The structural basis for homotropic and heterotropic cooperativity of midazolam metabolism by human cytochrome P450 3A4.
The structural basis for homotropic and heterotropic cooperativity of midazolam metabolism by human cytochrome P450 3A4.
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DOI：
10.1021/bi200924t
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发表时间：
2011-12-20
期刊：
影响因子：
2.9
通讯作者：
Atkins, William M.
中科院分区：
文献类型：
--
作者：
Roberts, Arthur G.;Yang, Jing;Halpert, James R.;Nelson, Sidney D.;Thummel, Kenneth T.;Atkins, William M.
人细胞色素 P450 3A4 (CYP3A4) 代谢大部分临床相关药物，并且经常表现出复杂的稳态动力学，涉及结合配体之间的同向和异向协同作用。在之前的研究中，镇静咪达唑仑 (MDZ) 的羟基化通过在较高药物浓度下 1'-OH-MDZ 与 4-OH-MDZ 的比率降低而表现出同向协同性。在这项研究中，MDZ 与抗癫痫药物卡马西平 (CBZ) 表现出异方协同作用，其特征在于 1'-OH-MDZ 与 4-OH-MDZ 的比率降低。为了揭示 MDZ 协同作用的结构基础，使用纵向 T1 NMR 弛豫和 AutoDock 4.2 的分子对接来探测与 CYP3A4 结合的 MDZ 和 CBZ。在模拟退火过程中，使用从纵向 T1 NMR 弛豫计算出的距离将分子限制在 CYP3A4 的无底物 X 射线晶体结构上。这些模拟表明，两个 MDZ 分子或一个 MDZ 分子和一个 CBZ 分子在 CYP3A4 活性位点内呈现堆叠构型。无论哪种情况，MDZ 分子 4 位的质子都比 1'-CH3 基团的质子更接近血红素。相比之下，MDZ 单分子的分子对接表明，该分子优先定向为 1'-CH3 位置比 4-位更靠近血红素。这项研究首次通过基于 NMR 的模型对人类细胞色素 P450 的异向性和同向性协同性进行了详细的分子分析。通过堆叠分子之间的直接相互作用实现配体结合的协同性可能代表同向和异向协同性的共同基序。
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 anti-epileptic drug carbamazepine (CBZ) with characteristic decreases in the 1′-OH-MDZ to 4-OH-MDZ ratios. To unravel the structural basis of MDZ cooperativity, MDZ and CBZ bound to CYP3A4 were probed using longitudinal T1 NMR relaxation and molecular docking with AutoDock 4.2. The distances calculated from the longitudinal T1 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′-CH3 group. In contrast, molecular docking of a single molecule of MDZ revealed that the molecule was preferentially oriented with the 1′-CH3 position closer to the heme than the 4-position. 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.
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