DISCOVERY OF CHEMICAL PROBES FOR METHYL-LYSINE READERS

描述（由申请人提供）：多细胞生物已经进化出复杂的机制，以实现基因的差异和细胞类型特异性表达。表观遗传学是指DNA在不同细胞类型中以及在发育和分化期间如何访问的这些可遗传变化。表观基因组的模板是染色质--组蛋白、RNA和DNA的复合物，它在每个细胞内以适当的可接近状态有效地包装基因组。染色质的状态，以及因此对遗传密码的访问，在很大程度上受到组蛋白和DNA的特定化学修饰以及其他蛋白质和蛋白质复合物对这些标记的识别的调节。我们对染色质功能的理解还处于起步阶段，化学生物学可以在推进科学知识和评估治疗机会方面发挥核心作用。具体而言，细胞渗透剂，调节染色质状态调节的高质量化学探针在表观遗传学，肿瘤学，发育生物学，神经学，干细胞命运和再生医学领域具有重要意义。组蛋白中赖氨酸残基甲基化状态的识别是染色质调控中的关键事件。例如，不同的赖氨酸甲基化标记（KMe）与活性（组蛋白3，赖氨酸4二甲基化，即-H3 K4 Me 2）和抑制的转录状态（H3 K9 Me 2）相关。人类基因组中超过200种甲基赖氨酸结合蛋白代表了一组相对未开发的小分子干预靶点。该计划的总体目标是开发甲基赖氨酸结合域的药理学探针，以开拓特定域的验证和可以评估选择性和作用机制问题的测定框架。我们已经发现了用于甲基赖氨酸读取器L3 MBTL 3和L3 MBTL 1的小分子配体，其具有低微摩尔至纳摩尔Kd值（ITC）。L3 MBTL 3的配体也证明对全细胞中L3 MBTL 3- GFP蛋白定位的影响。我们建议对这些系列进行优化，以提供高质量的化学探针。恶性脑肿瘤（MBT）重复序列是ca. 100个氨基酸，存在于9种人类蛋白质中，识别组蛋白的单赖氨酸和二甲基赖氨酸修饰。对于MBT结构域，或者实际上任何其他甲基赖氨酸结合结构域，没有高质量的化学探针。目前对MBT结构域拮抗作用的生物学后果的理解表明，拮抗剂可用于去分化、沉默基因的再表达和细胞重编程。我们将在甲基赖氨酸阅读器体外拮抗作用、靶向阅读器的基于细胞的定位、全基因组选择性指纹分析和阅读器拮抗作用的生物学后果之间建立牢固的联系。在这项研究过程中开发的探测器将免费提供给学术界，没有使用限制或知识产权限制。

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.