dextromethorphan and 7-methoxy-4-(aminomethyl)coumarin

The polymorphic human debrisoquine hydroxylase, cytochrome P450 2D6 (CYP2D6), is one of the most important phase I drug metabolising enzymes. It is responsible for metabolising a large number of compounds that mostly share similarity in having a basic N-atom and an aromatic moiety. In homology modelling studies, it has been suggested that in fixation of this aromatic moiety, there may be an important role for phenylalanine 120 (Phe(120)). In this study, the role of Phe(120) in ligand binding and catalysis was experimentally examined by mutating it into an alanine. Strikingly, this substitution led to a completely abolished 7-methoxy-4-(aminomethyl)-coumarin (MAMC) O-demethylating activity of CYP2D6. On the other hand, bufuralol metabolism was hardly affected (K(m) of 1-hydroxylation mutant: 1.2 microM, wild-type: 2.9 microM, 4-hydroxylation mutant: 1.5 microM, and wild-type: 3.2 microM) and neither was affected dextromethorphan O-demethylation (K(m) mutant: 1.2 microM, wild-type: 2 microM, k(cat) mutant: 4.5 min(-1), and wild-type: 3.3 min(-1)). However, the Phe(120)Ala mutant also formed 3-hydroxymorphinan, the double demethylated form of dextromethorphan, which was not detected using wild-type CYP2D6. 3,4-Methylenedioxymethamphetamine (MDMA) was demethylenated by both mutant and wild-type CYP2D6 to 3,4-dihydroxymethamphetamine (3,4-OH-MA K(m) of mutant: 55 microM and wild-type: 2 microM). In addition, the mutant formed two additional metabolites; 3,4-methylenedioxyamphetamine (MDA) and N-hydroxy-3,4-methylenedioxymethamphetamine (N-OH-MDMA). Inhibition experiments of dextromethorphan O-demethylation showed a decreased affinity of the Phe(120)Ala mutant for quinidine (IC(50) mutant: 240 nM and wild-type, 40 nM), while IC(50)s for quinine were equal (1 microM). These data indicate the importance of Phe(120) in the selectivity and regiospecificity in substrate binding and catalysis by CYP2D6.

Topics: Alanine; Coumarins; Cytochrome P-450 CYP2D6; Dextromethorphan; Humans; Models, Biological; Models, Molecular; Mutagenesis; N-Methyl-3,4-methylenedioxyamphetamine; Phenylalanine

The ligand-binding characteristics of rat and human CYP2D isoforms, i.e., rat CYP2D1-4 and human CYP2D6, were investigated by measuring IC(50) values of 11 known CYP2D6 ligands using 7-methoxy-4-(aminomethyl)coumarin (MAMC) as substrate. Like CYP2D6, all rat CYP2D isozymes catalyzed the O-demethylation of MAMC with K(m) and V(max) values ranging between 78 and 145 microM and 0.048 and 1.122 min(-1), respectively. To rationalize observed differences in the experimentally determined IC(50) values, homology models of the CYP2D isoforms were constructed. A homology model of CYP2D6 was generated on the basis of crystallized rabbit CYP2C5 and was validated on its ability to reproduce binding orientations corresponding to metabolic profiles of the substrates and to remain stable during unrestrained molecular dynamics simulations at 300 K. Twenty-two active site residues, sharing up to 59% sequence identity, were identified in the CYP2D binding pockets and included CYP2D6 residues Phe120, Glu216, and Asp301. Electrostatic potential calculations displayed large differences in the negative charge of the CYP2D active sites, which was consistent with observed differences in absolute IC(50) values. MD studies on the binding mode of sparteine, quinidine, and quinine in CYP2D2 and CYP2D6 furthermore concurred well with experimentally determined IC(50) values and metabolic profiles. The current study thus provides new insights into differences in the active site topology of the investigated CYP2D isoforms.

Topics: Alcohol Oxidoreductases; Amino Acid Sequence; Animals; Aryl Hydrocarbon Hydroxylases; Binding Sites; Coumarins; Cytochrome P-450 CYP2D6; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 2; Enzyme Inhibitors; Humans; Isoenzymes; Kinetics; Ligands; Mixed Function Oxygenases; Models, Molecular; Molecular Sequence Data; Protein Binding; Quinidine; Quinine; Rabbits; Rats; Sequence Homology, Amino Acid; Sparteine; Species Specificity

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.

Topics: Calibration; Cell Line; Coumarins; Cytochrome P-450 CYP2D6; Dextromethorphan; Humans; Kinetics; Microsomes; Microsomes, Liver; Recombinant Proteins; Sensitivity and Specificity; Spectrometry, Fluorescence; Substrate Specificity