Supporting Information for:

Using a Homology Model of Cytochrome P450 2D6 to Predict Substrate Site of Metabolism

Rayomand J. Unwalla, Jason B. Cross, Sumeet Salaniwal, Adam D. Shilling, Louis Leung, John Kao, and Christine Humblet

Contents:

Page 2 - Figure S1. Ramachandran plot of the CYP2D6 homology model.

Page 4 - Detailed description of the docking results for each substrate


Figure S1. Ramachandran plot of the CYP2D6 homology model.

Detailed description of the docking results for each substrate

Carteolol. Carteolol, a cardiac and antihypertensive drug, is hydroxylated to 8-hydroxycarteolol by CYP2D6 [60]. The third top scoring pose for (R)-carteolol placed the SoM 3.97 Å from the oxeme, and the internal SoM to basic nitrogen distance was 5.31 Å. A salt bridge was formed between the basic nitrogen and the side chain of Glu-216 (2.56 Å) and there was no tight ring packing with hydrophobic side chains in the pocket. The amide group was oriented towards the heme ring while the hydroxyl group was packed against the phenyl side chain of Phe-483 and not making any hydrogen bonds to the protein. These observations suggest that even though this pose correctly predicted the SoM for (R)-carteolol, there is significant uncertainty whether this represents the bioactive conformation.

The top scoring pose of (S)-carteolol was within the SoM to oxeme cut-off distance (3.52 Å), with an internal SoM to basic nitrogen distance was 4.67 Å, indicating a very compacted conformation. A salt bridge was present between the basic nitrogen and Glu-216 (2.69 Å). The hydroxyl group formed a hydrogen bond with the side chain of Glu-216 (2.97 Å) and was oriented to form a hydrogen bond with the Ser-217 side chain (3.89 Å), although the distance was slightly long. There was tight ring packing of the (S)-cartelol core scaffold against the heme ring, Phe-120, Val-370, Phe-483, and Leu-484, with the amide group deeply buried and forming no hydrogen bonds with the protein.

Codeine. Codeine or methylmorphine is a narcotic pain medicine used to treat mild to moderate pain. It was docked into the homology model for additional validation. It is metabolized by CYP2D6 via O-demethylation [61]. The fourth top scoring pose placed the SoM within the cut-off at 3.60 Å; this pose also had the N-methyl group in an (R) configuration that oriented the basic proton towards the Glu-216 side chain. Higher scoring poses outside the cut-off tended to have the codeine molecule twisted or flipped in the binding pocket so that the N-methyl group was facing the oxeme. The internal SoM to basic nitrogen distance was 7.83 Å and this distance is quite rigidly defined based on the relative inflexibility of the substrate’s scaffold. The salt bridge distance between the basic nitrogen and Glu-216 was 2.44 Å and there was tight packing of the codeine ring scaffold between the heme, Phe-120, Phe-483, and Leu-484. The hydroxyl group formed a hydrogen bond with the backbone nitrogen of Leu-484 (3.07 Å).

Debrisoquine. Debrisoquine is an antihypertensive drug that has been extensively examined in metabolism studies. The major metabolite of debrisoquine is 4(S)-OH debrisoquine. Minor metabolites have also been reported formed via hydroxylation at 1 and 3 positions [62]. Other studies have also reported hydroxylation at positions 7, 6 and 8 of the aromatic ring [63]. In our docking study, we did not identify any poses within the 4.5 Å cutoff that corresponded to the 1-OH debrisoquine metabolite, although several had a SoM to oxeme distance of ~5 Å. In addition, even for docking poses that placed the 3-carbon within the cutoff, the 4-carbon was always closer to the oxeme due to the orientation of the amidine group towards the Glu-216 side chain.

The 11th top scoring pose had a 4-carbon to oxeme distance of 4.15 Å and the basic nitrogen was positioned 2.62 Å from Glu-216, with monodentate coordination. The distance between the 4-carbon and basic nitrogen in debrisoquine was 4.80 Å, and due to limited conformational flexibility of the ring system this distance varied only slightly in other docked poses. The aromatic ring of debrisoquine packed against the side chains of Phe-120 and Phe-483, but was positioned away from the heme ring in most poses. The pose with the shortest oxeme to 4-carbon distance (3.10 Å) had tighter packing of the aromatic ring of debrisoquine with the heme and the distance from the basic nitrogen to Glu-216 was 2.68 Å and 2.82 Å (bidentate coordination). Higher scoring poses that did not place a SoM within the cutoff tended to have the same general pose, but were slightly more distant from the heme/oxeme complex.

