Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia 30602, United States.
Biochemistry. 2011 Dec 20;50(50):10804-18. doi: 10.1021/bi200924t. Epub 2011 Nov 22.
Human cytochrome P450 3A4 (CYP3A4) metabolizes a significant portion of clinically relevant drugs and often exhibits complex steady-state kinetics that can involve homotropic and heterotropic cooperativity between bound ligands. In previous studies, the hydroxylation of the sedative midazolam (MDZ) exhibited homotropic cooperativity via a decrease in the ratio of 1'-OH-MDZ to 4-OH-MDZ at higher drug concentrations. In this study, MDZ exhibited heterotropic cooperativity with the antiepileptic drug carbamazepine (CBZ) with characteristic decreases in the 1'-OH-MDZ to 4-OH-MDZ ratios. To unravel the structural basis of MDZ cooperativity, we probed MDZ and CBZ bound to CYP3A4 using longitudinal T(1) nuclear magnetic resonance (NMR) relaxation and molecular docking with AutoDock 4.2. The distances calculated from longitudinal T(1) NMR relaxation were used during simulated annealing to constrain the molecules to the substrate-free X-ray crystal structure of CYP3A4. These simulations revealed that either two MDZ molecules or an MDZ molecule and a CBZ molecule assume a stacked configuration within the CYP3A4 active site. In either case, the proton at position 4 of the MDZ molecule was closer to the heme than the protons of the 1'-CH(3) group. In contrast, molecular docking of a single molecule of MDZ revealed that the molecule was preferentially oriented with the 1'-CH(3) position closer to the heme than position 4. This study provides the first detailed molecular analysis of heterotropic and homotropic cooperativity of a human cytochrome P450 from an NMR-based model. Cooperativity of ligand binding through direct interaction between stacked molecules may represent a common motif for homotropic and heterotropic cooperativity.
人细胞色素 P450 3A4（CYP3A4）代谢了很大一部分临床上相关的药物，并且通常表现出复杂的稳态动力学，其中可能涉及结合配体之间的同型变构和异型变构协同作用。在以前的研究中，镇静剂咪达唑仑（MDZ）的羟化作用表现出同型变构协同作用，即在较高药物浓度下，1'-OH-MDZ 与 4-OH-MDZ 的比例降低。在这项研究中，MDZ 与抗癫痫药物卡马西平（CBZ）表现出异型变构协同作用，其 1'-OH-MDZ 与 4-OH-MDZ 的比例特征性降低。为了解 MDZ 协同作用的结构基础，我们使用纵向 T1 核磁共振（NMR）弛豫和 AutoDock 4.2 分子对接来探测与 CYP3A4 结合的 MDZ 和 CBZ。从纵向 T1 NMR 弛豫计算出的距离用于模拟退火过程中，将分子约束到 CYP3A4 的无底物 X 射线晶体结构中。这些模拟表明，要么是两个 MDZ 分子，要么是一个 MDZ 分子和一个 CBZ 分子，在 CYP3A4 活性位点内呈现堆叠构型。在任何一种情况下，MDZ 分子中 4 位的质子都比 1'-CH3 基团的质子更接近血红素。相比之下，单个 MDZ 分子的分子对接表明，该分子优先取向，其 1'-CH3 位置比 4 位更接近血红素。这项研究从基于 NMR 的模型提供了对人细胞色素 P450 同型变构和异型变构协同作用的第一个详细分子分析。通过堆叠分子之间的直接相互作用，配体结合的协同作用可能代表同型变构和异型变构协同作用的常见模式。
