Homology Modeling of Rat and Human Cytochrome P450 2D (CYP2D) Isoforms and Computational Rationalization of Experimental Ligand-Binding Specificities

J. Med. Chem., 46 (1), 74 -86, 2003. 10.1021/jm0209578 S0022-2623(02)00957-3
Web Release Date: December 5, 2002

Homology Modeling of Rat and Human Cytochrome P450 2D (CYP2D) Isoforms and Computational Rationalization of Experimental Ligand-Binding Specificities

Jennifer Venhorst, Antonius M. ter Laak, Jan N. M. Commandeur, Yoshihiko Funae, Toyoko Hiroi, and Nico P. E. Vermeulen*

Department of Pharmacochemistry, Faculty of Sciences, Division of Molecular Toxicology, Leiden/Amsterdam Center for Drug Research (LACDR), Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands, and Laboratory of Chemistry, Osaka University Medical School, 1-4-54, Asahimachi, Abeno-ku, Osaka 545, Japan

Received June 10, 2002

Abstract:

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 K_m and V_max values ranging between 78 and 145 M 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 potential calculations displayed large differences in the negative charge of the CYP2D active sites, which was consistent with observed differences in absolute IC₅₀ values. MD studies on the binding mode of sparteine, quinidine, and quinine in CYP2D2 and CYP2D6 furthermore concurred well with experimentally determined IC₅₀ values and metabolic profiles. The current study thus provides new insights into differences in the active site topology of the investigated CYP2D isoforms.

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