Mechanism-Based Discovery of Protectants Against Iron-Dependant Oxidative Injury

基于机制的铁依赖性氧化损伤保护剂的发现

• 批准号: 8255042
• 负责人: Justin M Schwartz
• 金额: $ 6.72万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-09 至 2012-09-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8255042
• 关键词: Acetaminophen; Antioxidants; Apoptosis; Blood; Brain Hypoxia-Ischemia; Calcium; Cell Death; Cell Survival; Cells; Cellular Stress; Cessation of life; Chelating Agents; Chemistry; Complications of Diabetes Mellitus; Confocal Microscopy; Cytoprotection; Cytoprotective Agent; Cytosol; Deferoxamine Methanesulfonate; Development; Dextrans; Diabetes Mellitus; Disease; Doxycycline; Egtazic Acid; Evaluation; Event; Fluorescent Probes; Goals; Hepatic; Hepatocyte; Hydroxyl Radical; Hypoxia; Injury; Intervention; Iron; Ischemia; Lead; Liver Mitochondria; Lysosomes; Mass Spectrum Analysis; Mediating; Membrane; Membrane Potentials; Mentors; Metalloproteases; Minocycline; Mitochondria; Modeling; Morbidity - disease rate; Myocardial Infarction; Necrosis; Neutral Red; Oxidative Stress; Pathway interactions; Permeability; Plastoquinone; Process; Production; Propidium Diiodide; Rattus; Reaction; Reactive Oxygen Species; Reperfusion Injury; Reperfusion Therapy; Rhodamine 123; Role; Screening procedure; Starch; Stress; Stroke; Testing; Tetracyclines; Time; Tissues; Toxic effect; Ubiquinone; United States; Work; base; calcium uniporter; cell injury; clinically relevant; cytotoxicity; dextran; fluorexon; fluorophore; inhibitor/antagonist; mitochondrial dysfunction; mitochondrial membrane; mortality; novel; novel therapeutic intervention; pharmacophore; prevent; research study; rhod 2-AM; success; uptake

DESCRIPTION (provided by applicant): Ischemia-reperfusion (I/R) injury by way of heart attacks, strokes and diabetes is one of the major causes of morbidity and mortality in the United States. Production of reactive oxygen species (ROS) after reintroduction of oxygenated blood to ischemic tissue is a major precipitating event causing necrosis and apoptosis in I/R injury. The most reactive and toxic ROS is the hydroxyl radical (OH7) formed by the iron- catalyzed Fenton reaction. The majority of chelatable iron capable of catalyzing the Fenton chemistry is normally contained in lysosomes. Based on preliminary studies, it is probable that iron-dependent radical chemistry during oxidative stress leads to lysosomal membrane disruption and release of chelatable iron into the cytosol. Mitochondria then take up this iron via the electrogenic calcium uniporter to promote intramitochondrial OH7 formation, the mitochondrial permeability transition (MPT) and ultimately cell death. The overall goal of the project to better characterize this process and to develop novel and potentially clinically relevant interventions to block this pathophysiological pathway and prevent lethal cell injury. To achieve this, two specific aims are proposed: 1. Screening of tetracycline-derived compounds to test the hypothesis that cytoprotective compounds are blockers of the mitochondrial calcium uniporter. In preliminary experiments, two tetracycline-derived compounds were shown to be protective against hypoxic and I/R injury. This cytoprotection was associated with inhibition of the mitochondrial electrogenic Ca2+/Fe2+ uniporter. Accordingly, a larger panel of tetracycline-derived compounds will be screened to confirm the association of cytoprotection with uniporter inhibition and to identify a pharmacophore for the cytoprotective compounds. Through use of chelators and confocal microscopy of iron- and calcium-indicating fluorophores, the specific roles of iron and calcium uptake to mitochondrial dysfunction during oxidative stress will be determined. 2. Synthesis and evaluation of lysosomal targeted antioxidants (LTAs) as cytoprotectants against iron- dependant oxidative injury. Antioxidant protection of lysosomes should prevent lysosomal disintegration and iron release and thereby decrease cytosolic iron available for mitochondrial uptake and ROS formation during oxidative stress. As no such LTA currently exists, LTAs will be synthesized and tested for their ability to localize in lysosomes and prevent lysosomal membrane disruption and iron release after acetaminophen- induced oxidative stress. LTAs and cytoprotective tetracycline-derived compounds will also be tested for their ability to protect against acetaminophen toxicity and I/R injury. Overall, these studies will lead to a better understanding of the interrelationships of lysosomal and mitochondrial dysfunction in oxidative, I/R and hepatotoxic stress and lead to new therapeutic interventions to minimize cell and tissue damage. PUBLIC HEALTH RELEVANCE: Ischemia/reperfusion injury occurring in heart attack, stroke and complications of diabetes is one of the leading causes of suffering and death in the United States. Iron appears to predispose these diseases. My project proposes to better understand the role of iron mobilization from lysosomes to mitochondria in ischemia/reperfusion injury and to develop novel therapies to mitigate or eliminate damage from this condition.

