Photoreactive warheads for covalent fragment libraries

该项目将在医疗保健技术EPSRC主题下进行，并与化学生物学和生物化学，化学反应动力学和机制以及合成有机化学的研究领域保持一致。制药管道需要识别有效的工具来解剖、询问和验证新的生物靶点。虽然光反应共价片段文库为在分离的蛋白质中发现这些工具提供了一个有效的平台，但由于光交联水平低，它们在活细胞环境中的使用受到限制。我们将通过研究这些弹头的化学反应动力学和机制来解决这一不足，并为有效和选择性地捕获蛋白质定义新的基团。推进和展示这种弹头发展的特定靶蛋白将是棕榈酰转移酶zDHHC酶。这个家族有23个成员，负责半胱氨酸残基的酰化，影响底物蛋白的功能、定位和运输。我们将针对这些蛋白质的配体结合域，目的是提供工具，使鉴定每个家族成员的目标底物成为可能，并开发一种方法来确定新化合物的选择性（如果有的话）。这将需要鉴定混杂探针，能够结合每个zDHHC酶，并通过质谱法确定标记水平。这将代表一个简单而有效的竞争性结合试验的基础，以筛选新的命中物质并在单个实验中建立选择性概况。这些工具的开发将为研究zDHHC酶提供一个新颖而强大的平台。此外，这种新型弹头将应用于对感兴趣的替代蛋白质家族的解剖和询问。

This project will be conducted within the Healthcare Technologies EPSRC theme and aligned with the research areas of Chemical Biology and Biological Chemistry, Chemical Reaction Dynamics and Mechanisms, and Synthetic Organic Chemistry.The pharmaceutical pipeline requires the identification of effective tools for the dissection, interrogation and validation of new biological targets. Whilst photoreactive covalent fragment libraries provide an effective platform for the discovery of these tools in isolated proteins, their use in live cell environments is limited due to low levels of photo-crosslinking. We will address this shortfall by investigating the chemical reaction dynamics and mechanism of these warheads and define novel groups for the efficient and selective capture of proteins.The specific target proteins to advance and showcase this warhead development will be the palmitoyl transferase zDHHC enzymes. This 23-member family are responsible for the acylation of cysteine residues, which influences the function, localisation and trafficking of substrate proteins. We will target the ligand binding domain of these proteins with the aim of providing tools to enable the identification of target substrates for each family member examined and develop a method to determine selectivity (if any) of novel compounds. This will require the identification of promiscuous probes, able to bind each zDHHC enzyme and to determine the level of labelling through mass spectrometry. This will represent the foundation of a simple and effective competitive binding assay to screen for new hit matter and establish selectivity profiles in a single experiment.The development of each of these tools will provide a novel and powerful platform with which to investigate the zDHHC enzymes. In addition, the novel warheads will find application in the dissection and interrogation of alternative protein families of interest.