Discovering and Exploiting Selectivity within Tandem Bromodomains

项目总结 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 在组蛋白和转录因子上结合乙酰赖氨酸的两个溴结构域。投注的重要性-- PAN-BET溴域抑制剂在I/II期很好地评价了人类疾病中受调控的转录 针对多种癌症的临床试验和针对患有冠状动脉疾病的2型糖尿病患者的III期试验。 尽管取得了这些成就，但仍存在几个关键问题。例如，BET蛋白被定位 在超增强子上不成比例地，基因组区域含有大量增强基因的元素簇 抄写。这种本地化的基础是未知的，但很重要，因为超级增强子是丰富的 在具有致癌潜力的基因座上。我们未发表的数据支持串联溴结构域起作用的假设 作为依赖乙酰化的染色质重组的支架；例如，将启动子与其 相应的远端增强子驱动转录(焦点1)。然而，串联溴结构域的能力 以乙酰化依赖的方式支架核小体和转录因子还没有被证明。 我们采用一种创新的结构和生物物理方法来研究Brd4在维持 染色质构象促进增强子驱动的致癌基因转录。这一机制 染色质重组，如果是真的，是范式的转变，并将对串联的研究产生广泛的影响 组蛋白结合域。我们还假设新陈代谢的变化导致不同的翻译后 被溴域“读”到的组蛋白的修饰。然而，更广泛的酰化和蛋白质结合 对溴域的特异性知之甚少。我们已经开始在我们最近的 重点介绍代谢衍生的酰化反应和邻近修饰如何调整BET的出版物 溴结构域与组蛋白结合。为了继续解决这个广泛的新陈代谢问题，我们正在使用 生物物理、结构生物学和蛋白质组学技术研究BET溴域酰化和 将酰基辅酶A代谢与转录联系起来的蛋白质选择性(焦点2)。为了帮助我们进行机械化的调查， 我们正在消除BET溴域生物学研究中的一个关键障碍：缺乏抑制剂和化学物质 选择性地针对单个BET蛋白的探针。目前，所有现有的BET抑制剂都针对Brd2，Brd3， BRD4和BRDT，具有相同的纳摩尔效力。这种缺乏选择性可能是导致 记忆丧失和淋巴毒性最近与现有的PAN-BET抑制剂有关。我们正在克服 这些障碍用一种新的基于片段的配基发现和化学生物学策略来发现 选择性Brd4抑制剂通过共价靶向Brd4中的独特半胱氨酸(焦点3)。这些化学工具 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 在开发针对癌症和糖尿病的Brd4轴的治疗药物方面也可能有用。

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 Our unpublished data support the hypothesis that tandem bromodomains act as a scaffold for acetylation-dependent reorganization of chromatin; for instance, joining promotors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