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MDMA (3,4-methylenedioxymethamphetamine). MDMA is a commonly abused drug that leads to feelings of euphoria and enhanced sociability [64]. It is metabolized by CYP2D6 via demethylenation, to 3,4-OH methamphetamine (3,4-OH-MA) with a preference for the (S)-MDMA enantiomer [65]. The top scoring pose of (S)-MDMA had an oxeme to SoM distance of 2.96 Å, with an internal distance between the SoM and basic nitrogen of 6.35 Å. The basic nitrogen formed a salt bridge with Glu-216 (2.70 Å) and the aromatic ring packed tightly between the heme ring, Phe-120, Phe-483, and Leu-484 side chains.

The top scoring pose of (R)-MDMA had an oxeme to SoM distance within the cut-off, and was slightly longer (3.19 Å) than seen for the (S) enantiomer. The internal SoM to basic nitrogen distance was 6.77 Å. This enantiomer exhibited the same ring packing as (S)-MDMA and also formed a salt bridge between the basic nitrogen and the side chain of Glu-216 (2.68 Å).

2-Methoxyamphetamine

2-Methoxyamphetamine is metabolized by CYP2D6 via O-demethylation as well as hydroxylation at the 5-carbon, and to a lesser extent by hydroxylation at the 3-carbon.[66; 67] The top scoring (R)-2-methoxyamphetamine docked pose placed the O-demethylation SOM 2.68 Å from the oxeme and ninth top scoring pose placed the 5-carbon SOM within the cutoff, at 3.17 Å. The internal distances between the basic nitrogen and SOMs were 5.52 Å for O-demethylation and 6.51 Å for 5-hydroxylation. In both cases, the aromatic ring was packed between the heme ring, Phe-120, and Phe-483, but the pose indicating O-demethylation was packed tighter into the hydrophobic pocket. The distance between the basic nitrogen and Glu-216 side chain was 2.73 Å in the case of O-demethylation and 2.58 Å for 5-hydroxylation. The third highest scoring pose placed the 3-carbon SOM 4.24 Å from the oxeme, but otherwise adopted a geometry similar to that seen for the pose consistent with O-demethylation.

The top scoring (S)-2-methoxyamphetamine docked pose had an oxeme to O-demethylation SOM distance slightly within the cut-off at 4.36 Å and the third top scoring pose placed the 5-carbon 3.47 Å from the oxeme. The internal distance between the basic nitrogen and SOMs were 3.70 Å for O-demethylation and 5.32 Å for 5-hydroxylation. Aromatic ring packing into the hydrophobic pocket was similar to that seen for (R)-methoxyphenamine. A salt bridge was formed between the basic nitrogen and side chain of Glu-216, with a distance of 2.78 for O-demethylation and 2.76 Å for 5-hydroxylation. Inspection of an alternate pose with a short oxeme to O-demethylation SOM distance showed a binding mode in which the 2-methoxyamphetamine substrate was more extended in the active site, with a basic nitrogen to SOM distance of 6.42 Å. The aromatic ring of this pose fit into the hydrophobic pocket nearly perpendicular compared to the highest scoring pose and it also formed a salt bridge with Glu-216 (2.70 Å). The 6th highest scoring pose corresponded to hydroxylation at the 3-carbon, with a SOM to oxeme distance of 3.99 Å.

Metoprolol. Metoprolol is a β-blocker commonly used to treat heart failure [68]. Metoprolol metabolism by CYP2D6 occurs via two mechanisms; O-demethylation, which is selective (but not exclusive) for the R-enantiomer, and α-hydroxylation, which shows a slight preference for the S-enantiomer [44].

The second docked pose of (R)-metoprolol placed the O-demethylation SoM 3.51 Å from the oxeme with the basic nitrogen forming a bidentate salt bridge to Glu-216 (3.08 Å and 3.42 Å). The internal basic nitrogen to O-demethylation distance was 10.37 Å and there was a hydrogen bond formed between the hydroxyl group and the Asp-301 side chain (3.04 Å). The ninth top scoring pose placed the α-carbon 3.86 Å from the oxeme and had an internal basic nitrogen to α-hydroxylation SoM distance of 8.62 Å. The phenyl group was placed near the hydrophobic pocket, but was not packed tightly against the side chains of Phe-120 and Phe-483. The basic nitrogen formed a monodentate salt bridge with Glu-216 (2.81 Å) and there were no apparent hydrogen bonds between the hydroxyl group and the protein.

