Discovering and Exploiting Selectivity within Tandem Bromodomains

发现和利用串联布罗莫结构域内的选择性

基本信息

批准号：
10241303
负责人：
Brian Christopher Smith
金额：
$ 23.1万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10241303
关键词：
Acetylation Achievement Acyl Coenzyme A Acylation Address Binding Binding Proteins Biology Biophysics Bromodomain Cell model Chemicals Chromatin Coronary Arteriosclerosis Cysteine DNA Sequence Data Diabetes Mellitus Disease model Distal Elements Enhancers Epigenetic Process Family Genetic Genetic Transcription Genomic Segment Goals Histones Individual Knowledge Ligands Link Location Lymphoid Lysine Malignant Neoplasms Memory Loss Metabolic Metabolism Modification Molecular Conformation Non-Insulin-Dependent Diabetes Mellitus Nucleosomes Oncogenic Pharmaceutical Preparations Phase I/II Clinical Trial Post-Translational Protein Processing Proteins Proteomics Publications Research Rodent Model Role Specificity Structure Techniques Tertiary Protein Structure Toxic effect Writing base biophysical techniques combinatorial human disease inhibitor/antagonist innovation member nanomolar novel phase III trial promoter scaffold side effect small molecule structural biology therapeutic target tool transcription factor

项目摘要

PROJECT SUMMARY Members of the bromodomain and extra-terminal domain (BET) family (Brd2, Brd3, Brd4, Brdt) each contain two bromodomains that bind acetyl-lysines on histones and transcription factors. The importance of BET- regulated transcription in human disease is well appreciated with pan-BET bromodomain inhibitors in phase I/II clinical trials for multiple cancers and phase III trials for type 2 diabetes subjects with coronary artery disease. Despite these achievements, several critical questions remain. For example, BET proteins are localized disproportionately at super-enhancers, genomic regions with large clusters of elements that enhance gene transcription. The basis of this localization is unknown but important given that super-enhancers are enriched at loci with oncogenic potential. Our unpublished data support the hypothesis that tandem bromodomains act as a scaffold for acetylation-dependent reorganization of chromatin; for instance, joining promotors with their corresponding distal enhancers to drive transcription (Focus 1). However, the ability of tandem bromodomains to scaffold nucleosomes and transcription factors in an acetylation-dependent manner has not been shown. We take an innovative structural and biophysical approach to investigate the role of Brd4 in maintaining chromatin conformations that facilitate enhancer-driven oncogenic gene transcription. This mechanism of chromatin reorganization, if true, is paradigm shifting and would have broad impact on studies of tandem histone-binding domains. We also hypothesize that metabolic changes induce distinct post-translational modifications on histones that are “read” by bromodomains. Yet, the broader acylation and protein binding specificity of bromodomains is poorly understood. We have begun to address this knowledge gap in our recent publication that highlights how metabolically-derived acylations and neighboring modifications tune BET bromodomain binding to histones. To continue to address this broad metabolic question, we are using biophysical, structural biology, and proteomic techniques to investigate BET bromodomain acylation and protein selectivity in linking acyl-CoA metabolism with transcription (Focus 2). To aid our mechanistic inquiries, we are removing a critical barrier in the study of BET bromodomain biology: the lack of inhibitors and chemical probes that selectively target individual BET proteins. Currently, all existing BET inhibitors target Brd2, Brd3, Brd4, and Brdt with equal nanomolar potency. This lack of selectivity may be responsible for the side effects of memory loss and lymphoid toxicity recently associated with existing pan-BET inhibitors. We are overcoming these barriers with a novel fragment-based ligand discovery and chemical biology strategy to discover selective Brd4 inhibitors by covalently targeting a unique cysteine within Brd4 (Focus 3). These chemical tools will be necessary to distinguish the differential activities of BET proteins in cell and rodent models of disease and may also be useful in developing therapeutics targeting the Brd4 axis in cancer and diabetes.

项目概要溴结构域和末端外结构域 (BET) 家族的成员（Brd2、Brd3、Brd4、Brdt）各自包含结合组蛋白和转录因子上的乙酰基赖氨酸的两个溴结构域 BET-的重要性。 I/II 期泛 BET 布罗莫结构域抑制剂对人类疾病中的转录调控受到高度重视针对多种癌症的临床试验以及针对患有冠状动脉疾病的 2 型糖尿病受试者的 III 期试验。尽管取得了这些成就，但仍然存在一些关键问题，例如 BET 蛋白是局部的。不成比例地出现在超级增强子中，具有增强基因的大簇元素的基因组区域这种定位的基础尚不清楚，但鉴于超级增强子的丰富，这一点很重要。我们未发表的数据支持串联溴结构域发挥作用的假设。作为染色质乙酰化依赖性重组的支架，例如将启动子与其连接相应的远端增强子驱动转录（焦点 1）。尚未显示以乙酰化依赖性方式支撑核小体和转录因子。我们采用创新的结构和生物物理方法来研究 Brd4 在维持促进增强子驱动的致癌基因转录的染色质构象。染色质重组如果属实，将是范式转变，将对串联研究产生广泛影响我们还发现代谢变化会诱导不同的翻译后作用。然而，更广泛的酰化和蛋白质结合是由溴结构域“读取”的组蛋白的修饰。我们对溴结构域的特异性知之甚少，我们最近已经开始解决这一知识空白。出版物强调了代谢衍生的酰化和邻近修饰如何调整 BET 为了继续解决这个广泛的代谢问题，我们正在使用溴结构域与组蛋白的结合。生物物理学、结构生物学和蛋白质组学技术来研究 BET 溴结构域酰化和连接酰基辅酶A代谢与转录的蛋白质选择性（焦点2）为了帮助我们进行机制研究，我们正在消除 BET 溴结构域生物学研究中的一个关键障碍：缺乏抑制剂和化学物质选择性靶向单个 BET 蛋白的探针目前，所有现有的 BET 抑制剂都靶向 Brd2、Brd3、 Brd4 和 Brdt 具有相同的纳摩尔效力，这种选择性的缺乏可能是造成副作用的原因。最近，我们正在克服与现有泛 BET 抑制剂相关的记忆丧失和淋巴毒性。通过基于片段的新型配体发现和化学生物学策略来发现这些障碍通过共价靶向 Brd4 内的独特半胱氨酸来选择性 Brd4 抑制剂（焦点 3）。将有必要区分细胞和啮齿动物疾病模型中 BET 蛋白的差异活性也可能有助于开发针对癌症和糖尿病的 Brd4 轴的疗法。