Development of chemical probes for DNA modifying enzymes

DNA修饰酶化学探针的开发

• 批准号: 1923239
• 金额: --
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1923239
• 关键词: Development; chemical; probes; DNA; modifying

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.

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。这种修饰发生在DNA或组蛋白上，经常导致蛋白质表达水平的变化。这些蛋白质表达水平的时空变化对于决定细胞命运以及哺乳动物发育的基本过程非常重要。我们感兴趣的一种特殊修饰是DNA的（去）甲基化，由DNA甲基转移酶（dnmt）、赖氨酸去甲基化酶（kdm）和10 - 11易位酶（TETs）等酶进行。考虑到DNA甲基化水平波动的重要性，获得能够选择性抑制特定目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酶抑制剂。特别是，我们将重点关注环肽抑制剂，已知其具有很强的结合亲和力和出色的靶标特异性，特别是在结构相似的蛋白质上，并且之前已在我们的小组中成功使用。我们最初的研究将集中在人类TET酶上，迄今为止还没有选择性抑制剂的报道。为这个特殊的酶家族开发选择性抑制剂是极具挑战性的，因为它们是铁（II）依赖性双加氧酶的一个大超家族的一部分，所有这些酶都有高度保守的活性位点。虽然这些酶的广谱抑制剂是可用的，但我们小组只报道了选择性抑制剂的工作，并且尚未确定具有足够选择性的化合物。我们将根据已有的构效关系数据设计抑制剂，并使用标准的固相肽合成方案进行合成。同时，我们计划优化目标酶（人类TET1， TET2和TET3）的蛋白质生产，以使其生产足够数量，然后进行生化和结构表征（酶动力学，x射线晶体学等），以扩大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对酶进行测试，结果将用于设计新的合成靶标。当确定了足够有效和选择性的化合物时，将通过引入非天然氨基酸来优化其物理化学性质，特别是溶解度和细胞渗透性。以细胞为基础的测定将用于评估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的生产及其生化表征，所有这些都应该使我们能够提出有关目标的功能和活性的问题。

项目成果

First-in-Class Inhibitors of the Ribosomal Oxygenase MINA53.

• DOI: 10.1021/acs.jmedchem.1c00605
• 发表时间: 2021-12-09
• 期刊: Journal of medicinal chemistry
• 影响因子: 7.3
• 作者: Nowak RP;Tumber A;Hendrix E;Ansari MSZ;Sabatino M;Antonini L;Andrijes R;Salah E;Mautone N;Pellegrini FR;Simelis K;Kawamura A;Johansson C;Passeri D;Pellicciari R;Ciogli A;Del Bufalo D;Ragno R;Coleman ML;Trisciuoglio D;Mai A;Oppermann U;Schofield CJ;Rotili D
• 通讯作者: Rotili D