MECHANISMS OF CYTOCHROME P450 ALLOSTERY

许多药物-药物相互作用起源于新陈代谢，其结果是激活或抑制 细胞色素P450(CYP)依赖的药物清除。对CYP依赖的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佳的变构。这种变构包括单个底物的非双曲线稳态动力学曲线(同质变构)，以及一种药物对另一种药物的动力学曲线的变化(异质变构)。导致变构的分子机制尚未确定，尽管已经提出了几种不同的模型，包括在单个流体活性中心内的多个配体结合，在这个大的活性中心的离散亚中心内的多个配体结合，以及依赖于配体的持久蛋白质构象平衡。在这里，非稳态动力学方法将被用来挑战这些模型，并在现有的CYP3A4和CYP2C9结构模型的背景下理解CYP变构。具体目标是：1) 确定配体结合的基本步骤、配体依赖的铁自旋态平衡或配体依赖的总蛋白质构象变化是否对睾酮、芘和金丝桃素与细胞色素P3A4的同质作用是差异化变构作用；2)通过顺磁平移的1H-核磁共振谱确定咖啡因与对乙酰氨基酚或咪达唑仑和α-纳豆香料与细胞色素P3A4的异构性作用的变构机制，并启动SAR-BY-核磁共振，通过15N-Phe标记的均匀去氢标记的细胞色素P3A4中的配基和结合部位的图谱；3)通过监测构象动力学 4)通过定点突变和催化转换实验绘制细胞色素P3A4/细胞色素b5结合表面的图谱，并确定特定的界面残基是否对细胞色素b5依赖的变构效应起作用。此外，在可能的情况下，将对CyperyF进行平行实验，CyPeryF是一种可溶的同系物，可以进行X射线结晶学分析。从长远来看，将通过合作寻找[CyperyF.配体]络合物的x射线结构，以便将变构效应与 CYP结构。

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.