Molecular Probes of the Mechanisms of Cytocrhome P450

描述(申请人提供)：该计划的中心主题涉及药物代谢酶细胞色素P450和细胞信号蛋白一氧化氮合酶(NOS)的作用机制的研究。金属卟啉模型化合物也被研究，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，活性铁卟啉物种的合成和表征，作为假定的酶中间体的模型，并将这些物种的相互转化联系起来，以实现对这些蛋白质的分子理解。细胞色素P450是参与药物解毒和激素代谢的中心蛋白质，而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。合成的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，这表明它们被用作药剂。我们的努力旨在提供机制和动力学信息的基础，这些信息可以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，如缺血再灌注、脓毒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。这些过程的阐述将有助于设计用于催化分解过氧亚硝酸根和这些其他物种的金属络合物，而蛋白质酪氨酸硝化的研究将阐明蛋白质在氧化应激条件下是如何受损的。通过对N-羟基精氨酸氧化的研究，旨在阐明一氧化氮合酶的一系列作用机制，并寻找有助于合理开发一氧化氮合酶抑制剂的新的氧化过程。发展了快速动力学技术来研究这些物种的反应活性。一个中心问题是如何调节亲核和亲电途径之间的金属卟啉中心的化学，这对于理解P450介导的各种过程是必不可少的。合成和半合成磷脂组件被用来模拟和理解细胞色素c在触发脂质氧化和细胞程序性死亡(细胞凋亡)中所起的作用的更大范围的事件。

DESCRIPTION (provided by applicant): The central theme of this program involves studies of the mechanisms of action of the drug-metabolizing enzymes cytochrome P450 and the cell signaling protein nitric oxide synthase (NOS). Metalloporphyrin model compounds are also investigated 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. Cytochrome P450 is the central protein involved in drug detoxification and hormone metabolism while 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. 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 stress. The studies N-hydroxyarginine oxidation aims to illuminate the range of mechanisms for NOS and to seek out new oxidation processes that may help with the rational development of NOS inhibitors. Rapid kinetic techniques have been developed to study the reactivity of these species. A central question is how to modulate the chemistry of metalloporphyrin centers between nucleophilic and electrophilic pathways that are essential to understand the variety of P450 mediated processes. Synthetic and semi-synthetic phospholipid assemblies 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).