Active Site Of Cytochrome P450 2D6 Research Articles

Pharmacophore, QSAR, and Binding Mode Studies of Substrates of Human Cytochrome P450 2D6 (CYP2D6) Using Molecular Docking and Virtual Mutations and an Application to Chinese Herbal Medicine Screening

The highly polymorphic human cytochrome P450 2D6 (CYP2D6) metabolizes about 25% of currently used drugs. In this study, we have explored the interaction of a large number of substrates (n = 120) with wild-type and mutated CYP2D6 by molecular docking using the CDOCKER module. Before we conducted the molecular docking and virtual mutations, the pharmacophore and QSAR models of CYP2D6 substrates were developed and validated. Finally, we explored the interaction of a traditional Chinese herbal formula, Fangjifuling decoction, with CYP2D6 by virtual screening. The optimized pharmacophore model derived from 20 substrates of CYP2D6 contained two hydrophobic features and one hydrogen bond acceptor feature, giving a relevance ratio of 76% when a validation set of substrates were tested. However, our QSAR models gave poor prediction of the binding affinity of substrates. Our docking study demonstrated that 117 out of 120 substrates could be docked into the active site of CYP2D6. Forty one out of 117 substrates (35.04%) formed hydrogen bonds with various active site residues of CYP2D6 and 53 (45.30%) substrates formed a strong π-π interaction with Phe120 (53/54), with only carvedilol showing π-π interaction with Phe483. The active site residues involving hydrogen bond formation with substrates included Leu213, Lys214, Glu216, Ser217, Gln244, Asp301, Ser304, Ala305, Phe483, and Phe484. Furthermore, the CDOCKER algorithm was further applied to study the impact of mutations of 28 active site residues (mostly non-conserved) of CYP2D6 on substrate binding modes using five probe substrates including bufuralol, debrisoquine, dextromethorphan, sparteine, and tramadol. All mutations of the residues examined altered the hydrogen bond formation and/or aromatic interactions, depending on the probe used in molecular docking. Apparent changes of the binding modes have been observed with the Glu216Asp and Asp301Glu mutants. Overall, 60 compounds out of 130 from Fangjifuling decoction matched our pharmacophore model for CYP2D6 substrates. Fifty four out of these 60 compounds could be docked into the active site of CYP2D6 and 24 of 54 compounds formed hydrogen bonds with Glu216, Asp301, Ser304, and Ala305 in CYP2D6. These results have provided further insights into the factors that determining the binding modes of substrates to CYP2D6. Screening of high-affinity ligands for CYP2D6 from herbal formula using computational models is a useful approach to identify potential herb-drug interactions.

Ligand- and Protein-Based Modeling Studies of the Inhibitors of Human Cytochrome P450 2D6 and a Virtual Screening for Potential Inhibitors from the Chinese Herbal Medicine, Scutellaria baicalensis (Huangqin,Baikal Skullcap)

We have previously examined the binding patterns of various substrates to human cytochrome P450 2D6 (CYP2D6) using a series of molecular modeling methods. In this study, we further explored the binding modes of various types of inhibitors to CYP2D6 using a combination of ligand- and protein-based modeling approaches. Firstly, we developed and validated a pharmacophore model for CYP2D6 inhibitors, which consisted of two hydrophobic features and one hydrogen bond acceptor feature. Secondly, we constructed and validated a quantitative structure-activity relationship (QSAR) model for CYP2D6 inhibitors which gave a poor to moderate prediction accuracy. Thirdly, a panel of CYP2D6 inhibitors were subject to molecular docking into the active site of wild-type and mutated CYP2D6 enzyme. We demonstrated that 8 residues in the active site (Leu213, Glu216, Ser217, Gln244, Asp301, Ser304, Ala305, and Phe483) played an important role in the binding to the inhibitors via hydrogen bond formation and/or π-π stacking interaction. Apparent changes in the binding modes of the inhibitors have been observed with Phe120Ile, Glu216Asp, Asp301Glu mutations in CYP2D6. Finally, we screened for potential binders/inhibitors from the Chinese herbal medicine Scutellaria baicalensis (Huangqin, Baikal Skullcap) using the established pharmacophore model for CYP2D6 inhibitors and molecular docking approach. Overall, 18 out of 40 compounds from S. baicalensis were mapped to the pharmacophore model of CYP2D6 inhibitors and most herbal compounds from S. baicalensis could be docked into the active site of CYP2D6. Our study has provided insights into the molecular mechanisms of interaction of synthetic and herbal compounds with human CYP2D6 and further benchmarking studies are needed to validate our modeling and virtual screening results.

