Evaluation of a novel high-throughput assay for cytochrome P450 2D6 using 7-methoxy-4-(aminomethyl)-coumarin.


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.

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@article{Venhorst2000EvaluationOA, title={Evaluation of a novel high-throughput assay for cytochrome P450 2D6 using 7-methoxy-4-(aminomethyl)-coumarin.}, author={Jennifer Venhorst and Rob C A Onderwater and John H. N. Meerman and Nico P. E. Vermeulen and Jan N. M. Commandeur}, journal={European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences}, year={2000}, volume={12 2}, pages={151-8} }