Biochemical mechanisms of beta cell protection through bromodomain inhibition

项目总结 自身免疫性糖尿病的特征是胰岛及其周围的炎症反应，随后是 选择性破坏产生胰岛素的β细胞。自身免疫性糖尿病的低符合率 同卵双胞胎提示表观遗传因素在糖尿病发病中的重要作用，但知之甚少 和进步。溴结构域是组蛋白和转录上赖氨酸乙酰化的表观遗传“阅读器” 因子；溴域与乙酰化组蛋白/蛋白结合调节细胞类型依赖的转录 举止。非肥胖型糖尿病(NOD)小鼠早期使用溴域抑制剂和 最近发现BET家族(Brd2-4)可以抑制自身免疫性糖尿病的发生。 BET抑制与抗炎和促增殖表型相关的保护作用 巨噬细胞和β细胞；然而，其机制尚不清楚。我们假设 Brd4调节胰岛β-细胞增殖和巨噬细胞炎症，并抑制Brd2和Brd3 是PAN-BET抑制剂在自身免疫性疾病中的责任。与这一假设一致的是，泛赌注 抑制与学习和记忆受损以及免疫系统功能降低有关。AS 表观遗传学干预对自身免疫性糖尿病是一种新的治疗靶点 人口，必须将偏离目标的影响降至最低。抑制BRD4作为自身免疫的一种治疗策略 糖尿病将在三个具体目标中进行检查：目标1)检验Brd4抑制可预防的假设 巨噬细胞的激活和炎性介质的产生可破坏β细胞。研究将建立 根据我们的初步数据显示，PAN-BET抑制剂可以减少巨噬细胞炎症的产生 IL-1、β、一氧化氮等介质。目的2)验证Brd4抑制保护β细胞 细胞因子通过刺激DNA损伤修复途径和保护线粒体来介导损伤 损坏。研究将使用缺乏Brd2、Brd3和Brd4的胰岛素瘤细胞和化学抑制剂来探索 BET蛋白在调节β细胞对炎性介质反应及活化中的作用 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Brd4抑制剂 用于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Brd4特有的特定氨基酸残基的共价靶向选择性地抑制Brd4。生化的， 将使用分子、免疫学、细胞生物学、遗传学和化学生物学方法来 研究抑制BET保护β-细胞及其发育的分子和细胞途径 选择性靶向BET转录调控因子家族的新工具和试剂。我们的长期目标 是为了阐明BET溴域和BET的转录调控的细胞类型特异性机制 开发新的疗法来选择性地抑制单个BET蛋白的活性，作为治疗的第一步 设计策略以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.