Yu Ai-Ming, Idle Jeffrey R, Herraiz Tomas, Küpfer Adrian, Gonzalez Frank J
Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Pharmacogenetics. 2003 Jun;13(6):307-19. doi: 10.1097/01.fpc.0000054094.48725.b7.
The objective of this investigation was to screen for potential endogenous substrates for CYP2D6. Using recombinant CYP2D6, together with hepatic microsomes from CYP2D6-transgenic mice, human liver microsomes, and a specific anti-CYP2D6 monoclonal antibody, it was ascertained that CYP2D6 does not significantly metabolize the endogenous phenylethylamines 2-phenylethylamine, octopamine, synephrine, 3-methoxy-p-tyramine, 4-methoxy-m-tyramine, metanephrine, and normetanephrine, nor the indolethylamines tryptamine, serotonin, 6-methoxytryptamine, and melatonin, nor the beta-carbolines harman, norharman and tryptoline. However, the indolethylamines 5-methoxy-N,N-dimethyltryptamine (5-MDMT) and pinoline (6-methoxy-1,2,3,4-tetrahydro-beta-carboline) showed relatively high affinity for CYP2D6 in a spectral binding assay (K(s) 28 +/- 5, and 0.5 +/- 0.3 microm (mean +/- SEM), respectively) and were O-demethylated only by CYP2D6 in a panel of 15 recombinant common human P450s. Pinoline and 5-MDMT O-demethylase activities were 35- and 11-fold greater in liver microsomes from CYP2D6-humanized mice, respectively, than those in liver microsomes from control mice. Moreover, the increased activities were completely inhibited by an anti-CYP2D6 monoclonal antibody. Kinetic analysis with recombinant CYP2D6 gave K(m) and k(cat) values for 5-MDMT and pinoline O-demethylations of 12 +/- 1 microm and 65 +/- 1 min(-1) and 1.8 +/- 0.3 microm and 26 +/- 1 min(-1), respectively. These two substrates can be added to 5-methoxytryptamine, which we have recently reported to be an endogenous CYP2D6 substrate. CYP2D6 is therefore a relatively highly specific, high-affinity, high-capacity 5-methoxyindolethylamine O-demethylase. Polymorphic cytochrome CYP2D6 may therefore exert an influence on mood and behavior by the O-demethylation of these 5-methoxyindolethylamines found in the brain and pineal gland. These processes may also impact on mental and neurological health. The findings may open new vistas for the determination of CYP2D6 phenotype.
本研究的目的是筛选细胞色素P450 2D6（CYP2D6）的潜在内源性底物。利用重组CYP2D6，结合来自CYP2D6转基因小鼠的肝微粒体、人肝微粒体以及一种特异性抗CYP2D6单克隆抗体，确定CYP2D6不会显著代谢内源性苯乙胺类物质2-苯乙胺、章鱼胺、辛弗林、3-甲氧基-对-酪胺、4-甲氧基-间-酪胺、变肾上腺素和去甲变肾上腺素，也不会代谢吲哚乙胺类物质色胺、血清素、6-甲氧基色胺和褪黑素，以及β-咔啉类物质哈尔满、去氢哈尔满和色醇。然而，吲哚乙胺类物质5-甲氧基-N,N-二甲基色胺（5-MDMT）和松果林（6-甲氧基-1,2,3,4-四氢-β-咔啉）在光谱结合试验中对CYP2D6表现出相对较高的亲和力（解离常数K(s)分别为28±5和0.5±0.3 μmol/L（平均值±标准误）），并且在一组15种重组常见人细胞色素P450中仅被CYP2D6进行O-去甲基化反应。松果林和5-MDMT的O-去甲基酶活性在CYP2D6人源化小鼠的肝微粒体中分别比对照小鼠肝微粒体中的活性高35倍和11倍。此外，活性的增加被抗CYP2D6单克隆抗体完全抑制。用重组CYP2D6进行动力学分析得出，5-MDMT和松果林O-去甲基化反应的米氏常数K(m)和催化常数k(cat)值分别为12±1 μmol/L和65±1 min⁻¹以及1.8±0.3 μmol/L和26±1 min⁻¹。这两种底物可以添加到5-甲氧基色胺中，我们最近报道5-甲氧基色胺是一种内源性CYP2D6底物。因此，CYP2D6是一种相对高度特异性、高亲和力、高催化能力的5-甲氧基吲哚乙胺O-去甲基酶。因此，多态性细胞色素CYP2D6可能通过对在脑和松果体中发现的这些5-甲氧基吲哚乙胺进行O-去甲基化反应而对情绪和行为产生影响。这些过程也可能影响精神和神经健康。这些发现可能为CYP2D6表型的测定开辟新的前景。
