Development of Chemical Tools to Probe and Inhibit Iron-Induced Oxidative Stress

描述（由申请人提供）：氧化应激与多种疾病有关，包括糖尿病、炎症、缺血后再灌注、UV暴露、动脉粥样硬化、衰老、黄斑变性和神经变性.脂质过氧化、DNA羟基化和蛋白质修饰是氧化应激的标志物，其主要由与高活性羟基自由基（OH）的反应引起，OH本身是铁催化的与氧物种反应的产物。虽然铁是健康细胞的重要和有益成分，但这种有害的反应表明细胞中不稳定的铁会引起严重的损伤。我们的长期目标是开发药用铁螯合剂，选择性地抑制这种有害的铁促进氧化应激部位的损伤，同时避免通常与铁螯合治疗相关的毒性。除了作为潜在的有用的药剂，所提出的前螯合剂也将作为荧光探针，以可视化铁在活细胞中的氧化应激中的作用。为了实现这些目标，本提案侧重于合成（具体目标1）和表征（具体目标2）新的前螯合剂家族所需的化学，其中掩蔽基团阻断了其他高亲和力铁螯合剂的关键金属结合功能。掩蔽基团是硼酸酯，因为这些基团被过氧化氢（氧化应激的一种组分）脱保护。在具体目标3中，我们开发了荧光类似物来探测活细胞内铁促进的氧化应激。一旦合成和表征确定了其体外特性，将在细胞培养物中测试这类新分子，以验证其通过在氧化应激源处去除铁来保护细胞免受氧化损伤的功效（具体目标4）。目前可用的螯合疗法面临改变健康金属分布和抑制关键金属酶的毒副反应。在不存在过氧化氢的情况下，所提出的掩蔽螯合剂将是无害的旁观者，不会干扰有益的金属。然而，增加过氧化氢水平的疾病状况会激活并暴露一种有效的螯合剂，该螯合剂可以螯合并去除作为OH生成来源的铁。与典型的抗氧化剂不同，只有在有害自由基产生后才能中和它们，有效的铁螯合剂可以通过禁用来源完全消除它们的产生。本文提出的分子代表了一种有前途的新策略，对许多人类疾病具有潜在影响，特别是那些正常金属离子稳态受损或发生异常金属积累的疾病。像帕金森病和老年性黄斑变性这样的退行性疾病只是两个例子。公共卫生相关性：超过一百万美国人患有帕金森病，这是一种进行性神经变性，没有已知的治疗方法，估计每年在美国的直接和间接费用为250亿美元。虽然其原因尚不清楚，但新出现的假说暗示铁诱导的氧化应激是神经元损伤的来源。本文提出的新的前螯合剂分子被设计为保护细胞免受这种类型的损伤，因此代表了一种有前途的新策略，其不仅有可能影响帕金森病，而且还有可能影响其他神经退行性疾病、年龄相关性黄斑变性和与氧化应激相关的各种其他人类疾病。

DESCRIPTION (provided by applicant): Oxidative stress is implicated in a wide variety of diseases, including diabetes, inflammation, post- ischemia reperfusion, UV exposure, atherosclerosis, aging, macular degeneration, and neurodegeneration. Lipid peroxidation, DNA hydroxylation, and protein modification are markers of oxidative stress that result primarily from reactions with the highly reactive hydroxyl radical, OH, itself a product of iron-catalyzed reactions with oxygen species. Whereas iron is an essential and beneficial component of healthy cells, this deleterious reactivity suggests that labile iron in the cell provokes serious damage. Our long-term goals are to develop medicinal iron chelating agents that selectively inhibit this deleterious iron-promoted damage at the site of oxidative stress while avoiding toxicity commonly associated with iron chelation therapy. In addition to being potentially useful medicinal agents, the proposed pro-chelators will also serve as fluorescent probes to visualize the role of iron in oxidative stress in living cells. In order to achieve these goals, this proposal focuses on the chemistry required to synthesize (Specific Aim 1) and characterize (Specific Aim 2) new families of pro-chelators in which a masking group blocks key metal-binding functionalities of otherwise high-affinity iron chelators. The masking groups are boronate esters, since these groups are deprotected by hydrogen peroxide, a component of oxidative stress. In Specific Aim 3 we develop fluorescent analogs to probe iron-promoted oxidative stress inside living cells. Once the synthesis and characterization establishes their in vitro properties, this new class of molecules will be tested in cell culture to validate their efficacy for protecting cells against oxidative damage by removing iron at the source of oxidative stress (Specific Aim 4). Currently available chelation therapies face toxic side reactions that alter healthy metal distribution and inhibit critical metalloenzymes. In the absence of hydrogen peroxide, the proposed masked chelators will be innocuous bystanders that will not interfere with beneficial metals. Disease conditions that increase hydrogen peroxide levels, however, activate and unmask a potent chelator that sequesters and removes the iron that is the source of OH generation. Unlike typical antioxidants that neutralize harmful free radicals only after they are produced, effective iron chelators can eliminate their production altogether by disabling the source. The molecules proposed herein represent a promising new strategy with potential impact on a number of human diseases, especially those where normal metal ion homeostasis is impaired or where aberrant metal accumulation takes place. Degenerative diseases like Parkinson's disease and age-related macular degeneration are just two examples. PUBLIC HEALTH RELEVANCE: Over a million Americans suffer from Parkinson's disease, a progressive form of neurodegeneration with no known cure that is estimated to cost $25 billion per year in direct and indirect costs in the United States. Although its cause is not known, emerging hypotheses implicate iron-induced oxidative stress as a source of neuronal damage. The new pro-chelator molecules proposed herein are designed to protect cells against precisely this type of damage, and therefore represent a promising new strategy with the potential to impact not only Parkinson's disease, but also other neurodegenerative conditions, age-related macular degeneration, and a wide variety of other human diseases associated with oxidative stress.