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Metabolism of coumarin by human P450s: A molecular modelling study
Toxicology in Vitro 20 (2006) 256–264 www.elsevier.com/locate/toxinvit
Metabolism of coumarin by human P450s: A molecular modelling study David F.V. Lewis
a,*
, Yuko Ito b, Brian G. Lake
c
a
b
School of Biomedical and Molecular Sciences, University of Surrey, Stag Hill, Guildford, Surrey GU2 7XH, UK Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka-city, Fukuoka 820-8502, Japan c BIBRA International Limited, Woodmansterne Road, Carshalton, Surrey SM5 4DS, UK Received 1 June 2005; accepted 1 August 2005 Available online 12 September 2005
Abstract The oxidative metabolism of coumarin via several human cytochrome P450 (CYP) enzymes from families CYP1, CYP2 and CYP3 is rationalized in terms of molecular modelling studies carried out on the key interactions with various amino acid residues in the relevant active sites. The findings from modelling by homology with the CYP2C5 crystallographic template are in agreement with the known metabolism of coumarin in human P450s from the CYP1, CYP2 and CYP3 families, which has been published recently, and with independently reported information from site-directed mutagenesis studies. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Coumarin; Metabolism; Molecular modelling; P450
1. Introduction Cytochrome P450 (CYP) enzymes are ubiquitous catalysts of oxidative metabolism in biological systems (Lewis, 2001). These haem-thiolate enzymes have been shown to be present in all five biological kingdoms and in virtually all species, notable exceptions being some of the archaebacteria such as E. coli, and the sequences of over 2700 individual CYP genes have been reported in the literature to date. In humans, 57 individual P450s have been identified and most of these are associated with endogenous metabolism (Nelson, 2002; Lewis, 2004). However, enzymes of the CYP1, CYP2 and CYP3 families are generally regarded as repre-
Abbreviations: CYP, cytochrome P450 when referring to a specific enzyme or family/subfamily; P450, enzymes in either a general or nonspecific manner; SRS, substrate recognition site(s). * Corresponding author. Tel.: +44 1483 686477; fax: +44 1483 300803. E-mail address: [email protected] (D.F.V. Lewis). 0887-2333/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2005.08.001
senting the major catalysts for xenobiotic and drug metabolism in man (Rendic, 2002). Coumarin (1,2-benzopyrone) occurs naturally in several plant families and essential oils, and is widely used as a fragrance ingredient. The toxicology of coumarin merits attention because it exhibits marked species differences in both metabolism and hepatotoxicity (Lake, 1999). Although considered a non-genotoxic agent, coumarin has been shown to produce tumours in rodent species (reviewed in Lake, 1999). Coumarin can be metabolised by a number of pathways including 3,4epoxidation, 3-hydroxylation and 7-hydroxylation (Fig. 1). Coumarin-3,4-epoxide is unstable and rearranges, with the loss of carbon dioxide, to o-hydroxyphenylacetaldehyde, which can be further metabolised to o-hydroxyphenylethanol and o-hydroxyphenylacetic acid (Born et al., 1997; Lake, 1999). Many studies have demonstrated that coumarin 3,4-epoxidation is the major pathway of coumarin metabolism in rat and mouse, with this route being responsible for the known toxicity of coumarin in rodents (Born et al., 2000, 2003; Kaighen
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O CH2 CHO
CYP1A CYP2E1 CYP2A6
O coumarin
O CYP3A4
O O coumarin-3,4-epoxide OH
HO
O O 7-hydroxycoumarin
OH o-hydroxyphenylacetaldehyde
O O 3-hydroxycoumarin
Fig. 1. Metabolism of coumarin in man by P450 enzymes (Note: There is some disagreement between the findings of Zhuo et al. (1999) and those of Born et al. (2002) on the formation of 3-hydroxycoumarin via CYP3A4, where the latter report this pathway and the former report generation of o-hydroxyphenylacetaldehyde).
