Discovery of Small Molecule MBT Domain Antagonists

描述（由申请人提供）：挑战领域和特定挑战主题的陈述研究领域：本申请涉及广泛的挑战领域(6)：使能技术和特定挑战主题06-OD-105：表观遗传调节剂的化学调节剂的鉴定。项目摘要/摘要：多细胞生物已经进化出复杂的机制来实现基因的差异表达和细胞类型特异性表达。表观遗传学是指基因组在不同细胞类型以及发育和分化过程中如何进入的可遗传变化。这种能力允许细胞之间的功能特化，即使每个细胞包含相同的基因组。在过去的十年里，产生这些可遗传变化的细胞机制一直是科学研究的重点，因为在生物学乃至人类健康领域，表观遗传学都可能发挥着重要作用书写表观基因组的模板是染色质——组蛋白、RNA和DNA的复合体，它有效地将基因组包装在每个细胞内合适的可接近状态。染色质的状态以及遗传密码的获取在很大程度上是由组蛋白和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来调节的。组蛋白和DNA最重要的修饰包括：组蛋白赖氨酸和精氨酸甲基化；赖氨酸乙酰化作用;DNA胞嘧啶甲基化；组蛋白sumomylation，泛素化，adp -核糖基化和磷酸化许多这些修饰创造了一个结合位点来招募其他蛋白质，这些蛋白质可以“读取”这些标记，并导致细胞类型和环境适当的基因表达或抑制。鉴于染色质调控对细胞生物学的广泛重要性，产生这些修饰的酶（“书写者”）、识别它们的蛋白质（“读取者”）和去除它们的酶（“擦除者”）是操纵的关键目标，以便进一步了解组蛋白密码及其在生物学和人类疾病中的作用。事实上，组蛋白去乙酰化酶的小分子抑制剂已经被证明在治疗癌症方面很有用对于生物医学界来说，为组蛋白编码者、解读者和擦除者进一步开发化学探针是一项重大的挑战和机遇。细胞渗透小分子化学探针调控染色质状态，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具有重要意义。[3-5]一套有效的、选择性的、特性良好的、细胞渗透性强的染色质调控小分子探针的“工具箱”的创建，将使有关染色质状态的生物学假设能够在基于细胞和动物的人类生物学和疾病模型中得到有信心的检验。鉴于组蛋白密码动态变化的新证据，小分子工具将在评估这种生物学的真实功能分析中发挥独特的作用。事实上，结构基因组学联盟（SGC）最近宣布他们打算重点关注表观遗传学和化学探针的创造，我们将在这方面与他们合作。恶性脑肿瘤（MBT）重复序列是约100个氨基酸的结构域，存在于11种识别组蛋白单赖氨酸和二甲基赖氨酸修饰的人类蛋白质中目前还没有已知的MBT结构域的小分子结合物。本研究旨在开发人类和果蝇MBT结构域蛋白识别甲基赖氨酸的有效拮抗剂，以便在细胞和体内模型中探索与正常和疾病生物学相关的阻断这种识别的生物学后果。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编程中有用。[7-9]纳入果蝇MBT结构域将使这一重要模型系统的功能研究成为可能为了发现和表征高质量的MBT结构域探针：将开发所有人类和果蝇MBT结构域蛋白的检测方法；通过集中筛选、虚拟筛选和基于结构的设计将产生小分子命中；命中将优化效力，选择性和细胞活性与他们的体外轮廓一致。在这项研究过程中开发的探针将免费提供给学术生物界，没有使用限制或知识产权限制。这项资助将继续聘用两名由启动基金资助的博士后研究人员，该基金将于2010年到期。

DESCRIPTION (provided by applicant): Statement of the Challenge Area and the specific Challenge Topic Research Area: This application addresses Broad Challenge Area (6): Enabling Technologies and Specific Challenge Topic 06-OD-105: Identification of chemical modulators of epigenetic regulators. Project Summary/Abstract: Multicellular organisms have evolved elaborate mechanisms to enable differential and cell-type specific expression of genes. Epigenetics refers to these heritable changes in how the genome is accessed in different cell-types and during development and differentiation. This capability permits specialization of function between cells even though each cell contains the same genome. Over the last decade, the cellular machinery that creates these heritable changes has been the subject of intense scientific investigation as there is no area of biology or indeed, human health where epigenetics may not play a fundamental role.[1] 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. The most important modifications of histones and DNA include: histone lysine and arginine methylation; lysine acetylation; DNA cytosine methylation; and histone sumoylation, ubiquitination, ADP-ribosylation and phosphorylation.[2] Many of these modifications create a binding site to recruit other proteins which can 'read' these marks and lead to cell-type and environmentally appropriate gene expression or repression. Given the wide-spread importance of chromatin regulation to cell biology, the enzymes which produce these modifications (the 'writers'), the proteins that recognize them (the 'readers'), and the enzymes that remove them (the 'erasers') are critical targets for manipulation in order to further understand the histone code and its role in biology and human disease. Indeed, small molecule inhibitors of histone de-acetylases have already proven useful in the treatment of cancer.[3] A systematic and therapeutically unbiased approach to further development of chemical probes for the writers, readers and erasers of the histone code is a major challenge and opportunity for the biomedical community. Cell penetrant, small molecule 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.[3-5] 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. Given the emerging evidence of dynamic changes in the histone code, small molecule tools will be uniquely useful in assessing this biology in authentic, functional assays. Indeed, the Structural Genomics Consortium (SGC) has recently announced their intention to focus heavily on epigenetics and creation of chemical probes and we will be collaborating with them in this endeavor. The malignant brain tumor (MBT) repeat is a structural domain of ca. 100 amino acids and occurs in 11 human proteins which recognize mono- and dimethyl-lysine modifications of histones.[6] There are no known small molecule binders of MBT domains. This proposal specifically aims to develop potent antagonists of methyl-lysine recognition by human and Drosophila 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. 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.[7-9] Inclusion of Drosophila MBT domains will enable functional studies in this important model system.[8] In order to discover and characterize high-quality probes for MBT domains: assays will be developed for all human and Drosophila MBT domain containing proteins; small molecule hits will be generated by focused screening, virtual screening and structure based design; hits will be optimized for potency, selectivity and cellular activity consistent with their in vitro profile. The probes developed in the course of this research would be made freely available to the academic biology community with no restrictions on use or intellectual property constraints. Funding from this grant will enable the continued employment of two postdoctoral researchers who are currently funded by start-up funds which will expire in 2010. PUBLIC HEALTH RELEVANCE: This proposal aims to develop small, drug-like molecules targeted to proteins that regulate how the DNA code is accessed and utilized in the different cell-types of the body. This is a novel area for exploration with many potential applications in the fields of epigenetics, oncology, developmental biology, neurology, stem cell fate and regenerative medicine. The chemical probes designed, synthesized and validated in this proposal will have applications in the discovery of molecular targets to treat diseases such as cancer and in the development of safe stem cell based therapeutics.