Molecular Probes of the Mechanisms of Cytocrhome P450

该计划的中心主题涉及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的作用机制的研究。我们还研究金属卟啉模型化合物，以阐明这些酶的过程是如何发生的。主要方法包括酶-底物相互作用的动力学和机理研究， 和表征的活性铁卟啉物种作为模型的推定酶的中间体，并涉及这些物种的相互转化对这些蛋白质的分子理解。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。我们已经发现一些铁卟啉在抑制蛋白质被过氧亚硝酸根硝化方面具有高度的生物活性。 其他金属卟啉已经成为P450作用的有效仿生物，这使得容易产生有用数量的药物代谢物，这通常是药物开发的瓶颈。细胞色素P450是参与药物解毒和激素代谢的中心蛋白，而相关的一氧化氮合酶是信号分子一氧化氮和过氧亚硝酸盐的来源。合成金属卟啉可以 作为探针，以诊断的方式干预这些过程。因此，这些代理人可能被证明是重要的工具，用于阐述生物学的超氧化物，过氧亚硝酸盐和NO。这些相同的金属卟啉已显示出令人印象深刻的活动，在动物表明其作为药物制剂在退行性疾病，如糖尿病，心肌病和年龄相关的疾病的应用。我们的努力 旨在提供可应用于体外模型、细胞培养研究和特定疾病状态如缺血-再灌注、败血症和自身免疫性疾病的整体动物模型的机制和动力学信息的基础。实验的目的是确定形成了什么反应中间体，以及它们的生物靶点可能是什么。这些过程的详细说明将有助于设计 金属络合物的催化分解过氧亚硝酸盐和这些其他物种，而蛋白质酪氨酸硝化的研究将阐明蛋白质是如何在氧化和亚硝化应激条件下被破坏。新型的快速动力学分析已被开发出来，以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， 提供中等复杂性的系统，用于建模和理解细胞色素c在触发脂质氧化和程序性细胞死亡（细胞凋亡）中的作用的更大规模事件。

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).