and Williams, 1961; Lake et al., 1989, 1992, 1994; Lovell et al., 1999). In contrast, both in vivo and in vitro studies have demonstrated that 7-hydroxylation is the major pathway of coumarin metabolism in humans, with o-hydroxyphenylacetic acid (derived from coumarin 3,4-epoxidation) being only a minor urinary metabolite of coumarin (Meineke et al., 1998; Pelkonen et al., 2000; Rautio et al., 1992; Shilling et al., 1969; van Iersel et al., 1994). With respect to the P450s involved in coumarin metabolism in humans, the role of CYP2A6 in coumarin 7-hydroxylation has been extensively studied (Lake, 1999; Pelkonen et al., 2000; Shimada et al., 1996). More recently, information has become available on the human P450s involved in coumarin 3,4-epoxidation and 3-hydroxylation. In a kinetic study, Zhuo et al. (1999) reported that cDNA-expressed CYP1A1, CYP1A2, CYP2B6, CYP2E1 and CYP3A4 could catalyse the metabolism of coumarin to the 3,4-epoxidation pathway metabolite o-hydroxyphenylacetaldehyde, whereas cDNA-expressed CYP2A6 only formed 7-hydroxycoumarin. The metabolism of coumarin to o-hydroxyphenylacetaldehyde by cDNA-expressed CYP1A1, CYP1A2 and CYP2E1 was also reported by Born et al. (2002) who, moreover, observed that coumarin could be metabolised to 3-hydroxycoumarin by CYP3A4 and to a lesser extent by other P450s. The present study involves the use of molecular modelling procedures, including protein homology modelling, for the investigation of the likely modes of binding between coumarin and the relevant human P450 enzymes which catalyze the different pathways of its metabolism.
2. Methods Human cytochrome P450 models of CYP1A1, CYP1A2, CYP2A6 and CYP3A4 were constructed by homology with the CYP2C5 (Williams et al., 2000) crys-
tallographic template (pdb code: 1dt6) using a multiple sequence alignment which has been reported previously (Lewis et al., 2003a,b,c,d,e). The full details of the homology modelling process has also been published recently (Lewis, 2002a,b) and, consequently, this will only be outlined here. Essentially, this involves changing the relevant amino acid residues in the template structure according to those required by the alignment with the target sequence. Following the insertion of residues via loop-searching of the protein databank, and deletion of any other residues required by the alignment, the raw structure is then energy minimized to achieve a low energy protein conformation which is also consistent with the known constraints of protein geometries. Each energy-minimized P450 model is then probed using known, selective substrates of the relevant enzyme, such that a template of superimposed molecules is built up for each active site region. The published information from site-directed mutagenesis and substrate metabolism is employed for assisting this process, together with interactive docking (using DOCK) in connection with a dynamic hydrogen bond functionality in the Sybyl Software Suite (Tripos Associates, St. Louis, Missouri). Atomic partial charges were calculated using the Gasteiger–Marsili method, and Sybyl also has an interactive docking feature which simultaneously calculates steric and electrostatic energies as the substrate is moved within the enzyme binding site. Sybyl Biopolymer was used for homology modelling of the P450 enzymes from the rabbit CYP2C5 crystal structure, whereas other features of the Sybyl software were employed for energy minimization and substrate docking. All molecular modelling was performed on a Silicon Graphics Indigo2 IMPACT 10000 graphics workstation operating under Unix. We have also employed AutoDock, version 3.05 (Morris et al., 1998) for automated docking of coumarin in the various P450 models. This technique has been compared with other similar algorithms such as FlexX and GOLD (de Graaf et al., 2005) and appears to perform satisfactorily in comparison with the other methods.
