Molecular Basis of Human Cytochrome P450 3A Function

项目主任/首席调查员(最后、第一、中间)：Halpert，James R. 项目总结(见说明)： 拟议研究的长期目标是确定非典型肺炎的机制基础。 成人肝脏和肠道中的主要P450--人细胞色素P4503A4氧化底物的动力学。这种酶 和相关的CYP3A5具有特殊的药理学和毒理学意义，因为它们能够 代谢各种不同结构、大小和形状的治疗和环境制剂。 在许多底物上表现出的非米氏行为是一个主要的混淆因素 药物-药物相互作用的体外-体内外推和预测。中心假说是细胞色素P3A4 协作性揭示了涉及三级结构调制的真正变构调节机制 酶的低聚作用，以及与氧化还原伙伴的相互作用。建立在新概念和 在本奖项期间开发的方法学，CYP3A4的分子机制 协作性将通过包括压力扰动在内的各种生物物理方法进行进一步测试 光谱学、快速动力学、荧光共振能量转移和时间分辨荧光 光谱分析、稳态动力学和高级结合分析。这些方法将是 应用于纯化的CYP3A4野生型、关键活性位点突变体和允许定点突变的新突变体 荧光探针的掺入。对肌萎缩侧索硬化的机制基础和生理作用的认识 CYP3A4的变构调节将为理解其机制提供必要的关键信息。 药物不良反应和药物间相互作用。个人的具体目标是： 1)探讨底物和效应剂诱导的构象转变的分子机制。 参与协同作用机制的细胞色素P3A4 2)研究CyP3A4对蛋白质相互作用的调控，包括其寡聚作用。 药物底物和效应物 3)探讨还原型谷胱甘肽和其他潜在的生理效应因子对协同作用的影响。 CYP3A4在微粒体和模型膜中的催化效率和功能异质性 相关性(请参阅说明)： 细胞色素P450 3A分解人类接触到的各种化合物，包括 毒品、环境污染物和工业化学品。这项研究将使我们能够理解 P450如何结合和代谢具有非常不同化学结构的化合物。所获得的信息将 帮助避免药物相互作用，并帮助预测个体对药物的反应。 项目/

Program Director/Principal Investigator (Last, First, Middle): Halpert, James R. PROJECT SUMMARY (See instructions): The long-term objective of the proposed research is to determine the mechanistic basis for the atypical kinetics of substrate oxidation by human CYP3A4, the major P450 in adult liver and intestine. This enzyme and the related CYP3A5 are of particular pharmacological and toxicological significance due to their ability to metabolize a vast array of therapeutic and environmental agents of diverse structures, sizes, and shapes. The non-Michaelis-Menten behavior exhibited with a number of substrates is a major confounding factor in vitro-in vivo extrapolations and predictions of drug-drug interactions. The central hypothesis is that CYP3A4 cooperativity reveals a true allosteric regulatory mechanism that involves modulation of the tertiary structure of the enzyme, its oligomerization, and interactions with redox partners. Building on new concepts and methodologies developed during the current award period, the molecular mechanisms of CYP3A4 cooperativity will be further tested by a variety of biophysical approaches including pressure perturbation spectroscopy, rapid kinetics, fluorescence resonance energy transfer, and time-resolved fluorescence spectroscopy along with steady-state kinetics and advanced binding assays. These approaches will be applied to purified CYP3A4wild-type, key active site mutants, and new mutants allowing site-directed incorporation of fluorescent probes. The knowledge of the mechanistic basis and physiological role of allosteric regulation of CYP3A4 will provide key information necessary for understanding the mechanisms of adverse drug effects and drug-drug interactions. The individual specific aims are: 1) To probe the molecular mechanisms of substrate- and effector-induced conformational transitions in CYP3A4 involved in the mechanisms of cooperativity 2) To investigate the modulation of protein-protein interactions of CYP3A4, including its oligomerization, by drug substrates and effectors 3) To probe the effect of reduced glutathione and other potential physiological effectors on cooperativity, catalytic efficiency, and functional heterogeneity of CYP3A4 in microsomes and model membranes RELEVANCE (See instructions): Cytochromes P450 3A break down a wide variety of compounds to which humans are exposed, including drugs, environmental contaminants, and industrial chemicals. The research will enable us to understand how P450s bind and metabolize compounds of very different chemical structure. The information gained will help avoid drug interactions and help predict individual response to medications. PROJECT/