficient method for analysing the species present in complex mixtures involving E3 ligases, PROTACs, and substrate proteins. This is due to its ability to report on multiple binding stoichiometries present in dynamic protein mixtures, including species populated to a low extent. When coupled with ion mobility, which is a complementary gas-phase technique, different conformations of proteins or protein complexes can also be separated. The aim of this PhD project is to further develop native ion mobility mass spectrometry methods for the analysis of PROTACs and other protein degraders, which have strong potential as therapies for cancer and other diseases.