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3. Results and discussion Fig. 2 shows how coumarin may fit within the putative active site of human CYP1A1 for metabolism at the experimentally observed position. It should be recognized that, in man, CYP1A1 is largely an extrahepatic enzyme, in contrast with the situation in experimental rodent species. The coumarin molecule is able to become orientated for 3,4-epoxidation by interactions with complementary amino acid residues which place the 3,4 bond directly above the haem moiety. It would appear to be a combination of hydrogen bond and p–p stacking contacts with key amino acids within the haem environment which position the coumarin substrate for 3,4epoxidation. Some of these have been probed using site-directed mutagenesis in CYP1A subfamily enzymes, and are known to affect the binding of other typical substrates. Table 1 shows the positions of some of these residues in an alignment between the substrate recognition sites (SRS regions) of CYP1A1, CYP1A2, CYP2A6 and CYP3A4, which has been based on the CYP2C5 crystallographic template used to generate homology models of these enzymes. From this table, it can be appreciated that there are certain similarities and also differences between the four matched regions of these enzymes. In fact, such comparisons between the relevant substrate recognition sites help to explain both the substrate
selectivity and altered regioselectivity of these P450s, particularly that of coumarin in this instance. For example, the presence of a phenylalanine residue at alignment position 205 (SRS2) tends to be associated with the binding of aromatic rings of typical substrates, and this residue is present in all of the listed enzymes except CYP3A4. Consequently, there tends to be a p–p stacking interaction between coumarin and the phenylalanine at position 205 in CYP1A1, CYP1A2, CYP2A6 and CYP2E1. However, in the case of CYP3A4, coumarin appears to be able to bind with an alternative phenylalanine residue. Apart from this aromatic p–p stacking information, at least one hydrogen bond-forming amino acid residue is apparently able to interact with coumarin in these P450s, and this tends to determine the site of metabolism in the substrate. For example, the asparagine residue at alignment position 290 in CYP2A6 can form a hydrogen bond with coumarin which orientates the molecule for 7-hydroxylation, as the relevant hydrogen atom in coumarin lies directly above the haem iron in the modelled enzyme–substrate complex (Lewis and Lake, 2002). Consequently, homology modelling of the relevant P450s can play a useful role in rationalizing the regioselectivity of these enzymes towards certain substrates, such as coumarin. Fig. 3 shows how coumarin may become orientated for 7-hydroxylation within the putative
Fig. 2. The putative active site of human CYP1A1 showing a possible orientation of coumarin for 3,4-epoxidation. Key amino acid residues are labelled according to their alignment position (see Table 1) with the CYP2C5 crystallographic template, and hydrogen bonds are shown in dashed lines between coumarin and the protein.
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Table 1 Similarities and differences between SRS regions in human P450 sequences SRS 1
Alignment position
CYP 1A1 1A2 2A6 2E1 3A4
100 L L Q L R
SRS 2
Alignment position
CYP 1A1 1A2 2A6 2E1 3A4
200 N K M L N
SRS 4
Alignment position
CYP 1A1 1A2 2A6 2E1 3A4
L N L A I
SRS 5
Alignment position
CYP 1A1 1A2 2A6 2E1 3A4
I S G I F
SRS 6
Alignment position
CYP 1A1 1A2 2A6 2E1 3A4
M L V L L
# T T T A F
Y Y A P P
L N M F T
D S D T P
T T S S K
L I L L F
L L W H V
N H G E K
# N E I N L
# F F F F L
G V Q H R
E E F L F
V T T L D
V A S S F
210 G S T T L
# G G I F F
* A A G A A
* G G G G G
F F T T Y
D D E E E
* T T T T T
V V V T T
L F V L I
360 P P P P –
F F M S M
T T S N R
# I I L L L
P P A P E
H H R H R
P P P P L
G G V I G
L L G G G
F F F F I
# L L I V A
I I K I S
470 Y Y H H L
S T F H F
# T T F F L
N D K R M
110 Q Q Y R S
T T D – P
N T L N K
290 D D N D I
# I I L A G
F S V F G
M M A G L
G G G D K
S S G G A
M L V I I
# S T V I S
# F F F F I
S S S N A
300 T T S S S
K K T C Q
–: denotes gap in the alignment with CYP2C5. *: denotes conserved residues in SRS4. #: potential substrate contact residue (as encountered in CYP2C5 substrate-bound crystal structure). SRS: substrate recognition site, a term coined by Gotoh (1992) in the analysis of CYP2 family sequences. Residues which have been probed using site-directed mutagenesis are emboldened (the ones in CYP2A6 correspond to those which have been probed for the other orthologues, such as CYP2A4 and CYP2A5). References: Lewis et al. (2003a), Domanksi et al. (2001), Hadjokas et al. (2002) and Domanski and Halpert (2001).
