Dissecting Signalling Pathways with PROTAC Chemical Probes.

蛋白水解靶向嵌合体（PROTACs）是一种异双功能分子，其中一个片段与目标蛋白（POI）相互作用，另一个片段与泛素连接酶的一个组分结合。这导致POI的多泛素化，然后以催化的方式经历不可逆的蛋白酶体降解。这种降低细胞蛋白水平的策略在生物学上引起了巨大的兴趣和兴奋，目前，有大量的研究旨在开发PROTACs靶向蛋白质和信号通路，这些蛋白质和信号通路以前被认为是用经典的蛋白质-蛋白质相互作用抑制剂无法治疗的。该项目旨在开发/表征新型细胞可渗透PROTACs，作为询问重要信号通路及其关键蛋白效应器的新型探针。例子包括Wnt/ β -连环蛋白信号通路和染色质重塑复合物，它们在人体生理学中起着多种关键作用。观察到它们的失调导致多种癌症类型，强调了这些关键作用。首先，候选人将专注于开发针对调控节点关键poi的PROTAC探针。这将涉及PROTACs的分子设计，化学合成和体外生物物理表征（NMR， ITC， MS）。第二个目标将集中于阐明新的PROTACs的细胞作用模式。它们的选择性和消耗poi的能力将通过WB和使用GFP结构的实时成像进行量化；以及它们的下游效应，通过监测已知对POI抑制有反应的下游基因的mRNA水平来确定。该项目建立在i)最近在Baud和Linclau实验室的概念验证结果表明，通过合成改变可以增强PROTAC的性质（例如溶解度/亲脂性）和选择性，绕过了该领域的一个重要的历史瓶颈；ii)在Giamas实验室的癌细胞生物学和信号传导方面的专业知识（例如，识别/破译与重要生物过程有关的新基因/蛋白质）。

Proteolysis targeting chimeras (PROTACs) are hetero-bifunctional molecules where one fragment interacts with the protein of interest (POI) and the other binds to a component of an ubiquitin ligase. This results in the poly-ubiquitination of the POI, which then undergoes irreversible proteasomal degradation, in a catalytic manner. This strategy to reduce cellular protein levels has generated huge interest and excitement in biology, and at present, there is significant research aimed at developing PROTACs to target proteins and signalling pathways previously thought undruggable using classical protein-protein interaction inhibitors. This project aims to develop/characterise new classes of cell permeable PROTACs, as novel probes to interrogate important signalling pathways and their key protein effectors. Examples include the Wnt/Beta-catenin signalling pathway and chromatin remodelling complexes, which play multiple pivotal functions in human physiology. These critical roles are underscored by the observation that their dysregulation leads to multiple cancer types. First, the candidate will focus on the development of PROTAC probes targeting key POIs at regulatory nodes. This will involve the molecular design, chemical synthesis and in vitro biophysical characterisation (NMR, ITC, MS) of the PROTACs. A second objective will focus on elucidating the cellular mode of action of the new PROTACs. Their selectivity and ability to deplete the POIs will be quantified by WB and live imaging using GFP constructs; and their downstream effects determined by monitoring mRNA levels of downstream genes known to respond to POI inhibition across a panel of cancer cell lines. This project builds on i)recent proof-of-concept results in the Baud and Linclau laboratories showing that it is possible to enhance PROTAC properties (e.g. solubility/lipophilicity) and selectivity through synthetic alteration, circumventing an important historical bottleneck in the field; and ii)expertise in cancer cell biology and signalling in the Giamas laboratory (e.g. identification/deciphering of new genes/proteins implicated in important biological processes).