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Design, synthesis, and characterization of 7-methoxy-4-(aminomethyl)coumarin as a novel and selective cytochrome P450 2D6 substrate suitable for high-throughput screening.
Chem Res Toxicol. 1999 Jul; 12(7):555-9.CR

Abstract

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.

Authors+Show Affiliations

Leiden/Amsterdam Center for Drug Research (LACDR), Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

10409393

Citation

Onderwater, R C., et al. "Design, Synthesis, and Characterization of 7-methoxy-4-(aminomethyl)coumarin as a Novel and Selective Cytochrome P450 2D6 Substrate Suitable for High-throughput Screening." Chemical Research in Toxicology, vol. 12, no. 7, 1999, pp. 555-9.
Onderwater RC, Venhorst J, Commandeur JN, et al. Design, synthesis, and characterization of 7-methoxy-4-(aminomethyl)coumarin as a novel and selective cytochrome P450 2D6 substrate suitable for high-throughput screening. Chem Res Toxicol. 1999;12(7):555-9.
Onderwater, R. C., Venhorst, J., Commandeur, J. N., & Vermeulen, N. P. (1999). Design, synthesis, and characterization of 7-methoxy-4-(aminomethyl)coumarin as a novel and selective cytochrome P450 2D6 substrate suitable for high-throughput screening. Chemical Research in Toxicology, 12(7), 555-9.
Onderwater RC, et al. Design, Synthesis, and Characterization of 7-methoxy-4-(aminomethyl)coumarin as a Novel and Selective Cytochrome P450 2D6 Substrate Suitable for High-throughput Screening. Chem Res Toxicol. 1999;12(7):555-9. PubMed PMID: 10409393.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Design, synthesis, and characterization of 7-methoxy-4-(aminomethyl)coumarin as a novel and selective cytochrome P450 2D6 substrate suitable for high-throughput screening. AU - Onderwater,R C, AU - Venhorst,J, AU - Commandeur,J N, AU - Vermeulen,N P, PY - 1999/7/20/pubmed PY - 1999/7/20/medline PY - 1999/7/20/entrez SP - 555 EP - 9 JF - Chemical research in toxicology JO - Chem Res Toxicol VL - 12 IS - 7 N2 - 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. SN - 0893-228X UR - https://www.unboundmedicine.com/medline/citation/10409393/Design_synthesis_and_characterization_of_7_methoxy_4__aminomethyl_coumarin_as_a_novel_and_selective_cytochrome_P450_2D6_substrate_suitable_for_high_throughput_screening_ L2 - https://doi.org/10.1021/tx980248q DB - PRIME DP - Unbound Medicine ER -