active site of CYP2A6 as outlined above, whereas Fig. 4 indicates that the positioning of coumarin in CYP1A2 is somewhat different from the situation in CYP2A6 due to the changed spacial arrangement of hydrogen bond forming residues. The putative active site of human CYP1A1 is closely related to that of CYP1A2, although some changes in certain residue positions exist, probably explaining the slightly different, but overlapping, substrate preferences of the two CYP1A subfamily enzymes. However, the route of coumarin metabolism is the same for both CYP1A1 and CYP1A2, as summarized in Table 2 which also provides information for CYP2A6, CYP2E1 and CYP3A4-mediated metabolism
of this substrate. As far as CYP3A4 is concerned, coumarin metabolism is directed towards either 3-hydroxylation or 3,4-epoxidation (Born et al., 2002; Zhuo et al., 1999), whereas the position in CYP2E1 mirrors that of the CYP1A-mediated situation where coumarin forms the 3,4-epoxide (see Table 2 and Fig. 1). In a previous study, we showed that the differences in coumarin metabolism in the rat and mouse may be due to a variation in the active site residues of rat CYP2A1 and mouse CYP2A4 and CYP2A5 (Lewis and Lake, 2002). For the two mouse orthologues, CYP2A4 and CYP2A5, it is likely that only a single amino acid residue change (L209F) is sufficient to alter the substrate
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Fig. 3. The putative active site of CYP2A6 showing an orientation of coumarin for 7-hydroxylation. Key amino acid residues are labelled according to their alignment position (see Table 1) with respect to the CYP2C5 crystallographic template. Hydrogen bonds are shown as dashed lines between coumarin and the protein.
selectivity of CYP2A4 from towards testosterone (as a 15a-hydroxylase) to that of coumarin (as a 7-hydroxylase) as encountered in CYP2A5 (Lindberg and Negishi, 1989). Consequently, it is interesting to note that the corresponding residue position in human CYP2A6 to Phe-209 in the mouse orthologue CYP2A5 is also a phenylalanine, and this is shown in Table 1 at alignment position 205 with respect to the CYP2C5 structural template. In fact, phenylalanine is present at this position for all of the human P450s discussed in this work apart from CYP3A4 where the corresponding residue at this point is leucine. However, if a phenylalanine is required for p–p stacking with the aromatic ring system of coumarin, this is afforded by one such residue at alignment position 102 in the CYP3A4 SRS1, as shown in Table 1. Therefore, it would appear that the presence of an aromatic amino acid in the active site region is probably essential for coumarin metabolism by the P450 concerned, although the actual position of coumarin oxygenation depends on other factors and this is probably caused by the disposition of a key hydrogen bond donor residue in the active site. Of the five human P450 enzymes under consideration, only CYP2A6 metabolizes coumarin at the 7-position and it is interesting to observe that the aspargine residue at position 290 (Table 1) may form a hydrogen bond with the substrate and thus orientate it for hydroxylation at the 7-position.