Residues Glutamate 216 and Aspartate 301 Are Key Determinants of Substrate Specificity and Product Regioselectivity in Cytochrome P450 2D6

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 site-directed 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 microm, k(cat) 5 min(-1)) along with nifedipine (K(m) 28 microm, k(cat) 2 min(-1)) and tolbutamide (K(m) 315 microm, 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.

Influence of phenylalanine-481 substitutions on the catalytic activity of cytochrome P450 2D6.

Homology models of the active site of cytochrome P450 2D6 (CYP2D6) have identified phenylalanine 481 (Phe(481)) as a putative ligand-binding residue, its aromatic side chain being potentially capable of participating in pi-pi interactions with the benzene ring of ligands. We have tested this hypothesis by replacing Phe(481) with tyrosine (Phe(481)-->Tyr), a conservative substitution, and with leucine (Phe(481)-->Leu) or glycine (Phe(481)-->Gly), two non-aromatic residues, and have compared the properties of the wild-type and mutant enzymes in microsomes prepared from yeast cells expressing the appropriate cDNA-derived protein. The Phe(481)-->Tyr substitution did not alter the kinetics [K(m) (microM) and V(max) (pmol/min per pmol) respectively] of oxidation of S-metoprolol (27; 4.60), debrisoquine (46; 2.46) or dextromethorphan (2; 8.43) relative to the respective wild-type values [S-metoprolol (26; 3.48), debrisoquine (51; 3.20) and dextromethorphan (2; 8.16)]. The binding capacities [K(s) (microM)] of a range of CYP2D6 ligands to the Phe(481)-->Tyr enzyme (S-metoprolol, 22.8; debrisoquine, 12.5; dextromethorphan, 2.3; quinidine, 0.13) were also similar to those for the wild-type enzyme (S-metoprolol, 10.9; debrisoquine, 8.9; dextromethorphan, 3.1; quinidine, 0.10). In contrast, the Phe(481)-->Leu and Phe(481)-->Gly substitutions increased significantly (3-16-fold) the K(m) values of oxidation of the three substrates [S-metoprolol (120-124 microM), debrisoquine (152-184 microM) and dextromethorphan (20-31 microM)]. Similarly, the K(s) values of the ligands to Phe(481)-->Leu and Phe(481)-->Gly mutants were also increased 3 to 10-fold (S-metoprolol, 33.2-41.9 microM; debrisoquine, 85-90 microM; dextromethorphan, 15.7-18.8 microM; quinidine 0.35-0.53 microM). However, contrary to a recent proposal that Phe(481) has the dominant role in the binding of substrates that undergo CYP2D6-mediated N-dealkylation routes of metabolism, the Phe(481)-->Gly substitution did not substantially decrease the capacity of the enzyme to N-deisopropylate metoprolol (wild-type, 1.12 pmol/min per pmol of P450; Phe(481)-->Gly, 0.71), whereas an Asp(301)-->Gly substitution decreased the N-dealkylation reaction by 95% of the wild-type rate. Overall, our results are consistent with the proposal that Phe(481) is a ligand-binding residue in the active site of CYP2D6 and that the residue interacts with ligands via a pi-pi interaction between its phenyl ring and the aromatic moiety of the ligand. However, the relative importance of Phe(481) in binding is ligand-dependent; furthermore, its importance is secondary to that of Asp(301). Finally, contrary to predictions of a recent homology model, Phe(481) does not seem to have a primary role in CYP2D6-mediated N-dealkylation.