描述（由申请人提供）：在美国，由心脏病发作、中风和糖尿病引起的缺血-再灌注（I/R）损伤是发病率和死亡率的主要原因之一。在缺血再灌注损伤中，氧合血回输到缺血组织后产生的活性氧（ROS）是导致坏死和凋亡的主要促发因素。最具活性和毒性的ROS是由铁催化的芬顿反应形成的羟基自由基（OH 7）。能够催化芬顿化学的大部分可螯合铁通常包含在溶酶体中。基于初步研究，氧化应激期间的铁依赖性自由基化学可能导致溶酶体膜破坏并将可螯合铁释放到胞质溶胶中。然后线粒体通过生电钙单向转运体吸收这种铁，以促进线粒体内OH 7形成、线粒体通透性转换（MPT）和最终细胞死亡。该项目的总体目标是更好地表征这一过程，并开发新的和潜在的临床相关干预措施，以阻断这一病理生理途径，防止致命的细胞损伤。为了实现这一目标，提出了两个具体目标：1。筛选四环素衍生化合物以检验细胞保护化合物是线粒体钙单向转运体阻断剂的假设。在初步实验中，两种四环素衍生的化合物显示出对缺氧和I/R损伤的保护作用。这种细胞保护作用与抑制线粒体产电Ca 2 +/Fe 2+单向转运体有关。因此，将筛选更大组的四环素衍生化合物以确认细胞保护与单向转运体抑制的关联并鉴定细胞保护化合物的药效团。通过使用螯合剂和共聚焦显微镜的铁和钙指示荧光团，铁和钙的摄取在氧化应激过程中线粒体功能障碍的具体作用将被确定。2.溶酶体靶向抗氧化剂（LTA）作为抗铁依赖性氧化损伤的细胞保护剂的合成和评价。溶酶体的抗氧化保护应防止溶酶体崩解和铁释放，从而减少氧化应激期间可用于线粒体摄取和ROS形成的细胞溶质铁。由于目前不存在此类LTA，因此将合成LTA并测试其定位于溶酶体中并防止对乙酰氨基酚诱导的氧化应激后溶酶体膜破坏和铁释放的能力。还将测试LTA和细胞保护性四环素衍生化合物防止对乙酰氨基酚毒性和I/R损伤的能力。总体而言，这些研究将导致更好地了解氧化，I/R和肝毒性应激中溶酶体和线粒体功能障碍的相互关系，并导致新的治疗干预措施，以尽量减少细胞和组织损伤。 公共卫生相关性：在美国，心脏病发作、中风和糖尿病并发症中发生的缺血/再灌注损伤是痛苦和死亡的主要原因之一。铁似乎易诱发这些疾病。我的项目旨在更好地了解铁从溶酶体动员到线粒体在缺血/再灌注损伤中的作用，并开发新的疗法来减轻或消除这种情况下的损害。