A Refined Substrate Model for Human Cytochrome P450 2D6

Abstract

Cytochromes P450 (P450s) constitute a large superfamily of heme-containing enzymes, capable of oxidizing and reducing a variety of substrates. Cytochrome P450 2D6 is a polymorphic member of the P450 superfamily and is absent in 5-9% of the Caucasian population as a result of a recessive inheritance of gene mutations. Recently, the importance of aspartic acid 301 (Asp301) for the catalytic activity of P450 2D6, as indicated by a preliminary homology model, was confirmed by site-directed mutagenesis experiments. In this study, the heme moiety and the I-helix containing Asp301 were incorporated into the previously derived substrate model for P450 2D6, in the spatial orientations as derived from a recently improved protein model for P450 2D6, thereby incorporating steric restrictions and orientational preferences into the substrate model. The direction of well-defined hydrogen bonds formed between Asp301 and basic nitrogen atoms of P450 2D6 substrates was incorporated into the substrate model as well. Also, the position(s) of the basic nitrogen atom(s) of the substrates was/were allowed more flexibility. This was established through the attachment of an aspartic acid residue (representing Asp301) to the (protonated) basic nitrogen atom(s) of the substrates and superimposing the C alpha- and C beta-atoms of this aspartic acid residue in the fitting procedure instead of the basic nitrogen atoms. A variety of 8 substrates of P450 2D6 (comprising 17 known P450 2D6 dependent metabolic pathways) has been incorporated successfully into this refined and more restrictive substrate model.

Genetic polymorphisms of cytochrome P450 2D6 (CYP2D6): clinical consequences, evolutionary aspects and functional diversity

“…Site directed mutagenesis experiments indicate that Asp301 is the negatively charged amino acid responsible for binding to the substrate nitrogen. 8 In addition, also Ser304, Thr309 and Val370 appear to be involved in substrate binding 9 although further models are required for confirmation.…”

The Molecular Basis of CYP2D6-Mediated N-Dealkylation: Balance between Metabolic Clearance Routes and Enzyme Inhibition

“…In drug discovery research it is of great interest to identify whether a compound is a substrate or not, and considerable effort has been put into the development of models to identify substrates and/or inhibitors of CYP2D6. Several pharmacophore models have been published identifying common features in CYP2D6 substrates and inhibitors (Koymans et al, 1992;de Groot et al, 1997de Groot et al, , 1999Lewis et al, 1997). The pharmacophore comprises basic nitrogen atom 5-7 or 10 Å from the site of oxidation, a flat hydrophobic region near the site of oxidation and a negative molecular electrostatic potential coplanar with this hydrophobic region (see examples in Fig.…”

Cyp2d6 Catalyzes 5-Hydroxylation of 1-(2-Pyrimidinyl)-Piperazine, an Active Metabolite of Several Psychoactive Drugs, in Human Liver Microsomes

“…It is not surprising to find that CYP2D6 catalyzes the 5-hydroxylation of 1-PP. Pharmacophore modeling found that typical CYP2D6 substrates often contain a cationic nitrogen 5 or 7 Å from their major sites of oxidation, ideally situated to form ion-pair interactions with CYP2D6 active-site aspartic acid (D301) (De Groot et al, 1997). 1-PP would be a preferred CYP2D6 substrate for the ion-pairing orientation.…”