MECHANISMS OF CYTOCHROME P450 ALLOSTERY

许多药物间相互作用起源于代谢，由激活或抑制 细胞色素P450（CYP 1A 1）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表征不佳的变构。这种变构包括单个底物的非双曲线稳态动力学曲线（同向变构），以及一种药物通过第二种药物的动力学曲线的改变（异向变构）。导致变构的分子机制尚未确定，尽管已经提出了几种对比模型，包括在单个流体活性位点内的多配体结合，在该大活性位点的离散子位点内的多配体结合，以及配体依赖的持久蛋白质构象平衡。在这里，非稳态动力学方法将被利用来挑战这些模型，并了解在现有的结构模型的背景下，CYP 3A 4和CYP 2C 9的代谢变构。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CYP 3A 4的同向效应是否是差异变构的; 2）通过顺磁位移1H-NMR谱测定，咖啡因和对乙酰氨基酚或咪达唑仑和α-萘酚香料与CYP 3A 4之间异变构效应的变构机制，并启动SAR-通过-NMR绘制均匀氘代、15 N-Phe-标记的CYP 3A 4中的配体结合位点; 3）通过以下途径监测CYP 3A 4的构象动力学 有限的蛋白水解/电喷雾质谱作为铁/亚铁氧化还原状态和配体结合的函数; 4）通过定点诱变和催化转换实验绘制CYP 3A 4/Cyt b5结合表面，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，将通过合作寻求[CYPeryF.配体]复合物的X射线结构，以将变构效应与 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.