P450 Allosterism and Drug Interactions

细胞色素P450‘S是代谢药物相互作用的主要来源。化合物的变构行为 细胞色素P450，包括主要的药物代谢亚型细胞色素P3A4，在体外和体内继续混乱 基于体外数据的药物相互作用的相关性和预测。其分子机制是 赋予变构的行为还不完全被理解，改进的机械模型可能会 改善预测性药物代谢。此外，尽管在体外的变构行为是众所周知的和广泛的 观察到，体内变构的例子是有限的。在做出贡献的多种机制中， 小分子药物在细胞色素P3A4外周效应位点的占位和构象变化 由电子转移伙伴诱导的细胞色素bs(Cyt Bs)，很可能会聚集在一套共同的 作用，其中活性部位通过蛋白质-蛋白质更有效地去溶或被‘良好包装’ 相互作用或多种药物结合。反过来，假设这会导致NADPH更有效的耦合 消耗和氧气减少，伴随着药物氧化，与减少的“解偶联”形成减少 氧气物种。这项建议的目的是：1)增加我们对变构CYP机制的理解 模型探测药物，以及2)完成CYP-Cytb5相互作用的比较和机制分析。 荧光探针尼罗红和TNS提供了活性中心水合和空间位阻的比较 当外周变构位点被占用或被清空时，CYP3A4的限制作用。质谱学和 计算模型提供了映射Cyt b5-CYP络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的效应器功能有关。其中每一种方法都将是 结合功能研究来表征决定反应效率的反应中间体 助熔剂通过催化循环。这些研究的一个主要目标是开发它们在 本计划项目4中与P-糖蛋白在概念上相似的研究。此外，重要的是 体内变构效应的研究仍然是推测的，这项提议的最终目标，目标3，将利用一种新的 体内、人体、临床研究探讨抗癫痫药物卡马西平与卡马西平的异向性作用 抗焦虑药物咪达唑仑。这项研究不仅试图在体内建立明确的原则证明 变构效应在人体细胞色素P450中的应用，也为以后的实验设计提供了一个可供开发的平台 活体研究。

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.