This becomes an aspartate in the CYP1A1, CYP1A2 and CYP2E1 sequences, and all of these enzymes are known to exhibit coumarin-3,4-epoxidase activity. In the case of CYP3A4, which mediates in 3-hydroxylation of coumarin (Born et al., 2002), there is a nonconservative change to isoleucine at this point (i.e. at the residue corresponding to position 290 in the alignment) and it appears that the carbonyl oxygen of coumarin can enter into hydrogen-bonded interactions with an active site serine at alignment position 113 (see Table 1) which corresponds to Ser-119 in CYP3A4. This residue has been the subject of site-directed mutagenesis experiments in CYP3A4, albeit with reference to other substrates of this enzyme (reviewed in Domanski and Halpert, 2001). However, the evidence suggests that Ser-119 would lie relatively close to the haem group as the hydrogen bond acceptor atoms on CYP3A4 sub˚ ngstroms from the site of metabstrates lie only a few A olism. Therefore, it is likely that coumarin binds via a hydrogen-bonded interaction with Ser-119 in CYP3A4, which serves to orientate the substrate for 3-hydroxylation. Fig. 5 shows how such an orientation may be achieved for coumarin binding to the putative active site of CYP3A4, where there is also the possibility of p–p stacking to a complementary aromatic amino acid residue, which can thus augment the hydrogen bond interaction between the substrate and Ser-119.
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261
Fig. 4. The putative active site of human CYP1A2 showing the substrate oritentated for 3,4-epoxidation. Key amino acid residues are labelled according to their alignment position (see Table 1) with the CYP2C5 crystallographic template. Hydrogen bonds are shown as dashed lines between coumarin and the protein.
Table 2 Metabolism of coumarin by human P450s CYP
Route of metabolism
References
1A1 1A2 2A6 2E1 3A4
3,4-Epoxidation 3,4-Epoxidation 7-Hydroxylation 3,4-Epoxidation 3-Hydroxylation and 3,4-epoxidation
Born et al. (2002) and Zhuo et al. (1999) Born et al. (2002) and Zhuo et al. (1999) Shimada et al. (1996), Pelkonen et al. (2000), Zhuo et al. (1999) and Lake (1999) Born et al. (2002) and Zhuo et al. (1999) Born et al. (2002) and Zhuo et al. (1999)
5
4 3
6
Coumarin structure showing sites of metabolism (arrowed) mediated by human P450s listed above. 2
7 8
O 1
O
Calculations based on substrate lipophilicity (using the log P value, where P is the octanol/water partition coefficient) and typical average energies for hydrogenbonded and p–p stacking interactions, generally provide reasonably satisfactory estimates for the overall enzyme–substrate binding energy, which agree well with experimentally determined values (Lewis, 2003). Table 3 provides a summary of these calculations based on an average value of 2 kcal mol1 for a typical hydrogen bond and 0.9 kcal mol1 for an aromatic p–p
stacking interaction between two six-membered aromatic rings. In the case of coumarin, there is no contribution from loss in bond rotational freedom due to the rigid planar structure of the molecule. Therefore, any differences in enzyme–substrate binding energy should be associated with the number of hydrogen-bonded and p–p stacking interactions, as the desolvation component will also likely be the same in each case, if one assumes that the hydrophobicity of the active sites are similar. Consequently, it is feasible to make reasonable
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Fig. 5. The putative site of CYP3A4 with coumarin positioned for 3-hydroxylation. Key amino acid residues are labelled according to their alignment position (see Table 1) with respect to the CYP2C5 crystallographic template. Hydrogen bonds are shown as dashed lines between coumarin and the protein.
