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

• 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
• 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

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 Our unpublished data support the hypothesis that tandem bromodomains act as a scaffold for acetylation-dependent reorganization of chromatin; for instance, joining promotors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.

项目摘要 布罗莫结构域和末端外结构域（BET）家族的成员（Brd 2、Brd 3、Brd 4、Brdt）各自含有 结合组蛋白和转录因子上的乙酰赖氨酸的两个溴结构域。BET的重要性- 在I/II期， 多种癌症的临床试验和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，但仍存在一些关键问题。例如，BET蛋白定位于 在超级增强子上不成比例，超级增强子是具有增强基因表达的大簇元件的基因组区域。 转录。这种定位的基础是未知的，但鉴于超级增强子富集， 在具有致癌潜力的位点。我们未发表的数据支持这一假设，串联溴结构域的行为 作为染色质乙酰化依赖性重组的支架;例如，将启动子与它们的 对应的远端增强子来驱动转录（焦点1）。然而，串联溴结构域的能力 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Brd 4在维持细胞凋亡中的作用。 促进增强子驱动的致癌基因转录的染色质构象。的这种机制 染色质重组，如果是真的，是范式转移，将对串联的研究产生广泛的影响， 组蛋白结合域。我们还假设代谢变化诱导不同的翻译后 被溴结构域“读取”的组蛋白修饰。然而，更广泛的酰化和蛋白质结合 溴结构域的特异性知之甚少。我们已开始解决这一知识差距， 一份出版物强调了代谢衍生的酰化和邻近修饰如何调节BET 布罗莫结构域结合组蛋白。为了继续解决这个广泛的代谢问题，我们正在使用 生物物理学、结构生物学和蛋白质组学技术来研究BET布罗莫结构域酰化， 连接酰基辅酶A代谢与转录的蛋白质选择性（焦点2）。为了帮助我们的机械调查， 我们正在消除BET布罗莫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Brd 2、Brd 3， Brd 4和Brdt具有相等的纳摩尔效力。这种选择性的缺乏可能是造成 记忆丧失和淋巴毒性最近与现有的泛BET抑制剂。我们正在克服 这些障碍与一个新的片段为基础的配体发现和化学生物学策略，以发现 通过共价靶向Brd 4内的独特半胱氨酸，获得选择性Brd 4抑制剂（焦点3）。这些化学工具 将有必要区分BET蛋白在细胞和啮齿动物疾病模型中的不同活性 并且还可用于开发靶向癌症和糖尿病中的Brd 4轴的治疗剂。