Onderwater R C, Venhorst J, Commandeur J N, Vermeulen N P
Leiden/Amsterdam Center for Drug Research (LACDR), Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Chem Res Toxicol. 1999 Jul;12(7):555-9. doi: 10.1021/tx980248q.
In this study, a selective substrate for cytochrome P450 2D6 was designed using a small molecule model developed by M. J. De Groot et al. [(1997) Chem. Res. Toxicol. 10, 41-48]. The substrate, 7-methoxy-4-(aminomethyl)coumarin (MAMC), and its putative O-demethylated metabolite 7-hydroxy-4-(aminomethyl)coumarin (HAMC) were synthesized, and their respective fluorescence properties were characterized. The selectivity of MAMC for P450 2D6 was characterized using microsomes containing single human P450 isoenzymes and human liver microsomes. Formation of the metabolic product HAMC was easily assessed in real time with fluorescence spectroscopy, since MAMC and HAMC excitation and emission wavelengths differed significantly. HPLC analysis confirmed that HAMC was the single metabolic product of MAMC and that HAMC formation accounts for the total increase in fluorescence. It was found that, in microsomes from yeast or lymphoblastoid cells selectively expressing P450 isoenzymes, MAMC was selective for P450 2D6 at a concentration of 25 microM with only P450 1A2 contributing significantly to the formation of HAMC. P450s 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and 3A5 were shown not to metabolize MAMC at a concentration of 25 microM. K(m) and v(max) values of MAMC for P450 2D6 were found to be 26.2 +/- 2.8 microM and 2.9 +/- 0.07 min(-)(1), respectively. For P450 1A2, MAMC was found to have a K(m) value of 29.7 +/- 6.2 microM and a v(max) of 0.57 +/- 0.07 min(-)(1). Formation of HAMC in human liver microsomes could be completely inhibited by quinidine, at a concentration of 0.5 microM selective for P450 2D6, and furafylline, at a concentration of 30 microM selective for P450 1A2. In conclusion, O-demethylation of 7-methoxy-4-(aminomethyl)coumarin is a rapid and easily determined parameter for P450 2D6 activity and, due to the fluorescent properties of the metabolite formed, may be a valuable new tool for high-throughput screening purposes.
在本研究中，使用M. J. 德格鲁特等人开发的小分子模型（[(1997) 《化学研究毒理学》10, 41 - 48]）设计了一种细胞色素P450 2D6的选择性底物。合成了底物7 - 甲氧基 - 4 - （氨基甲基）香豆素（MAMC）及其假定的O - 去甲基化代谢物7 - 羟基 - 4 - （氨基甲基）香豆素（HAMC），并对它们各自的荧光特性进行了表征。使用含有单个人P450同工酶的微粒体和人肝微粒体对MAMC对P450 2D6的选择性进行了表征。由于MAMC和HAMC的激发和发射波长有显著差异，代谢产物HAMC的形成可以通过荧光光谱轻松实时评估。高效液相色谱分析证实HAMC是MAMC的唯一代谢产物，并且HAMC的形成占荧光总增加量。结果发现，在选择性表达P450同工酶的酵母或淋巴母细胞微粒体中，MAMC在25 microM浓度下对P450 2D6具有选择性，只有P450 1A2对HAMC的形成有显著贡献。P450 2A6、2B6、2C8、2C9、2C19、2E1、3A4和3A5在25 microM浓度下不代谢MAMC。发现MAMC对P450 2D6的K(m)和v(max)值分别为26.2±2.8 microM和2.9±0.07 min⁻¹。对于P450 1A2，发现MAMC的K(m)值为29.7±6.2 microM，v(max)为0.57±0.07 min⁻¹。人肝微粒体中HAMC的形成可被0.5 microM对P450 2D6有选择性的奎尼丁和30 microM对P450 1A2有选择性的呋拉茶碱完全抑制。总之，7 - 甲氧基 - 4 - （氨基甲基）香豆素的O - 去甲基化是P450 2D6活性的一个快速且易于测定的参数，并且由于所形成代谢物的荧光特性，可能是用于高通量筛选目的的一种有价值的新工具。