Table 3 Estimation of binding energies for coumarin-P450 interactions CYP
DGdesol
DGhb
DGp–p
DGcalc bind
AutoDock
Km (lM)
DGexpt bind
1A1 1A2 2A6 2E1 3A4
1.9717 1.9717 1.9717 1.9717 1.9717
2.0 4.0 4.0 2.0 4.0
1.8 0.9 1.8 0.9 0
5.7717 6.8717 7.7717 6.8717 5.9717
5.28 5.81 5.70 5.61 5.98
51 19 6 12 54
6.0887 6.6969 7.4070 6.9800 6.0535
The agreement between experimental and calculated values for binding affinity (DGbind) shows an R2 of 0.95, for the five points. CYP, cytochrome P450 enzyme; DGcalc bind ¼ DGdesol þ DGhb þ DGp–p ; DGdesol = RT ln P, where P is the octanol/water partition coefficient; DGhb, average hydrogen bond energy of 2 kcal mol1 per hydrogen bond; DGp–p, average p–p stacking interaction energy of 0.9 kcal mol1 per p–p stack; DGexpt bind ¼ RT ln K m , where Km is the Michaelis constant (lM) taken from the literature (Zhuo et al., 1999); R, gas constant (1.9872 cal mol1 K1); T, absolute temperature (taken as 310 K); AutoDock, calculated binding energy (kcal mol1) between the coumarin substrate and each CYP using the AutoDock software (Morris et al., 1998).
estimates of overall binding affinity based on the likely contributions from the major contributing factors of desolvation, hydrogen bonding and p–p stacking according to the relationship: DGbind ¼ DGdesolv þ DGhb þ DGp–p where these terms have been described previously (Lewis, 2003) with the binding free energy (DGbind) being obtained from the Km value (Bauer et al., 2001). The results shown in Table 3, albeit based on a limited dataset, indicate that there is a very good agreement (R2 = 0.95) between calculated and experimental binding energies based on the above expression. This finding indicates that the overall method of estimating binding energies in this way is likely to be fairly sound, and may be employed for other P450 substrates in predicting their Km values provided that the likely interactions
within the enzyme active sites are either known, or can be inferred from molecular modelling investigations. The calculated binding energies obtained via AutoDock are listed in Table 3 and, apart from that of CYP3A4, are all somewhat lower than the experimentally determined values. It is thought that a possible explanation for this may lie in certain key interactions not being fully taken into account, particularly p–p stacking between nearby aromatic ring systems. The discrepancies for CYP1A1, CYP1A1 and CYP2E1 are approximately 1 kcal mol1, whereas that of CYP2A6 is nearer 2 kcal mol1 which suggests that one of the hydrogen bond interactions may not have been identified by the automated docking program. Taking these considerations into account, the agreement between AutoDock and the experimental data is quite satisfactory, and of a similar magnitude to that estimated
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from first principles via the additive method described above.
4. Conclusions The metabolism of coumarin by several human P450s can be rationalized in terms of the likely interactions between the substrate and enzyme active site in each case. In addition, it is possible to derive estimated values for the binding interaction energy which agree well with experimental findings. Automated docking of coumarin using the AutoDock software also produced satisfactory results although a discrepancy of about 1 kcal mol1 in the calculated binding energy was observed in most cases. It is anticipated, therefore, that a similar approach may be feasible for other P450–substrate interactions, and that there could be wider applicability of these techniques to other areas of ligand–receptor or enzyme– substrate binding interactions.
Acknowledgements The financial support of GlaxoSmithKline Research and Development Limited, Merck, Sharp & Dohme Limited, British Technology Group Limited and the University of Surrey, is gratefully acknowledged by one of us (DFVL). Yuko Ito would like to thank the Japanese government for the award of a visiting scientist scholarship as part of a PhD programme.
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Rautio, A., Kraul, H., Koji, A., Salmela, E., Pelkonen, O., 1992. Interindividual variability of coumarin 7-hydroxylase in healthy volunteers. Pharmacogenetics 2, 227–233. Rendic, S., 2002. Summary of information on human CYP enzymes: human P450 metabolism data. Drug Metabolism Reviews 34, 83– 448. Shilling, W.H., Crampton, R.F., Longland, R.C., 1969. Metabolism of coumarin in man. Nature 221, 664–665. Shimada, T., Yamazaki, H., Guengerich, F.P., 1996. Ethnic-related differences in coumarin 7-hydroxylation activities catalyzed by cytochromes P4502A6 in liver microsomes of Japanese and Caucasian populations. Xenobiotica 26, 395–403. van Iersel, M.L.P.S., Henderson, C.J., Walters, D.G., Price, R.J., Wolf, C.R., Lake, B.G., 1994. Metabolism of [3-14C] coumarin by human liver microsomes. Xenobiotica 24, 795–803. Williams, P.A., Cosme, J., Sridhar, V., Johnson, E.F., McRee, D.E., 2000. Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity. Molecular Cell 5, 121–131. Zhuo, X., Gu, J., Zhang, Q.-Y., Spink, D.C., Kaminsky, L.S., Ding, X., 1999. Biotransformation of coumarin by rodent and human cytochromes P450: metabolic basis of tissue-selective toxicity in olfactory mucosa of rats and mice. Journal of Pharmacology and Experimental Therapeutics 288, 463–471.
