Homology modeling of rat and human cytochrome P450 2D (CYP2D) isoforms and computational rationalization of experimental ligand-binding specificities

The ligand-binding characteristics of rat and human CYP2D isoforms, i.e., rat CYP2D1−4 and human CYP2D6, were investigated by measuring IC50 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 Km and Vmax values ranging between 78 and 145 μM and 0.048 and 1.122 min-1, respectively. To rationalize observed differences in the experimentally determined IC50 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 pote...