Molecular Basis of Human Cytochrome P450 3A Function

人细胞色素 P450 3A 功能的分子基础

• 批准号: 7617828
• 负责人: JAMES R HALPERT
• 金额: $ 40.33万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7617828
• 关键词: Active Sites; Adult; Award; Binding; Biological Assay; CYP3A4 gene; Chemicals; Cytochrome P450; Cytochromes b; Cytochromes b5; Development; Divalent Cations; Drug Interactions; Drug Kinetics; Enzymes; Fluorescence Resonance Energy Transfer; Heterogeneity; Human; In Vitro; Individual; Intestines; Kinetics; Knowledge; Laboratories; Ligand Binding; Ligands; Liver; Measures; Mediating; Methods; Modeling; Molecular; NADPH-Ferrihemoprotein Reductase; Pharmaceutical Preparations; Phospholipids; Play; Positioning Attribute; Property; Reaction; Research; Role; Salts; Shapes; Spectrum Analysis; Structure; Testing; Therapeutic; Therapeutic Agents; Work; base; cytochrome P450 3A; directed evolution; drug discovery; environmental agent; environmental chemical; enzyme activity; interest; mutant; oxidation; pressure; protein protein interaction

DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to determine the mechanistic basis for the atypical kinetics of substrate oxidation by human cytochromes P450 of the 3A subfamily. CYP3A4 is the major P450 in adult human liver and intestine, where it represents on average between 30 and 50% of the total P450. Because of the large number of therapeutic agents and environmental chemicals that can induce CYP3A4 expression and/or modulate the activity of the enzyme, a very significant potential for adverse drug reactions exists. Development of in vitro methods for predicting the pharmacokinetics and drug interaction potential of CYP3A4 substrates is often complicated by the non-Michaels Menten kinetics observed. Substantial work from this and other laboratories during the prior award periods has provided compelling evidence that such atypical results from simultaneous occupancy of a single active site by multiple ligands. At present, however, it is difficult to rationalize or predict interactions between two CYP3A4 substrates based on knowledge of their individual kinetic properties. Thus, a ligand can activate, inhibit, or have no effect on CYP3A4 depending on the particular substrate used in the catalytic assay and the specific product measured. In addition, the kinetic parameters, degree of cooperativity, and regioselectivity are influenced by such factors as NADPH-cytochrome P450 reductase, cytochrome b5, divalent cations, phospholipids, and salts. The central hypothesis is that conformational changes resulting from ligand binding and/or protein-protein interactions play a key role in atypical kinetics of CYP3A4-catalyzed oxidations. This will be tested by a variety of biophysical approaches including high-pressure perturbation spectroscopy, rapid kinetics, and fluorescence resonance energy transfer along with steady-state kinetics and binding assays. The specific aims are to: 1) elucidate the role of conformational heterogeneity of CYP3A4 in the mechanisms of cooperativity; 2) probe the involvement of oligomerization of CYP3A4 in the mechanisms of cooperativity; 3) determine the basis of altered cooperativity in available active-site mutants of CYP3A4 and new mutants to be created by directed evolution. The mechanistic information obtained about the binding of substrates and modulators to CYP3A4 should provide the intellectual framework required for rational assessment of inhibition and drug interaction potential, and thereby enhance drug discovery and therapy.

描述（由申请方提供）：拟定研究的长期目标是确定3A亚家族的人细胞色素P450对底物氧化的非典型动力学的机制基础。 CYP 3A 4是成人肝脏和肠道中的主要P450，平均占总P450的30%至50%。 由于大量治疗药物和环境化学物质可诱导CYP 3A 4表达和/或调节酶活性，因此存在非常显著的药物不良反应可能性。 用于预测CYP 3A 4底物的药代动力学和药物相互作用潜力的体外方法的开发通常由于观察到的非Michaels Menten动力学而变得复杂。 本实验室和其他实验室在先前授标期间的大量工作提供了令人信服的证据，证明这种非典型结果是由多个配体同时占据单个活性位点造成的。 然而，目前，很难合理化或预测两个CYP 3A 4底物之间的相互作用的基础上，他们各自的动力学特性的知识。 因此，根据催化试验中使用的特定底物和测量的特定产物，配体可以激活、抑制或不影响CYP 3A 4。 此外，动力学参数，程度的协同性，和区域选择性的影响因素，如NADPH-细胞色素P450还原酶，细胞色素b5，二价阳离子，磷脂，和盐。 中心假设是配体结合和/或蛋白质-蛋白质相互作用引起的构象变化在CYP 3A 4催化氧化的非典型动力学中起关键作用。 这将通过各种生物物理方法进行测试，包括高压微扰光谱法，快速动力学，荧光共振能量转移沿着稳态动力学和结合试验。 具体目标是：1）阐明CYP 3A 4的构象异质性在协同作用机制中的作用; 2）探索CYP 3A 4的寡聚化在协同作用机制中的参与; 3）确定CYP 3A 4的可用活性位点突变体和通过定向进化产生的新突变体的协同作用改变的基础。 关于底物和调节剂与CYP 3A 4结合的机制信息应提供合理评估抑制和药物相互作用潜力所需的知识框架，从而促进药物发现和治疗。