Metabolism of coumarin by human P450s: A molecular modelling study David F.V. Lewis
a,*
, Yuko Ito b, Brian G. Lake
c
a
b
School of Biomedical and Molecular Sciences, University of Surrey, Stag Hill, Guildford, Surrey GU2 7XH, UK Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka-city, Fukuoka 820-8502, Japan c BIBRA International Limited, Woodmansterne Road, Carshalton, Surrey SM5 4DS, UK Received 1 June 2005; accepted 1 August 2005 Available online 12 September 2005
Abstract The oxidative metabolism of coumarin via several human cytochrome P450 (CYP) enzymes from families CYP1, CYP2 and CYP3 is rationalized in terms of molecular modelling studies carried out on the key interactions with various amino acid residues in the relevant active sites. The findings from modelling by homology with the CYP2C5 crystallographic template are in agreement with the known metabolism of coumarin in human P450s from the CYP1, CYP2 and CYP3 families, which has been published recently, and with independently reported information from site-directed mutagenesis studies. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Coumarin; Metabolism; Molecular modelling; P450
1. Introduction Cytochrome P450 (CYP) enzymes are ubiquitous catalysts of oxidative metabolism in biological systems (Lewis, 2001). These haem-thiolate enzymes have been shown to be present in all five biological kingdoms and in virtually all species, notable exceptions being some of the archaebacteria such as E. coli, and the sequences of over 2700 individual CYP genes have been reported in the literature to date. In humans, 57 individual P450s have been identified and most of these are associated with endogenous metabolism (Nelson, 2002; Lewis, 2004). However, enzymes of the CYP1, CYP2 and CYP3 families are generally regarded as repre-
Abbreviations: CYP, cytochrome P450 when referring to a specific enzyme or family/subfamily; P450, enzymes in either a general or nonspecific manner; SRS, substrate recognition site(s). * Corresponding author. Tel.: +44 1483 686477; fax: +44 1483 300803. E-mail address: [email protected] (D.F.V. Lewis). 0887-2333/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2005.08.001
senting the major catalysts for xenobiotic and drug metabolism in man (Rendic, 2002). Coumarin (1,2-benzopyrone) occurs naturally in several plant families and essential oils, and is widely used as a fragrance ingredient. The toxicology of coumarin merits attention because it exhibits marked species differences in both metabolism and hepatotoxicity (Lake, 1999). Although considered a non-genotoxic agent, coumarin has been shown to produce tumours in rodent species (reviewed in Lake, 1999). Coumarin can be metabolised by a number of pathways including 3,4epoxidation, 3-hydroxylation and 7-hydroxylation (Fig. 1). Coumarin-3,4-epoxide is unstable and rearranges, with the loss of carbon dioxide, to o-hydroxyphenylacetaldehyde, which can be further metabolised to o-hydroxyphenylethanol and o-hydroxyphenylacetic acid (Born et al., 1997; Lake, 1999). Many studies have demonstrated that coumarin 3,4-epoxidation is the major pathway of coumarin metabolism in rat and mouse, with this route being responsible for the known toxicity of coumarin in rodents (Born et al., 2000, 2003; Kaighen
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257
O CH2 CHO
CYP1A CYP2E1 CYP2A6
O coumarin
O CYP3A4
O O coumarin-3,4-epoxide OH
HO
O O 7-hydroxycoumarin
OH o-hydroxyphenylacetaldehyde
O O 3-hydroxycoumarin
Fig. 1. Metabolism of coumarin in man by P450 enzymes (Note: There is some disagreement between the findings of Zhuo et al. (1999) and those of Born et al. (2002) on the formation of 3-hydroxycoumarin via CYP3A4, where the latter report this pathway and the former report generation of o-hydroxyphenylacetaldehyde).