The top scoring docked pose for (S)-metoprolol placed the α-hydroxylation SoM 4.00 Å from the oxeme, which was within the 4.5 Å cut-off. The internal distance between the α-hydroxylation SoM and basic nitrogen was 9.11 Å and the aromatic ring was not packed tightly into the hydrophobic pocket. A bidentate salt bridge was formed between the basic nitrogen and Glu-216 (2.64 Å and 3.14 Å), and there was a long interaction between the hydroxyl group and Asp-301 (3.82 Å). The 11th top scoring docked pose placed the O-demethylation SoM within the cutoff at 2.84 Å. The internal basic nitrogen to SoM distance was 8.22 Å and the aromatic ring was positioned closer to the hydrophobic pocket, but was not tightly packed. Two interactions were made between the substrate and Glu-216 side chain; a monodentate salt bridge with the basic nitrogen (2.60 Å) and a hydrogen bond with the hydroxyl group (2.78 Å).

Mexiletine. Mexiletine is a well charcterized antiarrhythmic drug that also has anesthetic and anticonvulsant properties [69], as well as being effective treatment for neuropathic pain [70]. Metabolism by CYP2D6 occurs by hydroxylation at the 4-carbon and methyl groups, and while there is some enantioselectivity, both stereoisomers do experience metabolism at both sites [71].

The top scoring pose of (R)-mexiletine had an oxeme to methyl SoM distance of 3.01 Å. The second pose placed the 4-hydroxylation SoM 3.23 Å from the oxeme. This was an excellent result, since both SoMs were identified by the two top scoring poses. The internal distance from the basic nitrogen to the SoMs were 5.73 Å to the methyl SoM and 6.56 Å to the 4-hydroxylation SoM. Both poses showed the aromatic group packed between the heme ring, Phe-120, Phe-483, and Leu-484, and although both poses also indicated a salt bridge between the basic nitrogen and Glu-216 (2.64 Å for methyl SoM, 2.61 Å for 4-hydroxylation SoM), the orientation of the substrate meant that the interaction vectors for each salt bridge were on different sides of the acidic side chain.

For (S)-mexiletine, the top scoring pose placed the 4-hydroxylation SoM 3.33 Å from the oxeme. The tenth pose identified the methyl hydroxylation SoM, placed 2.95 Å from the oxeme. Internal distances from the basic nitrogen to the SoMs were 6.69 Å for 4-hydroxylation and 5.80 Å for methyl hydroxylation. The aromatic ring packed into the same part of the hydrophobic pocket in each case, but was rotated relative to each other (the 4-hydroxylation pose placed the aromatic ring perpendicular to the Phe-120 side chain while the aromatic ring in methyl hydroxylation pose was nearly parallel with the Phe-120 side chain. Similar to the docked poses seen for the R-enantiomer, the salt bridges from the basic nitrogen of (S)-mexilitene to Glu-216 in each pose were on either side of the acidic side chain (2.67 Å for 4-hydroxylation, 2.81 Å for methyl hydroxylation).

Mianserin. Mianserin is a tetracyclic antidepressant with two metabolic pathways catalyzed by CYP2D6 [72]. Hydroxylation to 8-hydroxy mianserin is not enantioselective, while N-demethylation is a minor metabolite of (R)-mianserin [72]. The top scoring pose of (R)-mianserin was within the distance cut-off between the hydroxylation SoM and oxeme (4.34 Å). The internal distance between the hydroxylation SoM and the basic nitrogen was 7.01 Å with no appreciable conformational flexibility in the ring system. The basic nitrogen formed a salt bridge with Glu-216 (3.39 Å) and there was some packing of the ring scaffold into the hydrophobic pocket between Phe-120 and Phe-483. The second highest scoring pose oriented the N-methyl group 3.17 Å away from the oxeme, with the substrate flipped in the binding site relative to the previously described pose. There was some stacking between the aromatic rings in mianserin against Phe-120, but it was not deeply buried. Overall, this was an excellent result, since both SoMs were identified as the two top scoring poses.

The sixth docked pose for (S)-mianserin was the highest scoring within the oxeme to hydroxylation SoM cutoff (4.03 Å). The basic nitrogen was positioned between Asp-301 (3.76 Å) and Glu-216 (4.05 Å), but it was not oriented to make high quality contacts with either residue. There was some stacking with Phe-120, but this pose (and other similar poses that positioned the SoM relatively well) did not appear to be a good representation of a potential binding mode. Additional conformational sampling of the ring or the inclusion of explicit side chain flexibility could have potentially improved the pose geometry and docking score for this binding mode. Higher scoring poses were either flipped in the binding pocket with a salt bridge interaction between the basic nitrogen and Glu-216, similar to that expected for N-demethylation, or were slightly more distant from the oxeme than the cut-off allowed.