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
1
Figure S1. Ramachandran plot of the CYP2D6 homology model.
2
Detailed description of the docking results for each substrate
Carteolol. Carteolol, a cardiac and antihypertensive drug, is hydroxylated to 8hydroxycarteolol 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 Glu216 (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 Leu484, 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
3
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 3carbon 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
4
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.
.
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 Leu484 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).
5
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 3carbon.[66; 67] The top scoring (R)-2-methoxyamphetamine docked pose placed the Odemethylation SOM 2.68 Å from the oxeme and ninth top scoring pose placed the 5carbon 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 Odemethylation 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 5hydroxylation. 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 5hydroxylation. 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.
6
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
7
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.
8
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
9
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 Asp301 (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.
Tolterodine. Tolterodine was developed as a treatment for urinary incontinence and is
defined specifically as the (R) enantiomer [73]. CYP2D6 metabolism results in
hydroxylation at the 5-methyl position [73; 74]. The fifth highest scoring pose placed the
5-methyl group within the distance cut-off to the oxeme (2.59 Å). The internal distance
between the SoM and basic nitrogen was 6.61 Å and the basic nitrogen formed a salt
bridge with Glu-216 (2.97 Å). The 2-hydroxyl group was oriented facing the Phe-4832
side chain and formed no apparent hydrogen bonds, while the ring it is connected to was
packed between the heme, Phe-120, Val-370, Phe-483, and Leu-484 residues. The
10
unsubstituted phenyl group packed against the side chains of Leu-213, Val-308, and Leu484. Higher scoring poses that placed the SoM outside the distance cutoff to the oxeme
had the substituted phenyl ring flipped so that the 2-hydroxyl and 3-carbon positions
were oriented towards the oxeme.
Venlafaxine. Venlafaxine is a bicyclic antidepressant that is metabolized by CYP2D6
via O-demethylation [75]. This reaction is stereoselective, but not exclusive, for the Renantiomer [75]. The top scoring pose of (R)-venlafaxine placed the SoM 3.86 Å from
the oxeme and the internal distance between the basic nitrogen and SoM was 8.67 Å.
Two interactions were made with the Glu-216 side chain; a salt bridge with the basic
nitrogen (2.82 Å) and a hydrogen bond with the hydroxyl group (2.66 Å). The aromatic
ring was packed near the Phe-120 and Phe-483 side chains, while the cyclohexane ring
packed against the Leu-213 and Leu-484 side chains.
For (S)-venlafaxine, the top scoring pose also identified the SoM, placing the Odemethylation site 3.80 Å from the oxeme. The internal distance between the SoM and
basic nitrogen was 8.66 Å. Interactions to Glu-216 were similar to those found for the Renantiomer, with the basic nitrogen forming a salt bridge (2.84 Å) and the hydroxyl
forming a hydrogen bond (2.43 Å). While the aromatic ring was packed in the same
manner as seen with the R-enantiomer, the cyclohexane ring was placed adjacent to the
Phe-120 side chain.
11