Development of chemical probes for DNA modifying enzymes

该项目属于EPSRC化学生物学和生物化学研究领域。表观遗传修饰酶能够在不改变碱基DNA序列的情况下，通过各种机制传递化学信息。这些修饰发生在DNA或组蛋白上，通常会导致蛋白质表达水平的变化。蛋白质表达水平的这些时间和空间变化对于决定细胞命运以及对哺乳动物发育至关重要的过程非常重要。我们感兴趣的一种特殊修饰是DNA的(去)甲基化，由诸如DNA甲基转移酶(DNMT)、赖氨酸去甲基酶(KDM)和TEN-ELE1易位酶(TETS)等酶执行。鉴于DNA甲基化水平波动的重要性，获得能够选择性抑制特定靶点的化学工具对于评估酶的功能至关重要。因此，我们在这个项目中的目标是开发高选择性的酶抑制剂。特别是，我们将重点介绍环肽抑制剂，它们被认为具有很强的结合亲和力和良好的靶向性，特别是在结构相似的蛋白质上，以前曾在我们的团队中成功使用过。我们的初步研究将集中在人类Tet酶上，到目前为止还没有关于Tet酶的选择性抑制剂的报道。开发针对这一特殊酶家族的选择性抑制剂是极其具有挑战性的，因为它们是依赖Fe(II)的双加氧酶超家族的一部分，所有这些超家族都具有高度保守的活性部位。虽然这些酶的广泛抑制剂是可用的，但我们小组报告了唯一一项针对选择性抑制剂的工作，而且还没有确定具有足够选择性的化合物。我们将根据现有的构效关系数据设计我们的抑制剂，并使用标准的固相肽合成方案来合成它们。同时，我们计划优化目标酶(人类TET1、TET2和TET3)的蛋白质生产，以使它们能够产生足够的数量，然后进行它们的生化和结构表征(酶动力学、X射线结晶学等)。以扩大我们对酶及其活性部位的理解。这些化合物将使用已建立的酶分析方法针对酶进行测试，结果将用于设计新的合成靶标迭代。当一种具有足够效力和选择性的化合物被确定时，将通过引入非天然氨基酸来优化以改善其物理化学性质，特别是溶解性和细胞渗透性。将使用基于细胞的分析来评估这些化合物在细胞中的活性。总之，我们将集中于靶标选择性化学探针的开发，以及所需酶的活性构建体的生产及其生化特性，所有这些都应该使我们能够提出关于靶标的功能和活性的问题。

This project falls within the EPSRC Chemical Biology and Biological Chemistry research area.Epigenetic modifying enzymes are capable of relaying chemical information through various mechanisms without altering the base DNA sequence. The modifications occur on either DNA or histones, and often result in changes of protein expression levels. These temporal and spatial changes in protein expression levels are hugely important for determining cell fate, as well as processes which are fundamental in mammalian development. A particular modification of interest to us is the (de)methylation of DNA, carried out by enzymes such as DNA methyltransferases (DNMTs), lysine demethylases (KDMs), and ten-eleven translocation enzymes (TETs). Given the importance of fluctuations of DNA methylation levels, obtaining chemical tools that can selectively inhibit a particular target is crucial for evaluating the functions of the enzymes. As such, our goal in this project is to develop highly selective enzyme inhibitors. In particular, we will focus on cyclic peptide inhibitors, which are known to have strong binding affinity and excellent target specificity, particularly over structurally similar proteins, and have been previously successfully employed within our group. Our initial investigations will focus on the human TET enzymes, for which no selective inhibitors have been reported to date. Developing selective inhibitors for this particular family of enzymes is extremely challenging, as they are part of a large superfamily of Fe(II)-dependent dioxygenases, all of which have highly conserved active sites. While broad-range inhibitors of these enzymes are available, the only work towards a selective inhibitor has been reported by our group, and a sufficiently selective compound has yet to be identified. We will design our inhibitors based on pre-existing structure-activity relationship data and synthesise them using standard solid phase peptide synthesis protocols. In parallel, we plan to optimise protein production of the target enzymes (human TET1, TET2, and TET3) to allow their production in sufficient quantities, followed by their biochemical and structural characterisation (enzyme kinetics, X-ray crystallography, etc.) to broaden our understanding of the enzymes and their active sites. The compounds will be tested against the enzymes using established enzyme assays, and the results will be utilised to design new iterations of synthetic targets. When a sufficiently potent and selective compound is identified, optimisation to improve its physiochemical properties, particularly solubility and cell permeability, will be carried out by introducing unnatural amino acids. Cell-based assays will be employed to evaluate the activity of the compounds in cells. In summary, we will be focusing on the development of target-selective chemical probes, as well as production of active constructs of the desired enzymes and their biochemical characterisation, all of which should enable us to ask questions about the function and activity of the targets.