Cytochrome P450 2D6 (CYP2D6) metabolizes a wide range of therapeutic drugs. CYP2D6 substrates typically contain a basic nitrogen atom, and the active-site residue Asp-301 has been implicated in substrate recognition through electrostatic interactions. Our recent computational models point to a predominantly structural role for Asp-301 in loop positioning (Kirton, S. B., Kemp, C. A., Tomkinson, N. P., St.-Gallay, S., and Sutcliffe, M. J. (2002) Proteins 49, 216 -231) and suggest a second acidic residue, Glu-216, as a key determinant in the binding of basic substrates. We have evaluated the role of Glu-216 in substrate recognition, along with Asp-301, by sitedirected mutagenesis. Reversal of the Glu-216 charge to Lys or substitution with neutral residues (Gln, Phe, or Leu) greatly decreased the affinity (K m values increased 10 -100-fold) for the classical basic nitrogen-containing substrates bufuralol and dextromethorphan. Altered binding was also manifested in significant differences in regiospecificity with respect to dextromethorphan, producing enzymes with no preference for N-demethylation versus O-demethylation (E216K and E216F). Neutralization of Asp-301 to Gln and Asn had similarly profound effects on substrate binding and regioselectivity. Intriguingly, removal of the negative charge from either 216 or 301 produced enzymes (E216A, E216K, and D301Q) with elevated levels (50 -75-fold) of catalytic activity toward diclofenac, a carboxylate-containing CYP2C9 substrate that lacks a basic nitrogen atom. Activity was increased still further (>1000-fold) upon neutralization of both residues (E216Q/D301Q). The kinetic parameters for diclofenac (K m 108 M, k cat 5 min ؊1 ) along with nifedipine (K m 28 M, k cat 2 min ؊1) and tolbutamide (K m 315 M, k cat 1 min ؊1 ), which are not normally substrates for CYP2D6, were within an order of magnitude of those observed with CYP3A4 or CYP2C9. Neutralizing both Glu-216 and Asp-301 thus effectively alters substrate recognition illustrating the central role of the negative charges provided by both residues in defining the specificity of CYP2D6 toward substrates containing a basic nitrogen.Cytochromes P450 are a superfamily of heme-containing enzymes responsible for the oxidative metabolism of an extremely wide variety of substrates. Human cytochrome P450 2D6 (CYP2D6) 1 is one of the most important members of this family due to its central role in the metabolism of many drugs in common clinical use (1), such as opioids, antidepressants, neuroleptics, and various cardiac medications. CYP2D6 is polymorphic, giving rise to wide interindividual and ethnic differences in drug metabolism (2, 3). Inheritance of the defective gene results in the "poor metabolizer" phenotype that results in impaired drug oxidation reactions (4) and may be linked to altered disease susceptibility (5, 6). P450-drug and drug-drug interactions involving CYP2D6 ligands are a prime consideration in the development of new drugs, emphasizing the importance of a detailed understanding of the factors that gover...

Cytochrome P450 2D6 (CYP2D6) metabolizes approximately one third of the drugs in current clinical use. To gain insight into its structure and function, we have produced four different sets of comparative models of 2D6: one based on the structures of P450s from four different microorganisms (P450 terp, P450 eryF, P450 cam, and P450 BM3), another on the only mammalian P450 (2C5) structure available, and the other two based on alternative amino acid sequence alignments of 2D6 with all five of these structures. Principal component analysis suggests that inclusion of the 2C5 crystal structure has a profound effect on the modeling process, altering the general topology of the active site, and that the models produced differ significantly from all of the templates. The four models of 2D6 were also used in conjunction with molecular docking to produce complexes with the substrates codeine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP); this identified Glu 216 [in the F-helix; substrate recognition site (SRS) 2] as a key determinant in the binding of the basic moiety of the substrate. Our studies suggest that both Asp 301 and Glu 216 are required for metabolism of basic substrates. Furthermore, they suggest that Asp 301 (I-helix, SRS-4), a residue thought from mutagenesis studies to bind directly to the basic moiety of substrates, may play a key role in positioning the B'-C loop (SRS-1) and that the loss of activity on mutating Asp 301 may therefore be the result of an indirect effect (movement of the B'-C loop) on replacing this residue.

Although the residues that determine the preference of CYP2D6 (cytochrome P450 2D6) for compounds containing a basic nitrogen are well characterized, the contribution of other active site residues to substrate binding and orientation is less well understood. Our structural model of CYP2D6 identifies the aromatic residue Phe120 as a likely major feature of the active site. To examine the role of Phe120, mutants of CYP2D6 in which this residue has been substituted by alanine, leucine, tyrosine, serine, histidine, tryptophan or methionine residues have been prepared in bacterial membranes co-expressing human cytochrome NADPH cytochrome P450 oxidoreductase. The mutants have been characterized using the prototypical bufuralol 1' hydroxylase and dextromethorphan O- and N-demethylase activities of CYP2D6. Larger effects on K(m) values are observed for dextromethorphan O-demethylation than for bufuralol 1' hydroxylation, indicating that the Phe120 side chain is more important in dextromethorphan than in bufuralol binding. A role for this side chain in determining the regiospecificity of substrate oxidation was indicated by changes in the relative rates of O- and N-demethylation of dextromethorphan and, notably, by the formation of 7-hydroxy dextromethrophan, a novel dextromethorphan metabolite, in mutants in which it had been substituted. Computational studies of dextromethorphan binding to the active site of the Phe120-->Ala mutant were carried out to throw light on the way in which the removal of this side chain leads to different modes of ligand binding.

There has been much interest in the development of a predictive model of cytochrome P450 2D6 particularly because this enzyme is involved in the oxidation of at least 50 drugs. Previously we have described the combined use of homology modeling and molecular docking to correctly position a range of substrates in the CYP2D6 active site with the known sites of metabolism above the heme. Here, our approach identifies correctly the site of metabolism of the atypical (no basic nitrogen) cytochrome P450 2D6 substrate, spirosulfonamide. The same method is used to screen a small compound database for cytochrome P450 2D6 inhibition. A database containing 33 compounds from the National Cancer Institute database was docked into our cytochrome P450 2D6 homology model using the program GOLDv2.0. Experimental IC50 values for the 33 compounds were determined; comparison with the corresponding docked scores revealed a correlation with a regression coefficient of r2 = 0.61 (q2 = 0.59). The method was able to discriminate between tight and weak binding compounds and correctly identified several novel inhibitors. The results therefore suggest that our approach, which combines homology modeling with molecular docking, has produced a useful predictive in silico tool for cytochrome P450 2D6 inhibition, which is best used as one filter in a multifilter database screen.