title: Evaluation of a novel high-throughput assay for cytochrome P450 2D6 using 7-methoxy-4-(aminomethyl)-coumarin.
abstract: We recently reported on the design, synthesis and characterisation of a novel and selective substrate of human cytochrome P450 2D6 (CYP2D6), 7-methoxy-4-(aminomethyl)-coumarin (MAMC). Here, we describe a high-throughput microplate reader assay, which makes use of MAMC as a fluorescent probe for determining the inhibition and activity of CYP2D6 in heterologously expressed systems and human liver microsomes. The high-throughput screening (HTS) assay can be used both in an end-point and real-time configuration, and is easy to use, rapid and sensitive. In addition, end-point measurements by means of flow injection analysis have also successfully been performed. The HTS-assay was validated by performing inhibition experiments for several low- and high-affinity ligands (n=6) of CYP2D6, and comparing the findings to those obtained with the standard O-demethylation assay of dextromethorphan. The results indicate that all compounds tested display competitive inhibition in both the MAMC and dextromethorphan assay, and the K(i) values reveal a very good correlation (R(2)=0.984) between the two assays. To further demonstrate the usefulness of the HTS-assay, IC(50) values of a series of five N-substituted analogs of 3, 4-methylenedioxyamphetamine for CYP2D6 have been determined. The results obtained demonstrate that the current HTS-assay represents a significant improvement over previous assays, with a higher turnover of MAMC and a higher selectivity for CYP2D6.
authors: Leiden/Amsterdam Center for Drug Research (LACDR), Department of Pharmacochemistry, Division of Molecular Toxicology, Vrije Universiteit, De Boelelaan 1083, 1081 HV, Amsterdam, The Netherlands.
authors: J
authors: J
authors: Venhorst
authors: R C
authors: RC
authors: Onderwater
authors: J H
authors: JH
authors: Meerman
authors: N P
authors: NP
authors: Vermeulen
authors: J N
authors: JN
authors: Commandeur
mesh_terms: D002138:Calibration; D002460:Cell Line; D003374:Coumarins / Q000493:pharmacokinetics*; D019389:Cytochrome P-450 CYP2D6 / Q000032:analysis / Q000378:metabolism*; D003915:Dextromethorphan / Q000493:pharmacokinetics*; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology*; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity
chemical_list: C120605:7-methoxy-4-(aminomethyl)coumarin; D003374:Coumarins; D011994:Recombinant Proteins; D003915:Dextromethorphan; D019389:Cytochrome P-450 CYP2D6