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 nov el and selective substrate of human cytochrome P450 2D6 (CYP2D6), 7-methoxy -4-(aminomethyl)-coumarin (MAMC). Here, we describe a high-throughput micro plate reader assay, which makes use of MAMC as a fluorescent probe for dete rmining the inhibition and activity of CYP2D6 in heterologously expressed s ystems and human liver microsomes,The high-throughput screening (MTS) 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 o f how injection analysis have also successfully been performed. The MTS-ass ay was validated by performing inhibition experiments for several low- and high-affinity ligands (n = 6) of CYP2D6, and comparing the findings to thos e 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 Ki values reveal a ver y good correlation (R-2 = 0.984) between the two assays. To further demonst rate the usefulness of the MTS-assay, IC,, values of a series of five N-sub stituted analogs of 3,4-methylenedioxyamphetamine for CYP2D6 have been dete rmined. The results obtained demonstrate that the current MTS-assay represe nts a significant improvement over previous assays, with a higher turnover of MAMC and a higher selectivity for CYP2D6. (C) 2000 Elsevier Science B.V. All rights reserved.