Molecular Probes of the Mechanisms of Cytochrome P450

描述（由申请人提供）：该计划的中心主题涉及细胞色素P450酶和一氧化氮合酶（NOS）的作用机制的研究。主要方法包括酶-底物相互作用的动力学和机理研究，作为推定酶中间体模型的活性铁卟啉物种的合成和表征，以及将这些物种的相互转化与这些蛋白质的分子理解联系起来。细胞色素P450是参与药物解毒和激素代谢的中心蛋白，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在诊断中介入这些过程。因此，这些试剂可能被证明是阐明超氧化物、过氧亚硝酸盐和一氧化氮生物学的重要工具。这些金属卟啉在动物体内表现出令人印象深刻的活性，表明它们可以作为药物制剂。我们的努力旨在提供一个机制和动力学信息的基础，可以应用于体外模型，细胞培养研究和全动物模型的特定疾病状态，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了什么活性中间体，以及它们的生物学靶标可能是什么。这些过程的细化将有助于设计用于催化分解过氧亚硝酸盐和这些其他物种的金属配合物。对一氧化氮合酶的研究旨在阐明一氧化氮合酶的一系列机制，并寻求新的氧化过程，有助于合理开发一氧化氮合酶抑制剂。过氧铁（III）卟啉类、氧铁（IV）类和二氧亚铁类现在很容易在常温水溶液中得到。快速动力学技术已被用于研究这些物质的反应性。一个核心问题是如何调节亲核和亲电途径之间的这些复合物的化学性质，这对于理解P450和NOS介导的过程的多样性至关重要。合成和半合成磷脂组件用于模拟和理解P450和NOS作用的更大规模事件，并探测膜对活性氧和活性氮的渗透性。

DESCRIPTION (provided by applicant): The central theme of this program involves studies of the mechanisms of action of cytochrome P450 enzymes and nitric oxide synthase (NOS). 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 relating 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. The studies of nitric oxide synthase aim 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. Peroxoiron(III) porphyrin species, oxoiron(IV) species and ferrous-dioxygen species are now readily accessible in aqueous solution at ambient temperature. Rapid kinetic techniques have been developed to study the reactivity of these species. A central question is how to modulate the chemistry of these complexes between nucleophilic and electrophilic pathways that are essential to understand the variety of P450 and NOS - mediated processes. Synthetic and semi-synthetic phospholipid assemblies are used to model and understand the larger scale events in P450 and NOS action and to probe the permeability of membranes to reactive oxygen and nitrogen species.