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研究领域的声明：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：鉴定表观遗传调节剂的化学调节剂。项目摘要/摘要：多细胞生物已经进化出复杂的机制，使差异和细胞类型特异性表达的基因。表观遗传学是指在不同细胞类型中以及在发育和分化期间基因组如何访问的这些可遗传的变化。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。在过去的十年中，创造这些可遗传变化的细胞机制一直是激烈的科学研究的主题，因为在生物学或人类健康领域，表观遗传学可能不会发挥根本作用。[1]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。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; DNA胞嘧啶甲基化;组蛋白类泛素化、泛素化、ADP核糖基化和磷酸化。[2]这些修饰中的许多产生结合位点以招募可以“读取”这些标记的其他蛋白质，并导致细胞类型和环境适当的基因表达或抑制。鉴于染色质调节对细胞生物学的广泛重要性，产生这些修饰的酶（“写入器”），识别它们的蛋白质（“读取器”）和去除它们的酶（“擦除器”）是操纵的关键目标，以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可用于治疗癌症。[3]一个系统的和治疗公正的方法，以进一步发展化学探针的作家，读者和擦除组蛋白代码是一个重大的挑战和机会，生物医学界。细胞渗透剂是一种调节染色质状态的小分子化学探针，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等领域具有重要意义。[3-5]创建一个“工具箱”的有效的，选择性的，良好的特性和细胞渗透性的小分子探针的染色质调节将允许生物学假说有关染色质状态进行测试，在基于细胞和动物模型的人类生物学和疾病的信心。鉴于组蛋白密码动态变化的新证据，小分子工具将在真实的功能测定中评估这种生物学方面具有独特的用途。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，我们将在这奋进与他们合作。恶性脑肿瘤（MBT）重复序列是ca. 100个氨基酸，存在于11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。[6]没有已知的MBT结构域的小分子结合剂。该提议的具体目的是开发甲基赖氨酸被人和果蝇MBT结构域蛋白识别的有效拮抗剂，以允许在基于细胞的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。[7-9]果蝇MBT结构域的纳入将使在这个重要的模型系统的功能研究。[8]为了发现和表征MBT结构域的高质量探针：将针对所有人类和果蝇含MBT结构域的蛋白质开发测定法;将通过集中筛选、虚拟筛选和基于结构的设计生成小分子命中物;将针对与其体外特征一致的效力、选择性和细胞活性优化命中物。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。这笔赠款的资金将使两名博士后研究人员能够继续就业，他们目前由将于2010年到期的启动资金资助。 公共卫生相关性：该提案旨在开发针对蛋白质的小型药物样分子，这些蛋白质调节DNA代码如何在身体的不同细胞类型中获得和利用。这是一个新的探索领域，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许多潜在的应用。在该提案中设计、合成和验证的化学探针将在发现治疗疾病（如癌症）的分子靶点和开发安全的基于干细胞的疗法中具有应用。

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.