Influence of phenylalanine-481 substitutions on the catalytic activity of cytochrome P450 2D6

Homology models of the active site of cytochrome P450 2D6 (CYP2D6) have identified phenylalanine 481 (Phe481) as a putative ligand-binding residue, its aromatic side chain being potentially capable of participating in π-π interactions with the benzene ring of ligands. We have tested this hypothesis by replacing Phe481 with tyrosine (Phe481 → Tyr), a conservative substitution, and with leucine (Phe481 → Leu) or glycine (Phe481 → Gly), two non-aromatic residues, and have compared the properties of the wild-type and mutant enzymes in microsomes prepared from yeast cells expressing the appropriate cDNA-derived protein. The Phe481 → Tyr substitution did not alter the kinetics [Km (µM) and Vmax (pmol/min per pmol) respectively] of oxidation of S-metoprolol (27; 4.60), debrisoquine (46; 2.46) or dextromethorphan (2; 8.43) relative to the respective wild-type values [S-metoprolol (26; 3.48), debrisoquine (51; 3.20) and dextromethorphan (2; 8.16)]. The binding capacities [Ks (µM)] of a range of CYP2D6 ligands to the Phe481 → Tyr enzyme (S-metoprolol, 22.8; debrisoquine, 12.5; dextromethorphan, 2.3; quinidine, 0.13) were also similar to those for the wild-type enzyme (S-metoprolol, 10.9; debrisoquine, 8.9; dextromethorphan, 3.1; quinidine, 0.10). In contrast, the Phe481 → Leu and Phe481 → Gly substitutions increased significantly (3-16-fold) the Km values of oxidation of the three substrates [S-metoprolol (120-124µM), debrisoquine (152-184µM) and dextromethorphan (20-31µM)]. Similarly, the Ks values of the ligands to Phe481 → Leu and Phe481 → Gly mutants were also increased 3 to 10-fold (S-metoprolol, 33.2-41.9µM; debrisoquine, 85-90µM; dextromethorphan, 15.7-18.8µM; quinidine 0.35-0.53µM). However, contrary to a recent proposal that Phe481 has the dominant role in the binding of substrates that undergo CYP2D6-mediated N-dealkylation routes of metabolism, the Phe481 → Gly substitution did not substantially decrease the capacity of the enzyme to N-deisopropylate metoprolol (wild-type, 1.12pmol/min per pmol of P450; Phe481 → Gly, 0.71), whereas an Asp301 → Gly substitution decreased the N-dealkylation reaction by 95% of the wild-type rate. Overall, our results are consistent with the proposal that Phe481 is a ligand-binding residue in the active site of CYP2D6 and that the residue interacts with ligands via a π-π interaction between its phenyl ring and the aromatic moiety of the ligand. However, the relative importance of Phe481 in binding is ligand-dependent; furthermore, its importance is secondary to that of Asp301. Finally, contrary to predictions of a recent homology model, Phe481 does not seem to have a primary role in CYP2D6-mediated N-dealkylation.

Regioselective hydroxylation of debrisoquine by cytochrome P4502D6: implications for active site modelling

1. Debrisoquine, a prototypic probe substrate for human cytochrome P4502D6 (CYP2D6), is hydroxylated at the alicyclic C4-position by this enzyme. Phenolic metabolites of debrisoquine (5-, 6-, 7- and 8-hydroxydebrisoquine) have also been reported as in vivo metabolites, but the role of CYP2D6 in their formation is unclear. 2. As part of studies to develop a predictive model of the active site of CYP2D6 using pharmacophore and homology modelling techniques, it became important to determine the precise regioselective hydroxylation of debrisoquine by CYP2D6. 3. Data from studies with human liver microsomes and yeast microsomes containing cDNA-derived CYP2D6 demonstrated unequivocally that debrisoquine was hydroxylated by CYP2D6 at each aromatic site in the molecule, as well as at the alicyclic 4-position. The four phenolic metabolites amounted to > 60% of the total identified products and the pattern of regioselective hydroxylation (4-HD > 7-HD > 6-HD > 8-HD > 5-HD) was similar in both in vitro systems. 4. A pharmacophore model for CYP2D6 indicated that while the hydroxylation of debrisoquine at alternative positions could arise from the substrate adopting multiple binding orientations, the energy constraints for the aromatic hydroxylations were unfavourable. An alternative proposal involving essentially a single binding orientation and a mechanism of hydroxylation based on benzylic radical spin delocalization could satisfactorily rationalize all the hydroxylations of debrisoquine. 5. This latter proposal demonstrates the need to consider the mechanism of oxidation as well as the spatial orientation of the substrate in the development of a predictive model of the active site of CYP2D6.