Investigating new mechanisms for cysteine-to-lysine transfer

半胱氨酸残基是在多肽和蛋白质上进行选择性化学反应的方便部位。从构建抗体-药物偶联物作为靶向癌症疗法，到用于分析和阵列的带有多肽和蛋白质的表面功能化，这种“生物偶联”是许多应用所需的。然而，半胱氨酸生物偶联物可能会遇到一些关键的缺陷。例如，如果半胱氨酸以二硫键的形式存在于蛋白质中，那么它的永久修饰会导致这个关键结构基序的丢失。这个博士项目旨在克服现有半胱氨酸连接方法的局限性，并通过研究一种有前途的新方法来实现被称为半胱氨酸到赖氨酸转移(CLT)的位置选择性蛋白质修饰，从而在该领域提供新的机会。在CLT中，试剂最初连接到半胱氨酸残基上，然后转移连接到附近的赖氨酸氨基酸上。这项工作将探索前所未有的新试剂类别来实现这种反应活性，从而将发展我们对转移化学的基本理解，同时为构建蛋白质结合物提供强大的新平台；例如，作为预期的疗法，如抗体-药物结合物，这将是这项工作的关键目标应用。因此，这个项目属于EPSRC化学生物学和生物化学研究领域，介于物理科学和医疗技术的研究主题之间。该项目将涉及有机合成和化学生物学技术的广泛研究和培训。最初的反应发现阶段将包括试剂设计和合成规划，然后是多步合成，最后是在生物相容条件下的小分子反应研究。接下来，最有希望的试剂将在选择蛋白质的生物结合实验中进行探索，并进行相关的广泛的生物结合分析。在调查了试剂范围并优化了生物偶联方案后，最终的抗体偶联物的目标是构建证明可能在最先进水平上进行的改进。除了保留关键的二硫键，这将提高稳定性和均一性，建议这些方法将以受控的方式实现这些蛋白质前所未有的多功能，这将被证明是能够实现令人兴奋的新机会。

Cysteine residues represent convenient sites to carry out selective chemical reactions on peptides and proteins. Such 'bioconjugation' is desirable for many applications, from the construction of antibody-drug conjugates as targeted cancer therapies to the functionalisation of surfaces with peptides and proteins for assays and arrays. However, cysteine bioconjugates can suffer some key drawbacks. For example, if the cysteine is present in the protein in the form of a disulfide bond, then its permanent modification leads to loss of this key structural motif. This PhD project aims to overcome limitations with existing cysteine conjugation methods, and offer new opportunities in the field, by investigating a promising new approach to achieve site-selective protein modification referred to as 'cysteine-to-lysine transfer' (CLT). In CLT the reagents initially hook on to cysteine residues and then undergo transfer conjugation to a nearby lysine amino-acid. The work will explore unprecedented new reagent classes to achieve this reactivity, and thus will develop our fundamental understanding of transfer chemistries whilst providing powerful new platforms for the construction of protein conjugates; for example, as prospective therapeutics such as antibody-drug conjugates which will be a key target application of this work. As such this project is within the EPSRC Research Area of Chemical Biology and Biological Chemistry, and sits between the Research Themes of Physical Sciences and Healthcare Technologies.The project will involve extensive research and training in Organic Synthesis and Chemical Biology techniques. The initial reaction discovery phase will involve reagent design and synthetic planning, then multi-step synthesis, and finally small molecule reaction investigations in biologically compatible conditions. Next, the most promising reagents will be explored in bioconjugation experiments with select proteins, with associated extensive bioconjugate analysis. After investigating the reagent scope and optimising the bioconjugation protocols, final antibody conjugates aim to be constructed to demonstrate the improvements possible over the state-of-the art. In addition to the retention of key disulfide linkages, which will improve stability and homogeneity, it is proposed that the methods will enable unprecedented multifunctionalisation of these proteins in a controlled manner and this will be demonstrated to show exciting new opportunities are enabled.