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的福尔斯。表观遗传修饰酶能够通过各种机制传递化学信息而不改变DNA碱基序列。这些修饰发生在DNA或组蛋白上，并且通常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哺乳动物发育中的基本过程非常重要。我们感兴趣的一个特别的修饰是DNA的甲基化（去甲基化），由酶如DNA甲基转移酶（DNMT）、赖氨酸脱甲基酶（KDM）和10 - 11易位酶（THEX）进行。鉴于DNA甲基化水平波动的重要性，获得可以选择性抑制特定靶标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，我们将专注于环肽抑制剂，这是已知的具有很强的结合亲和力和优异的目标特异性，特别是在结构相似的蛋白质，并已成功地在我们的小组内使用。我们的初步研究将集中在人类泰特酶，迄今为止还没有报道选择性抑制剂。开发这种特定酶家族的选择性抑制剂极具挑战性，因为它们是Fe（II）依赖性双加氧酶的大超家族的一部分，所有这些酶都具有高度保守的活性位点。虽然这些酶的广泛的抑制剂是可用的，唯一的工作对一个选择性抑制剂已被我们的小组报告，和一个足够的选择性化合物尚未确定。我们将根据已有的结构-活性关系数据设计我们的抑制剂，并使用标准固相肽合成方案合成它们。与此同时，我们计划优化目标酶（人TET 1，TET 2和TET 3）的蛋白质生产，以允许它们以足够的量生产，然后进行其生物化学和结构表征（酶动力学，X射线晶体学等）。拓宽我们对酶及其活性位点的理解。这些化合物将使用已建立的酶测定法针对酶进行测试，结果将用于设计新的合成靶点迭代。当鉴定出足够有效和选择性的化合物时，将通过引入非天然氨基酸进行优化以改善其理化性质，特别是溶解度和细胞渗透性。将采用基于细胞的测定来评价化合物在细胞中的活性。总之，我们将专注于目标选择性化学探针的开发，以及所需酶的活性结构及其生化特性的生产，所有这些都将使我们能够询问有关目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.