Development of innovative label-free methods to investigate the binding properties and degradation efficiencies of PROTACs

摘要：蛋白水解靶向嵌合体(PROTAC)是一种小分子降解物，它将泛素E3连接酶连接到目标蛋白上，从而诱导其泛素化和降解。靶蛋白、E3连接酶和PROTAC分子之间必须形成一个三元复合体才能实现降解，这是大多数生物物理技术难以测量的。我们将开发新的、无标记的天然质谱学方法来研究类细胞环境中的PROTAC。这将在一个实验中揭示三元络合物形成、目标泛素化和目标降解之间的相互作用。这些方法将比目前用于推断PROTAC生物物理特性的方法更快，劳动强度更低，而且还将提供关于其降解效率的信息。因此，该项目将催化将PROTAC转化为临床疗法的进展，这将对癌症治疗产生影响。主要研究问题：该PHD项目的总体目标是开发、测试和简化新的质谱学工具，以(I)量化细胞裂解产物中三元复合体的形成，(Ii)确定目标蛋白质在PROTAC参与时泛化的效率，以及(Iii)测量目标蛋白质的降解率，所有这些都在一个单独的实验中完成。自然质谱学将解决以下关键问题：-PROTAC形成三元络合物的情况如何，PROTAC对给定目标的特异性如何，以及结合机制在细胞裂解液中是否协同？-对于给定的PROTAC，三元络合物形成、目标泛素化和目标降解之间的相互作用是什么？-如何将实验简化为高通量、无标记的方法，以最少的时间和样品要求揭示最大信息？

Short Summary: Proteolysis-targeting chimeras (PROTACs) are small molecule degraders that connect a ubiquitin E3 ligase to a target protein, thus inducing its ubiquitination and degradation. A ternary complex must be formed between the target protein, E3 ligase and PROTAC molecule to achieve degradation, which is difficult to measure with most biophysical techniques. We will develop novel, label-free native mass spectrometry methods to study PROTACs in cell-like environments. This will reveal the interplay between ternary complex formation, target ubiquitination and target degradation in a single experiment. These methods will be faster and less labour intensive than current methods used to deduce the biophysical characteristics of PROTACs, and furthermore will provide information on their degradation efficiencies. The project will therefore catalyse the progression of PROTACs into clinical therapies that will have an impact on cancer treatment. Key research questions: The overall aim of this PhD project is to develop, test and streamline new mass spectrometry tools to (i) quantify the formation of ternary complexes in cell lysates (ii) determine how efficiently the target proteins is ubiquitinated upon PROTAC engagement and (iii) measure the degradation rate of the target protein, all in a single experiment. The following key questions will be addressed with native mass spectrometry:- How well do PROTACs form ternary complexes, how specific are the PROTACs for a given target, and is the binding mechanism cooperative in the cell lysate?- What is the interplay between ternary complex formation, target ubiquitination and target degradation for a given PROTAC?- How can experiments be streamlined into high-throughput, label-free methods to reveal the maximum information with minimum time and sample requirements?