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

描述(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。将含氧血重新注入缺血组织后产生的活性氧物种(ROS)是导致I/R损伤中坏死和细胞凋亡的主要沉淀事件。最具活性和毒性的ROS是铁催化的Fenton反应生成的羟基自由基(OH7)。能够催化芬顿化学的大多数可螯合铁通常包含在溶酶体中。根据初步研究，氧化应激过程中铁依赖的自由基化学可能导致溶酶体膜的破坏和可螯合铁释放到细胞质中。然后，线粒体通过电生钙单一转运体吸收这些铁，促进线粒体内OH7的形成，线粒体通透性转变(MPT)，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，防止致命细胞损伤。为了实现这一目标，提出了两个具体的目标：1.筛选四环素类化合物，以验证细胞保护性化合物是线粒体钙单转运体阻滞剂的假设。在初步实验中，两个四环素类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抑制线粒体电生钙/铁单一转运体有关。因此，将筛选更多的四环素衍生化合物，以确认细胞保护与单转运体抑制的关联，并确定细胞保护化合物的药效团。通过使用螯合剂和指示铁和钙的荧光团的共聚焦显微镜，将确定铁和钙摄取在氧化应激期间线粒体功能障碍中的具体作用。2.抗铁依赖性氧化损伤细胞保护剂溶酶体靶向抗氧化剂的合成与评价。溶酶体的抗氧化保护应该防止溶酶体的解体和铁的释放，从而减少氧化应激过程中可用于线粒体摄取和ROS形成的胞内铁。由于目前还不存在这样的LTA，LTA将被合成并测试它们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防止溶酶体膜破裂和铁释放的能力。LTA和具有细胞保护作用的四环素衍生化合物也将接受测试，以确定它们抵御对乙酰氨基酚毒性和I/R损伤的能力。总体而言，这些研究将有助于更好地了解氧化、缺血/再灌注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，将细胞和组织损伤降至最低。 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/再灌注损伤是导致患者痛苦和死亡的主要原因之一。铁似乎是这些疾病的诱因。我的项目建议更好地了解从溶酶体到线粒体的铁动员在缺血/再灌注损伤中的作用，并开发新的治疗方法来减轻或消除这种情况下的损害。

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.