P450 Allosterism and Drug Interactions

细胞色素 P450 (CYP) 是代谢药物相互作用的主要来源。的变构行为 CYP，包括主要药物代谢亚型 CYP3A4，在体外和体内继续混淆 基于体外数据的相关性和药物相互作用的预测。分子机制 赋予变构行为尚未完全理解，改进的机械模型可能会 改善预测药物代谢。此外，尽管体外变构行为是众所周知且广泛的 据观察，体内变构的例子是有限的。在多种做出贡献的机制中， 小分子药物在 CYP3A4 外周效应位点的占据以及构象变化 由电子转移伙伴细胞色素 bs (Cyt bs) 诱导，可能会收敛于一组常见的 效应，其中活性位点通过蛋白质-蛋白质更有效地去溶剂化或“充分堆积” 相互作用或多种药物结合。反过来，假设这会导致 NADPH 更有效的偶联 消耗和 O2 减少，伴随药物氧化，“解偶联”减少形成还原态 氧物种。该提案旨在：1）增加我们对变构 CYP 机制的理解 模型探针药物，2) 完成 CYP-Cyt b5 相互作用的比较和机制分析。 荧光探针尼罗红和 TNS 提供了活性位点水合和空间位阻的比较 当外周变构位点被占据或空时，CYP3A4 的限制。质谱分析和 计算模型提供了绘制 Cyt b5-CYP 复合物中结合相互作用的方法以及 由此产生的构象变化与 Cyt bs 的效应器功能相关。这些方法中的每一种都将 与功能研究相结合，表征决定反应效率的反应中间体 通过催化循环的通量。这些研究的一个主要目标是开发它们的转化效用 本计划项目 4 中 P-糖蛋白概念上的类似研究。此外，重要性 体内变构效应的影响仍然是推测性的，该提案的最终目标，即目标 3，将利用一种新颖的 体内、人体、临床研究，探讨抗癫痫药物卡马扎平和 抗焦虑药咪达唑仑。这项研究不仅旨在为体内实验建立明确的原理验证 人类 CYP 的变构效应，同时也提供了一个可在其他领域利用的平台实验设计 体内研究。

Cytochrome P450's (CYPs) are a major source of metabolic drug interactions. The allosteric behavior of CYPs, including the major drug metabolizing isoform CYP3A4, continues to confound in vitro - In vivo correlations and the prediction of drug interactions based on in vitro data. The molecular mechanisms that confer allosteric behavior are incompletely understood, and improved mechanistic models are likely to improve predictive drug metabolism. Moreover, although allosteric behavior in vitro is well known and widely observed, examples of in vivo allosterism are limited. Among the multiple mechanisms that contribute, occupancy by small molecule drugs at a peripheral effector site of CYP3A4, and conformational changes induced by the electron transfer partner Cytochrome bs (Cyt bs), are likely to converge on a set of common effects, wherein the active site is more efficiently desolvated or 'well-packed' through protein-protein interactions or multiple drug binding. In turn this, hypothetically, leads to more efficient coupling of NADPH consumption and O2 reduction, concomitant with drug oxidation, with decreased 'uncoupling' to form reduced oxygen species. This proposal aims to: 1) increase our understanding of allosteric CYP mechanisms with model probe drugs, and 2) to complete a comparative, and mechanistic, analysis of CYP-Cyt b5 interactions. The fluorescent probes Nile Red and TNS provide a comparison of the active site hydration and steric constraints of CYP3A4 when the peripheral allosteric site is occupied or empty. Mass spectrometry and computational models provide methods to map binding interactions in Cyt b5-CYP complexes and the resulting conformational changes linked to effector functions of Cyt bs. Each of these approaches will be combined with functional studies to characterize reaction intermediates that determine the efficiency of the flux through the catalytic cycle. A major goal of these studies is to develop their translational utility in conceptually analogous studies with P-glycoprotein in Project 4 of this Program. In addition, the importance of in vivo allosteric effects remains speculative, and the final aim, aim 3, of this proposal will utilize a novel in vivo, human, clinical study to explore heterotropic effects between the antiepileptic drug carbamazapine and the antianxiety drug midazolam. This study not only seeks to establish definitive proof-of-principle for in vivo allosteric effects in human CYPs, but also to provide a platform experimental design to be exploited in other in vivo studies.