Biochemical mechanisms of beta cell protection through bromodomain inhibition

项目摘要 自身免疫性糖尿病的特征在于胰岛内和周围的炎症反应， 选择性破坏产生胰岛素的β细胞。自身免疫性糖尿病的低一致率， 单卵双胞胎表明表观遗传因素在糖尿病发病中的重要作用，但对其了解甚少 和进步。溴结构域是组蛋白上赖氨酸乙酰化和转录的表观遗传“读者” 溴结构域与乙酰化组蛋白/蛋白质的结合调节细胞类型依赖性转录 方式用布罗莫结构域抑制剂早期治疗非肥胖糖尿病（NOD）小鼠， 最近显示末端外（BET）家族（Brd 2 -4）抑制自身免疫性糖尿病的发展。 BET抑制的保护作用与抗炎和促增殖表型相关， 巨噬细胞和β细胞;然而，机制知之甚少。我们假设 Brd 4调节胰岛中β细胞增殖和巨噬细胞炎症， 是自身免疫性疾病中泛BET抑制剂的责任。与这一假设相一致，泛BET 抑制与受损的学习和记忆以及降低的免疫系统功能有关。作为 自身免疫性糖尿病的表观遗传干预代表了一种新的治疗靶点， 人口，脱靶效应必须最小化。Brd 4抑制作为自身免疫性疾病的治疗策略 糖尿病将在三个具体目标中进行检查：目标1）测试Brd 4抑制防止糖尿病的假设。 巨噬细胞活化和已知损伤β细胞的炎性介质的产生。研究将建立 我们的初步数据显示，泛BET抑制剂减弱巨噬细胞产生的炎性细胞因子， 介质如IL-1β和一氧化氮。目的2）测试Brd 4抑制保护β细胞免于 通过刺激DNA损伤修复途径和保护线粒体免受 损害研究将使用Brd 2、Brd 3和Brd 4缺陷的胰岛素瘤细胞和化学抑制剂来探索 BET蛋白在调节β细胞对炎症介质的反应和激活炎症介质中的作用。 促进β细胞从氧化应激中恢复的防御途径。目的3）开发选择性Brd 4抑制剂 有效治疗自身免疫性糖尿病我们提供证据来支持我们的创新方法， 使用共价靶向Brd 4特有的特定氨基酸残基选择性抑制Brd 4。生物化学， 分子、免疫学、细胞生物学、遗传学和化学生物学方法将用于 研究BET抑制剂保护β细胞的分子和细胞途径， 选择性靶向BET家族转录调节因子的新工具和试剂。我们的长期目标 阐明BET溴结构域转录调控的细胞类型特异性机制， 开发新的治疗剂以选择性地抑制单个BET蛋白的活性，作为治疗的初始步骤。 设计策略来阻止自身免疫性糖尿病的发展和进展。

PROJECT SUMMARY Autoimmune diabetes is characterized by an inflammatory reaction in and around pancreatic islets followed by selective destruction of insulin producing β-cells. Low concordance rates of autoimmune diabetes in monozygotic twins indicate an important but poorly understood role for epigenetic factors in diabetes initiation and progression. Bromodomains are epigenetic “readers” of lysine acetylation on histones and transcription factors; bromodomain binding to acetylated histones/proteins regulates transcription in a cell-type dependent manner. Early treatment of non-obese diabetic (NOD) mice with an inhibitor of the bromodomain and extraterminal (BET) family (Brd2-4) was recently shown to suppress development of autoimmune diabetes. The protective effects of BET inhibition correlated with anti-inflammatory and pro-proliferative phenotypes in macrophages and β-cells, respectively; however, the mechanisms are poorly understood. We hypothesize that Brd4 regulates β-cell proliferation and macrophage inflammation in islets, and that inhibition of Brd2 and Brd3 are liabilities of pan-BET inhibitors in autoimmune diseases. Consistent with this hypothesis, pan-BET inhibition is associated with impaired learning and memory as well as reduced immune system function. As epigenetic intervention in autoimmune diabetes represents a novel therapeutic target in a primarily pediatric population, off-target effects must be minimized. Brd4 inhibition as a therapeutic strategy in autoimmune diabetes will be examined in three specific aims: Aim 1) Test the hypothesis that Brd4 inhibition prevents macrophage activation and production of inflammatory mediators known to damage β-cells. Studies will build on our preliminary data showing that pan-BET inhibitors attenuate macrophage production of inflammatory mediators such as IL-1β and nitric oxide. Aim 2) Test the hypothesis that Brd4 inhibition protects β-cells from cytokine-mediated damage by stimulating DNA damage repair pathways and protecting mitochondria from damage. Studies will use insulinoma cells deficient in Brd2, Brd3, and Brd4 and chemical inhibitors to explore the role of BET proteins in the regulation of β-cell responses to inflammatory mediators and the activation of defense pathways that facilitate β-cell recovery from oxidative stress. Aim 3) Develop selective Brd4 inhibitors for effective treatment of autoimmune diabetes. We provide evidence to support our innovative approach to selectively inhibit Brd4 using covalent targeting of a specific amino acid residue unique to Brd4. Biochemical, molecular, immunological, cell biological, genetic, and chemical biological approaches will be used to investigate the molecular and cellular pathways through which BET inhibition protects β-cells and to develop novel tools and reagents to selectively target the BET family of transcriptional regulators. Our long-term goals are to elucidate the cell-type specific mechanisms of transcriptional regulation by BET bromodomains and develop novel therapeutics to selectively inhibit the activity of individual BET proteins as an initial step in the design of strategies to halt the development and progression of autoimmune diabetes.