MECHANISMS OF CYTOCHROME P450 ALLOSTERY

细胞色素 P450 变构机制

• 批准号: 6701456
• 负责人: WILLIAM M ATKINS
• 金额: $ 21.62万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6701456
• 关键词: X ray crystallography; acetaminophen; allosteric site; binding sites; caffeine; conformation; cytochrome P450; drug interactions; drug metabolism; electrospray ionization mass spectrometry; enzyme inhibitors; enzyme mechanism; ligands; midazolam; nuclear magnetic resonance spectroscopy; proteolysis; pyrenes; testosterone

Many drug-drug interactions are metabolic in origin, resulting from the activation or inhibition of Cytochrome P450 (CYP)-dependent drug clearance. A potential contribution to CYP-dependent drug interactions is the poorly characterized allostery that is widely observed in vitro, and also in vivo. This allostery includes non-hyperbolic steady state kinetic profiles for individual substrates (homotropic allostery), as well as alterations in the kinetic profiles of one drug by a second (heterotropic allostery). The molecular mechanisms leading to allostery are not defined, although several contrasting models have been proposed and include multiple ligand binding within a single fluid active site, multiple ligand binding within discrete subsites of this large active site, and ligand-dependent persistent protein conformational equilibria. Here, non-steady state kinetic methods will be exploited to challenge these models and to understand CYP allostery in the context of existing structural models for CYP3A4 and CYP2C9. The specific aims are: 1) to determine whether the elementary steps of ligand binding, ligand-dependent ferric spin state equilibrium, or ligand-dependent changes in gross protein conformation are differentially allosteric for the homotropic effects of testosterone, pyrene, and hypericin with CYP3A4; 2) to determine by paramagnetically shifted 1H-NMR spectra, the mechanism of allostery for the heterotropic effects between caffeine and acetaminophen or midazolam and alpha-naptho flavors with CYP3A4, and to initiate SAR-by-NMR to map ligand binding sites in uniformly deuterated, 15N-Phe-labeled CYP3A4; 3) to monitor conformational dynamics of CYP3A4 via limited proteolysis/electrospray mass spectrometry as a function of ferric/ferrous redox state and ligand binding; 4) to map the CYP3A4/Cyt b5 binding surface via site-directed mutagenesis and catatylic turnover experiments, and determine whether specific interfacial residues contribute to Cyt b5-dependent allosteric effects. In addition, where possible, parallel experiments will be performed with CYPeryF, a soluble homolog for which X-ray crystallographic analysis is possible. In the long term, x-ray structures of [CYPeryF.ligand] complexes will be sought, via collaboration, in order to correlate allosteric effects with CYP structure.

许多药物-药物相互作用本质上是代谢性的，是由激活或抑制 细胞色素 P450 (CYP) 依赖性药物清除。 CYP 依赖性药物相互作用的一个潜在贡献是在体外和体内广泛观察到的特征不明的变构。这种变构包括各个底物的非双曲稳态动力学曲线（同向变构），以及一种药物的动力学曲线被另一种药物改变（异向变构）。导致变构的分子机制尚未定义，尽管已经提出了几种对比模型，包括单个流体活性位点内的多个配体结合、该大活性位点的离散子位点内的多个配体结合以及配体依赖性持久蛋白质构象平衡。在这里，将利用非稳态动力学方法来挑战这些模型，并在 CYP3A4 和 CYP2C9 的现有结构模型的背景下理解 CYP 变构。具体目标是：1） 确定配体结合、配体依赖性铁自旋状态平衡或总蛋白质构象的配体依赖性变化的基本步骤对于睾酮、芘和金丝桃素与CYP3A4的同向效应是否具有差异变构； 2) 通过顺磁位移 1H-NMR 谱确定咖啡因和对乙酰氨基酚或咪达唑仑和 α-萘并香料与 CYP3A4 之间异方效应的变构机制，并启动 SAR-by-NMR 以绘制均匀氘化中的配体结合位点, 15N-Phe 标记的 CYP3A4; 3) 通过监测CYP3A4的构象动力学 有限蛋白水解/电喷雾质谱作为三价铁/亚铁氧化还原态和配体结合的函数； 4) 通过定点诱变和催化周转实验绘制 CYP3A4/Cyt b5 结合表面图，并确定特定的界面残基是否有助于 Cyt b5 依赖性变构效应。此外，在可能的情况下，将使用 CYPeryF 进行平行实验，CYPeryF 是一种可溶性同系物，可以进行 X 射线晶体学分析。从长远来看，将通过合作寻找 [CYPeryF.ligand] 复合物的 X 射线结构，以便将变构效应与 CYP结构。