Molecular Basis of Human Cytochrome P450 3A Function

项目主任/主要研究者（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的机制基础， 人CYP 3A 4（成人肝脏和肠中的主要P450）的底物氧化动力学。这种酶 和相关的CYP 3A 5具有特殊的药理学和毒理学意义，因为它们能够 代谢大量不同结构、大小和形状的治疗剂和环境剂。 许多底物表现出的非米氏行为是一个主要的混杂因素， 体外-体内外推和药物-药物相互作用的预测。中心假设是CYP 3A 4 协同性揭示了一个真正的变构调节机制，涉及三级结构的调制 的酶，其寡聚化，并与氧化还原伙伴的相互作用。建立在新的概念和 在本奖项期间开发的方法，CYP 3A 4的分子机制 协同性将通过包括压力扰动在内的各种生物物理方法得到进一步检验 光谱学、快速动力学、荧光共振能量转移和时间分辨荧光 光谱学沿着与稳态动力学和先进的结合分析。这些方法将 适用于纯化的CYP 3A 4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。对机械基础和生理作用的认识， CYP 3A 4的变构调节将为理解CYP 3A 4的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。具体目标如下： 1)探讨底物和效应子诱导的构象转变的分子机制， CYP 3A 4参与协同机制 2)研究CYP 3A 4的蛋白质-蛋白质相互作用的调节，包括其寡聚化， 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物对协同性的影响， 微粒体和模型膜中CYP 3A 4的催化效率和功能异质性 相关性（参见说明）： 细胞色素P450 3A分解人类接触的各种化合物，包括 药物、环境污染物和工业化学品。这项研究将使我们能够了解 P450如何结合和代谢化学结构非常不同的化合物。获得的信息将 有助于避免药物相互作用，并有助于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/