Molecular Probes of the Mechanisms of Cytocrhome P450

这个项目的中心主题是研究药物代谢酶细胞色素P450和其他血红素蛋白，如肌红蛋白和细胞色素c的作用机制。我们还研究了金属卟啉模型化合物，以阐明这些酶过程是如何发生的。主要的方法包括酶-底物相互作用的动力学和机理研究，合成 并表征活性铁卟啉物种作为假定的酶中间体的模型，并将这些物种之间的相互转化联系起来，以期从分子上理解这些蛋白质。在这个项目的过程中，模型化合物的另外两个应用也得到了发展。我们发现一些铁卟啉在抑制过氧亚硝酸根对蛋白质的硝化反应中具有很高的生物活性。 其他金属卟啉已经成为P450作用的有效仿生药物，因为它可以很容易地产生大量有用的药物代谢物，这往往是药物开发的瓶颈。细胞色素P450是参与药物解毒和激素代谢的中心蛋白质，而与之相关的一氧化氮合酶是信号分子一氧化氮和过氧亚硝酸根的来源。合成的金属卟啉可以 被用作探针，以诊断的方式干预这些过程。因此，这些试剂可能被证明是阐明超氧化物、过氧亚硝酸盐和NO生物学的重要工具。这些金属卟啉在动物身上表现出令人印象深刻的活性，表明它们作为药物应用于糖尿病、心肌病和年龄相关疾病等退行性疾病。我们的努力 旨在提供可应用于特定疾病状态的体外模型、细胞培养研究和整体动物模型的机制和动力学信息的基础，如缺血再灌注、败血症和自身免疫性疾病。实验的目的是确定形成什么活性中间体，以及它们的生物靶标可能是什么。这些流程的详细说明将有助于设计 蛋白质酪氨酸硝化的研究将阐明蛋白质在氧化和亚硝化胁迫条件下是如何受到损害的。开发了新型的快速动力学分析来研究在这些过程中观察到的反应性。细胞色素c与合成和半合成磷脂组装体的结合 提供了一个中等复杂性的系统，用于模拟和理解细胞色素c在触发脂质氧化和程序性细胞死亡(细胞凋亡)中所起的作用的更大范围的事件。

The central theme of this program involves studies of the mechanisms of action of the drug-metabolizing enzymes cytochrome P450 and other heme proteins such as myoglobin and cytochrome c. We also investigate metalloporphyrin model compounds to elucidate how these enzymatic processes occur. The principal approaches involve kinetic and mechanistic studies of enzyme-substrate interactions, the synthesis and characterization of reactive iron porphyrin species as models of putative enzymic intermediates and to relate the interconversions of these species toward a molecular understanding of these proteins. Two additional applications of the model compounds have evolved in the course of this project. We have discovered that some iron porphyrins are highly bioactive in suppressing protein nitration by peroxynitrite. Other metalloporphyrins have emerged as effective biomimics of P450 action, alowing the facile production of useful quantities of drug metabolites, often a bottleneck in drug development. Cytochrome P450 is the central protein involved in drug detoxification and hormone metabolism while the related nitric oxide synthase is the source of the signal molecules nitric oxide and peroxynitrite. Synthetic metalloporphyrins can be employed as probes to intervene in these processes in diagnostic ways. Thus, these agents may prove to be significant tools for elaborating the biology of superoxide, peroxynitrite and NO. These same metalloporphyrins have shown impressive activity in animals suggesting their application as pharmaceutical agents in degenerative diseases such as diabetes, cardiomyopathy ang age-related disorders. Our effort seeks to provide a foundation of mechanistic and kinetic information that can be applied to in vitro models, cell culture studies and whole animal models of specific disease states such as ischemia-reperfusion, sepsis and autoimmune diseases. Experiments are aimed at determining what reactive intermediates are formed and what their biological targets are likely to be. The elaboration of these processes will facilitate the design of metal complexes for the catalytic decomposition of peroxynitrite and these other species, while studies of protein tyrosine nitration will elucidate how proteins are damaged under conditions of oxidative and nitrosative stress. Novel types of rapid kinetic analysis have been developed to study the reactivity observed in these processes. Binding of cytochrome c to synthetic and semi-synthetic phospholipid assemblies, which afford a system of intermediate complexity, are used to model and understand the larger scale events in the role of cytochrome c in triggering lipid oxidation and programmed cell death (apoptosis).