Molecular Basis of Human Cytochrome P450 3A Function

描述(申请人提供)：拟议研究的长期目标是确定3A亚家族的人细胞色素P450氧化底物的非典型动力学的机制基础。CYP3A4是成人肝脏和肠道中的主要P450，平均占总P450的30%~50%。由于大量的治疗剂和环境化学物质可以诱导CYP3A4的表达和/或调节酶的活性，因此存在非常显著的药物不良反应的可能性。体外方法预测细胞色素P3A4底物的药代动力学和药物相互作用潜力的发展往往因观察到的非迈克尔曼腾动力学而变得复杂。该实验室和其他实验室在以前的授权期内所做的大量工作提供了令人信服的证据，证明这种非典型的结果是由多个配体同时占据一个活性部位造成的。然而，目前很难根据对两种底物各自的动力学性质的了解来合理化或预测两种底物之间的相互作用。因此，根据催化分析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CYP3A4。此外，NADPH-细胞色素P450还原酶、细胞色素b5、二价阳离子、磷脂和盐等因素对酶的动力学参数、协同程度和区域选择性均有影响。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CYP3A4催化的非典型氧化动力学中起着关键作用。这将通过各种生物物理方法进行测试，包括高压微扰光谱、快速动力学、荧光共振能量转移以及稳态动力学和结合分析。其具体目的是：1)阐明CyP3A4的构象异质性在协同作用机制中的作用；2)探讨CyP3A4寡聚参与协同作用的机制；3)确定现有的CYP3A4活性位点突变体和定向进化创建的新突变体的协同作用改变的基础。所获得的有关底物和调节剂与CYP3A4结合的机制信息应该为合理评估抑制和药物相互作用潜力提供所需的智力框架，从而促进药物发现和治疗。

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.