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

描述（由申请人提供）：由心脏病发作、中风和糖尿病引起的缺血再灌注（I/R）损伤是美国发病率和死亡率的主要原因之一。氧合血重新注入缺血组织后活性氧（ROS）的产生是I/R损伤中引起坏死和细胞凋亡的主要沉淀事件。最具活性和毒性的活性氧是由铁催化的芬顿反应形成的羟基自由基（OH7）。大多数能够催化芬顿化学的螯合铁通常包含在溶酶体中。根据初步研究，氧化应激期间铁依赖的自由基化学可能导致溶酶体膜破坏和可螯合铁释放到细胞质中。然后，线粒体通过电致钙单转运器吸收这些铁，促进线粒体内OH7的形成，线粒体通透性转变（MPT）并最终导致细胞死亡。该项目的总体目标是更好地描述这一过程，并开发新的、潜在的临床相关干预措施，以阻断这一病理生理途径，防止致命细胞损伤。为实现这一目标，提出了两个具体目标：筛选四环素衍生化合物，以验证细胞保护化合物是线粒体钙单转运体阻滞剂的假设。在初步实验中，两种四环素衍生化合物被证明对缺氧和I/R损伤具有保护作用。这种细胞保护作用与线粒体电致Ca2+/Fe2+单转运蛋白的抑制有关。因此，将筛选更大范围的四环素衍生化合物，以确认细胞保护与单转运蛋白抑制的关联，并确定细胞保护化合物的药效团。通过使用螯合剂和铁和钙指示荧光团的共聚焦显微镜，将确定氧化应激期间铁和钙摄取对线粒体功能障碍的具体作用。2. 溶酶体靶向抗氧化剂（LTAs）作为铁依赖性氧化损伤细胞保护剂的合成与评价。溶酶体的抗氧化保护应防止溶酶体解体和铁释放，从而减少氧化应激过程中线粒体摄取和ROS形成所需的胞质铁。由于目前不存在这样的LTA，我们将合成LTA并测试其在溶酶体中的定位能力，以及在对乙酰氨基酚诱导的氧化应激后阻止溶酶体膜破坏和铁释放的能力。还将测试LTAs和细胞保护性四环素衍生化合物对对乙酰氨基酚毒性和I/R损伤的保护能力。总的来说，这些研究将使我们更好地了解溶酶体和线粒体功能障碍在氧化应激、I/R和肝毒性应激中的相互关系，并导致新的治疗干预措施，以尽量减少细胞和组织损伤。

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.