Homology modeling of rat and human cytochrome P450 2D (CYP2D) isoforms and computational rationalization of experimental ligand-binding specificities
Homology modeling of rat and human cytochrome P450 2D (CYP2D) isoforms and computational rationalization of experimental ligand-binding specificities
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DOI：
10.1021/jm0209578
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发表时间：
2003-01-02
影响因子：
7.3
通讯作者：
Vermeulen, NPE
中科院分区：
文献类型：
--
作者：
Venhorst, J;ter Laak, AM;Vermeulen, NPE
大鼠和人CYP 2D亚型的配体结合特征，即，以7-甲氧基-4-（氨甲基）香豆素（MAMC）为底物，测定了11种已知的CYP 2D 6配体对大鼠CYP 2D 1 -4和人CYP 2D 6的IC 50值。与CYP 2D 6一样，所有大鼠CYP 2D同工酶催化MAMC的O-脱甲基化，Km和Vmax值分别为78和145 μ M以及0.048和1.122 min（-1）。为了使实验测定的IC 50值中观察到的差异合理化，构建了CYP 2D亚型的同源性模型。CYP 2D 6的同源性模型是在结晶的兔CYP 2C 5的基础上产生的，并根据其再现对应于底物代谢谱的结合方向的能力以及在300 K下无限制的分子动力学模拟期间保持稳定的能力进行了验证。在CYP 2D结合口袋中鉴别出，包括CYP 2D 6残基Phe 120、Glu 216和Asp 301。静电电位计算显示CYP 2D活性位点的负电荷存在较大差异，这与观察到的绝对IC 50值差异一致。此外，对Sparteine、奎尼丁和奎宁在CYP 2D 2和CYP 2D 6中的结合模式的MD研究与实验测定的IC 50值和代谢特征一致。因此，目前的研究提供了新的见解的差异，在活性位点的拓扑结构的研究CYP 2D亚型。
The ligand-binding characteristics of rat and human CYP2D isoforms, i.e., rat CYP2D1-4 and human CYP2D6, were investigated by measuring IC50 values of 11 known CYP2D6 ligands using 7-methoxy-4-(aminomethyl)coumarin (MAMC) as substrate. Like CYP2D6, all rat CYP2D isozymes catalyzed the O-demethylation of MAMC with K-m and V-max values ranging between 78 and 145 muM and 0.048 and 1.122 min(-1), respectively. To rationalize observed differences in the experimentally determined IC50 values, homology models of the CYP2D isoforms were constructed. A homology model of CYP2D6 was generated on the basis of crystallized rabbit CYP2C5 and was validated on its ability to reproduce binding orientations corresponding to metabolic profiles of the substrates and to remain stable during unrestrained molecular dynamics simulations at 300 K Twenty-two active site residues, sharing up to 59% sequence identity, were identified in the CYP2D binding pockets and included CYP2D6 residues Phe120, Glu216, and Asp301. Electrostatic potential calculations displayed large differences in the negative charge of the CYP2D active sites, which was consistent with observed differences in absolute IC50 values. MD studies on the binding mode of sparteine, quinidine, and quinine in CYP2D2 and CYP2D6 furthermore concurred well with experimentally determined IC50 values and metabolic profiles. The current study thus provides new insights into differences in the active site topology of the investigated CYP2D isoforms.