DISCOVERY OF CHEMICAL PROBES FOR METHYL-LYSINE READERS

描述(由申请人提供)：多细胞生物体已经进化出复杂的机制，使基因的差异和细胞类型特异性表达成为可能。表观遗传学是指DNA在不同类型的细胞中以及在发育和分化过程中如何获得这些可遗传的变化。在其上书写表观基因组的模板是染色质--组蛋白、RNA和DNA的复合体，在每个细胞内以适当的可访问状态有效地包装基因组。染色质的状态，从而获得遗传密码，在很大程度上受到组蛋白蛋白质和DNA的特定化学修饰的调节，以及其他蛋白质和蛋白质复合体对这些标记的识别。我们对染色质功能的了解还处于初级阶段，化学生物学可以在促进科学知识和评估治疗机会方面发挥核心作用。在表观遗传学、肿瘤学、发育生物学、神经学、干细胞命运和再生医学等领域，细胞穿透性、高质量的化学探针调控染色质状态具有重要意义。识别组蛋白中赖氨酸残基的甲基化状态是染色质调节中的一个关键事件。例如，不同的赖氨酸甲基化标记(KME)与活性状态(组蛋白3，赖氨酸4二甲基化，即-H3K4me2)和抑制转录状态(H3K9me2)相关。人类基因组中的200多种甲基赖氨酸结合蛋白代表了一组相对未被探索的小分子干预目标。该计划的主要目标是开发甲基赖氨酸结合结构域的药理探针，以开创特定结构域和分析框架的先河，在此框架中可以评估选择性和作用机制。我们发现了甲基赖氨酸阅读器的小分子配体L3MBTL3和L3MBTL1，具有低的微摩尔到纳摩尔的Kd值(ITC)。L3MBTL3的配体对L3MBTL3-GFP蛋白在整个细胞中的定位也有影响。我们建议对这些系列进行优化，以提供高质量的化学探针。恶性脑瘤(MBT)重复序列是一个由大约100个氨基酸组成的结构域，存在于9种人类蛋白质中，它们识别组蛋白的单甲基和二甲基赖氨酸修饰。目前还没有针对MBT结构域的高质量化学探针，也没有针对任何其他甲基赖氨酸结合域的高质量化学探针。目前对MBT结构域拮抗的生物学后果的了解表明，拮抗剂可能在去分化、沉默基因的重新表达和细胞重新编程方面有用。我们将在甲基赖氨酸阅读器的体外拮抗、靶向阅读器的细胞定位、全基因组选择性指纹识别和阅读器拮抗的生物学后果之间建立牢固的联系。在本研究过程中开发的探测器将免费提供给学术界，不受使用限制或知识产权限制。 与公共卫生相关：多细胞生物体已经进化出复杂的机制，能够实现基因的差异化和细胞类型特异性表达。这种能力允许细胞之间的功能专门化，即使每个细胞包含基本上相同的遗传密码。一种有效的、选择性的、特征良好的、细胞穿透的染色质调节小分子探针的创建将使有关染色质状态的生物学假说能够在基于细胞的和人类生物学和疾病的动物模型中进行信心测试。这项建议的具体目的是开发有效的含有人MBT结构域的蛋白质识别甲基赖氨酸的拮抗剂，以便能够在与正常和疾病生物学相关的细胞和体内模型中探索阻止这种识别的生物学后果。

DESCRIPTION (provided by applicant): Multicellular organisms have evolved elaborate mechanisms to enable differential and cell-type specific expression of genes. Epigenetics refers to these heritable changes in how DNA is accessed in different cell-types and during development and differentiation. The template upon which the epigenome is written is chromatin - the complex of histone proteins, RNA and DNA that efficiently package the genome in an appropriately accessible state within each cell. The state of chromatin, and therefore access to the genetic code, is largely regulated by specific chemical modifications to histone proteins and DNA, and the recognition of these marks by other proteins and protein complexes. Our understanding of chromatin function is in its infancy and chemical biology can play a central role in advancing scientific knowledge and assessing therapeutic opportunities. Specifically, cell penetrant, high-quality chemical probes that modulate the regulation of chromatin state are of great significance in the fields of epigenetics, oncology, developmental biology, neurology, stem cell fate and regenerative medicine. The recognition of the methylation-state of lysine residues in histones is a critical event in chromatin regulation. For example, different lysine methylation marks (KMe) are associated with active (histone 3, lysine 4 dimethylation, i.e. - H3K4Me2) and repressed transcriptional states (H3K9Me2). The more than 200 methyl-lysine binding proteins in the human genome represent a relatively unexplored set of targets for intervention with small molecules. The overarching objectives of this program are to develop pharmacological probes of methyl-lysine binding domains to pioneer both the validation of specific domains and the assay framework in which issues of selectivity and mechanism of action can be assessed. We have discovered small molecule ligands for the methyl-lysine readers, L3MBTL3 and L3MBTL1, with low micro-molar to nanomolar Kd values (ITC). Ligands for L3MBTL3 also demonstrate effects on L3MBTL3- GFP protein localization in whole cells. We propose the optimization of these series to provide high quality chemical probes. The malignant brain tumor (MBT) repeat is a structural domain of ca. 100 amino acids and occurs in 9 human proteins that recognize mono- and dimethyl-lysine modifications of histones. There are no high-quality chemical probes for MBT domains, or indeed, any other methyl-lysine binding domain. Current understanding of the biological consequences of MBT domain antagonism would suggest that antagonists may be useful in de-differentiation, re-expression of silenced genes and cellular reprogramming, We will establish a firm connection between methyl-lysine reader in vitro antagonism, cell-based localization of the targeted reader, genome-wide selectivity finger-printing and biological consequences of reader antagonism. The probes developed in the course of this research would be made freely available to the academic community with no restrictions on use or intellectual property constraints. PUBLIC HEALTH RELEVANCE: Multicellular organisms have evolved elaborate mechanisms to enable the differential and cell-type specific expression of genes. This capability permits specialization of function between cells even though each cell contains essentially the same genetic code. The creation of a 'tool-kit' of potent, selective, well-characterized and cell-penetrant small molecule probes of chromatin regulation will permit biological hypotheses concerning chromatin-state to be tested with confidence in cell-based and animal models of human biology and disease. This proposal specifically aims to develop potent antagonists of methyl-lysine recognition by human MBT domain containing proteins in order to permit exploration of the biological consequences of blocking this recognition in cell-based and in vivo models with relevance to normal and disease biology.