title: Homology modeling of rat and human cytochrome P450 2D (CYP2D) isoforms and computational rationalization of experimental ligand-binding specificities.
abstract: 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.
authors.affiliation: Department of Pharmacochemistry, Faculty of Sciences, Division of Molecular Toxicology, Leiden/Amsterdam Center for Drug Research, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Venhorst
authors.forename: Antonius M
authors.initials: AM
authors.lastname: ter Laak
authors.forename: Jan N M
authors.initials: JN
authors.lastname: Commandeur
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Funae
authors.forename: Toyoko
authors.initials: T
authors.lastname: Hiroi
authors.forename: Nico P E
authors.initials: NP
authors.lastname: Vermeulen
mesh_terms: D000429:Alcohol Oxidoreductases; D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry; D001665:Binding Sites; D003374:Coumarins / Q000737:chemistry; D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D000072467:Cytochrome P450 Family 2; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D006899:Mixed Function Oxygenases / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011802:Quinidine / Q000737:chemistry; D011803:Quinine / Q000737:chemistry; D011817:Rabbits; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013034:Sparteine / Q000737:chemistry; D013045:Species Specificity
chemical_list: C120605:7-methoxy-4-(aminomethyl)coumarin; D003374:Coumarins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008024:Ligands; D013034:Sparteine; D003577:Cytochrome P-450 Enzyme System; D011803:Quinine; D006899:Mixed Function Oxygenases; D000429:Alcohol Oxidoreductases; D001189:Aryl Hydrocarbon Hydroxylases; C504568:Cyp2d1 protein, rat; C119985:Cyp2d2 protein, rat; C115670:Cyp2d4 protein, rat; D019389:Cytochrome P-450 CYP2D6; D000072467:Cytochrome P450 Family 2; D011802:Quinidine