Fluorescence Polarization Probes for Universal Coverage of the Human Bromodomains

用于普遍覆盖人类布罗莫结构域的荧光偏振探针

• 批准号: 8981746
• 负责人: Konrad T. Howitz
• 金额: $ 22.29万
• 依托单位: REACTION BIOLOGY CORPORATION
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8981746
• 关键词: Acetylation; Address; Affect; Affinity; Amino Acid Sequence; Atherosclerosis; Biological; Biological Assay; Biological Factors; Biology; Bromodomain; Cells; Chalcones; Chemicals; Chromatin; Collection; Coupled; Cutaneous; DNA; DNA Sequence; Development; Disease; Enzymes; Epigenetic Process; FDA approved; Family; Flavones; Flavonoids; Fluorescence Polarization; Fluorescent Probes; Gene Activation; Gene Expression; Gene Expression Regulation; Genetic; Goals; Histone Acetylation; Histones; Human; Individual; Inflammation; Inherited; Investigation; Libraries; Lysine; Maintenance; Malignant Neoplasms; Mediating; Metabolic Diseases; Methylation; Modification; Morphologic artifacts; N-terminal; Nerve Degeneration; Non-Insulin-Dependent Diabetes Mellitus; Perception; Pharmaceutical Preparations; Phase; Plants; Play; Post-Translational Protein Processing; Process; Proteins; Reaction; Reader; Relative (related person); Research; Rhodamine B; Role; Scanning; Services; Source; Structure; System; T-Cell Lymphoma; Tail; Therapeutic Agents; Universal Coverage; Virus Diseases; Work; analog; assay development; base; epigenetic regulation; epigenomics; histone modification; inhibitor/antagonist; kinase inhibitor; melting; programs; public health relevance; screening

 DESCRIPTION (provided by applicant): Post-translational modification (PTM) of the lysines and other residues in the N-terminal tails of histones are known to have profound effects on the structure and function of chromatin, perhaps most notably on the regulation of gene expression. One of these 'epigenetic' histone modifications, the PTM lysine acetylation is generally associated with gene activation. Epigenetic regulation of gene expression plays significant roles in normal development, as well as in diseases, including cancer, neurodegeneration and metabolic disease. The 'perception' of the chromatin acetylation state is mediated by proteins that contain a type of modular "epigenetic reader" domain, a bromodomain. The human bromodomains, 61 different domains present in 46 distinct proteins, are the principal known class of acetyllysine readers. The bromodomains, particular a subclass known as the BET family are implicated in various disease states including cancer, inflammation, type II diabetes, atherosclerosis and viral infections. Investigation of the biological roles and disease connections of the BET family has been aided tremendously by the development of high affinity inhibitors, sometimes termed chemical "probes", which also show promise as possible therapeutic agents for cancer and other diseases. The development of probes for many of the non-BET bromodomains, however, has lagged and to some extent this may be due to deficiencies in the available assay systems, which can on the one hand be simple to use and relative artefact free, albeit not very quantitative (thermal melt/shift, aka Differential Scanning Fluorimetry) and on the other hand, highly sensitive and quantitative assays (AlphaScreen) that are expensive, relative difficult to implement and prone to numerous 'false hit' artefact when screening compound libraries. We propose to address this problem by developing a suite of simple to perform, quantitative but, inexpensive fluorescence polarization (FP) assays, based on fluorophor-conjugated bromodomain inhibitor compounds. Our goal is for these assays to be broadly applicable to the 61 human bromodomains. In order to accomplish that efficiently, we propose to screen for 'promiscuous' bromodomain inhibitors, so that relatively few of these, in fluorophor-conjugated form, will be needed to allow convenient assay of the entire set of human bromodomains. We argue, and have some preliminary evidence to suggest, that phenolic plant natural products, particularly the chalcones and flavones may be relatively rich in suitable inhibitor compounds. Hits from screening these and other compounds classes, along with already developed probe compounds will be coupled to fluorophors to produce FP probes and these will be used to develop the assays.

 描述（由申请人提供）：已知组蛋白 N 末端尾部的赖氨酸和其他残基的翻译后修饰 (PTM) 对染色质的结构和功能具有深远的影响，也许最显着的是对基因的调控PTM 赖氨酸乙酰化是这些“表观遗传”组蛋白修饰之一，通常与基因表达的表观遗传调控相关，在正常发育和正常发育中发挥着重要作用。在癌症、神经退行性疾病和代谢疾病等疾病中，染色质乙酰化状态的“感知”是由含有一种模块化“表观遗传读取器”结构域（人类溴结构域）的蛋白质介导的，该结构域存在于 46 个不同的结构域中。溴结构域，特别是被称为 BET 家族的一个亚类，与多种疾病状态有关，包括癌症、炎症、II 型糖尿病、动脉粥样硬化和病毒感染的生物学作用和疾病关系的研究。 高亲和力抑制剂（有时称为化学“探针”）的开发极大地帮助了 BET 家族的发展，这些抑制剂也显示出作为癌症和其他疾病的可能治疗剂的前景。然而，已经滞后，在某种程度上，这可能是由于可用测定系统的缺陷，一方面，该系统使用简单且相对无伪影，尽管不是很定量（热熔化/转移，又名差示扫描荧光测定法）和 另一方面，高灵敏度和定量分析（AlphaScreen）价格昂贵，相对难以实施，并且在筛选化合物库时容易出现大量“误击”伪影，我们建议通过开发一套易于执行的定量但不准确的方法来解决这个问题。 ，基于荧光团缀合的溴结构域抑制剂化合物的廉价荧光偏振 (FP) 测定，我们的目标是使这些测定广泛适用于 61 种人类溴结构域。筛选“混杂的”布罗莫结构域抑制剂，因此需要相对较少的荧光团缀合形式的抑制剂才能方便地测定整套人类布罗莫结构域，我们认为，并有一些初步证据表明，酚类。植物天然产物，特别是查酮和黄酮，可能含有相对丰富的合适的抑制剂化合物，通过筛选这些化合物和其他化合物类别，以及已经开发的探针化合物，将与荧光团偶联以产生。 FP 探针将用于开发检测方法。