Defining Nuclear H2O2 Regulation by Covalent Regulators

通过共价调节剂定义核 H2O2 调节

• 批准号: 10725269
• 负责人: Liron Bar-Peled
• 金额: $ 45.89万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10725269
• 关键词: Acrylamides; Aging; Biological; Biological Aging; Biological Assay; Biological Process; Biology; Caenorhabditis elegans; Cell Aging; Cell Line; Cell Nucleus; Cells; Chemical Agents; Chemicals; Cysteine; Cytosol; DNA Damage; Data; Detection; Development; Disease; Drug Metabolic Detoxication; Equilibrium; Fibroblasts; Genetic; Genetic Models; Genomic Instability; Goals; Golgi Apparatus; Homeostasis; Hydrogen Peroxide; Investments; Knowledge; Libraries; Link; Longevity; Maintenance; Malignant Neoplasms; Measures; Metabolic; Metabolic Diseases; Methods; Mitochondria; Neurodegenerative Disorders; Nuclear; Nucleic Acids; Organelles; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Phenotype; Play; Population; Process; Protein Overexpression; Proteins; Proteomics; Reactive Oxygen Species; Regulation; Reporter; Research; Resolution; Role; Sensitivity and Specificity; Series; Shapes; Signal Transduction; Specificity; Technology; Testing; chemoproteomics; chemotherapy; complex biological systems; genome integrity; in vivo; inhibitor; innovation; kinase inhibitor; loss of function; mouse model; next generation; pharmacologic; protein function; screening; senescence; sensor; small molecule; small molecule libraries; success; targeted treatment; tool

Project Summary Loss of metabolic homeostasis is through to contribute to aging and replicative senescence. Control of redox balance is critical for maintenance of metabolic homeostasis. Improper levels of reactive oxygen species (ROS) are thought to be an important contributor to multiple aging related diseases such as metabolic diseases, cancer and neurodegenerative disorders. At high levels, ROS modify nucleic acids–an important mechanism by which this group of reactive metabolites leads to genomic instability and replicative senescence. While there has been much investment into understanding how different ROS damage nucleic acids, surprisingly little is known about the pathways which generate, detoxify and sense nuclear ROS. This knowledge-gap hampers our understanding of the roles nuclear ROS plays during biological aging. A major obstacle to deciphering the biology of nuclear ROS is the inability to control the levels of ROS in a nucleus-specific manner, through defined mechanisms of action. Small molecules have been instrumental in biological breakthroughs often regulating biological processes at a level of specificity and precision not achievable with even the most advanced genetic models. The purpose of this application is to develop a suite of chemical probes that specifically increase nuclear ROS levels and characterize their corresponding protein targets. We will do so, by combining a nuclear-localized H2O2 sensor (HyPer7) with a chemical proteomic-guide small molecule screen. We have previously used these approaches to identify a small inhibitor that increases C elegans longevity by ~45% and characterized its target protein, providing support for the great utility of chemoproteomic screening approaches to study biological aging. Here, we will leverage a cysteine-focused small molecule library (5000+ chemically diverse chloroacetamide/acrylamides) to identify covalent probes that increase steady state nuclear H2O2 levels. We focus on cysteines given their critical role in protein function and the ability to identify covalent inhibitors that engage them using chemoproteomics. A preliminary screen of 270+ molecules has already furnished 9 compounds that specifically increase nuclear H2O2 levels but not at other compartments. We will subsequently use chemical proteomics to identify the corresponding protein target and determine their importance in regulating nuclear H2O2 levels and replicative senescence. The research proposed herein, takes full advantage of a series of recently developed methods: genetically encoded ROS reporters and chemical proteomics, which have previously been used in isolation, to be integrated into an effective approach to identify the pathways that control nuclear H2O2 levels. These studies will provide both a deeper understanding of the key pathways involved in nuclear ROS regulation and develop a much-needed suite of pharmacological agents to study how ROS in different compartments shapes biological aging.

项目摘要 代谢稳态的丧失会导致衰老和复制性衰老。氧化还原控制 平衡对于维持代谢稳态至关重要。活性氧（ROS）水平不当 被认为是多种衰老相关疾病的重要因素，如代谢疾病、癌症、 和神经变性疾病。在高水平下，ROS修饰核酸-一种重要的机制， 这组活性代谢物导致基因组不稳定性和复制性衰老。虽然已经 为了了解不同的ROS如何破坏核酸，人们投入了大量的资金，但令人惊讶的是， 产生、解毒和感知核ROS的途径。这种知识鸿沟阻碍了我们的理解 在生物衰老过程中细胞核活性氧的作用。破译核生物学的主要障碍 ROS是不能以细胞核特异性的方式控制ROS水平，通过确定的机制， 行动上小分子在生物学突破中发挥了重要作用，经常调节生物过程 其特异性和精确度即使是最先进的遗传模型也无法实现。目的 这项应用的目的是开发一套化学探针，专门增加核ROS水平， 表征其相应的蛋白质靶标。我们将通过结合核定位的H2 O2传感器 （Hyper 7）与化学蛋白质组学引导的小分子筛选。我们以前使用过这些方法 为了鉴定一种能使秀丽隐杆线虫寿命延长约45%的小抑制剂，并表征其靶蛋白， 为化学蛋白质组学筛选方法研究生物衰老的巨大效用提供支持。在这里， 我们将利用半胱氨酸为重点的小分子库（5000+化学多样性 氯乙酰胺/丙烯酰胺）以鉴定增加稳态核H2 O2水平的共价探针。我们 由于半胱氨酸在蛋白质功能中的关键作用以及识别共价抑制剂的能力， 用化学蛋白质组学吸引他们。对270多个分子的初步筛选已经提供了9个 这些化合物专门增加核H2 O2水平，但不增加其他隔室的水平。我们随后将 使用化学蛋白质组学来识别相应的蛋白质靶点，并确定它们在调节 核H2 O2水平和复制衰老。本文提出的研究充分利用了一系列 最近开发的方法：基因编码的ROS报告和化学蛋白质组学， 以前被孤立使用，被整合到一个有效的方法，以确定控制的途径， 核H2 O2水平。这些研究将使人们更深入地了解参与的关键途径 核活性氧调节，并开发一套急需的药理学药物，以研究活性氧如何在 不同的隔间形成生物老化。