and Williams, 1961; Lake et al., 1989, 1992, 1994; Lovell et al., 1999). In contrast, both in vivo and in vitro studies have demonstrated that 7-hydroxylation is the major pathway of coumarin metabolism in humans, with o-hydroxyphenylacetic acid (derived from coumarin 3,4-epoxidation) being only a minor urinary metabolite of coumarin (Meineke et al., 1998; Pelkonen et al., 2000; Rautio et al., 1992; Shilling et al., 1969; van Iersel et al., 1994). With respect to the P450s involved in coumarin metabolism in humans, the role of CYP2A6 in coumarin 7-hydroxylation has been extensively studied (Lake, 1999; Pelkonen et al., 2000; Shimada et al., 1996). More recently, information has become available on the human P450s involved in coumarin 3,4-epoxidation and 3-hydroxylation. In a kinetic study, Zhuo et al. (1999) reported that cDNA-expressed CYP1A1, CYP1A2, CYP2B6, CYP2E1 and CYP3A4 could catalyse the metabolism of coumarin to the 3,4-epoxidation pathway metabolite o-hydroxyphenylacetaldehyde, whereas cDNA-expressed CYP2A6 only formed 7-hydroxycoumarin. The metabolism of coumarin to o-hydroxyphenylacetaldehyde by cDNA-expressed CYP1A1, CYP1A2 and CYP2E1 was also reported by Born et al. (2002) who, moreover, observed that coumarin could be metabolised to 3-hydroxycoumarin by CYP3A4 and to a lesser extent by other P450s. The present study involves the use of molecular modelling procedures, including protein homology modelling, for the investigation of the likely modes of binding between coumarin and the relevant human P450 enzymes which catalyze the different pathways of its metabolism.
2. Methods Human cytochrome P450 models of CYP1A1, CYP1A2, CYP2A6 and CYP3A4 were constructed by homology with the CYP2C5 (Williams et al., 2000) crys-
tallographic template (pdb code: 1dt6) using a multiple sequence alignment which has been reported previously (Lewis et al., 2003a,b,c,d,e). The full details of the homology modelling process has also been published recently (Lewis, 2002a,b) and, consequently, this will only be outlined here. Essentially, this involves changing the relevant amino acid residues in the template structure according to those required by the alignment with the target sequence. Following the insertion of residues via loop-searching of the protein databank, and deletion of any other residues required by the alignment, the raw structure is then energy minimized to achieve a low energy protein conformation which is also consistent with the known constraints of protein geometries. Each energy-minimized P450 model is then probed using known, selective substrates of the relevant enzyme, such that a template of superimposed molecules is built up for each active site region. The published information from site-directed mutagenesis and substrate metabolism is employed for assisting this process, together with interactive docking (using DOCK) in connection with a dynamic hydrogen bond functionality in the Sybyl Software Suite (Tripos Associates, St. Louis, Missouri). Atomic partial charges were calculated using the Gasteiger–Marsili method, and Sybyl also has an interactive docking feature which simultaneously calculates steric and electrostatic energies as the substrate is moved within the enzyme binding site. Sybyl Biopolymer was used for homology modelling of the P450 enzymes from the rabbit CYP2C5 crystal structure, whereas other features of the Sybyl software were employed for energy minimization and substrate docking. All molecular modelling was performed on a Silicon Graphics Indigo2 IMPACT 10000 graphics workstation operating under Unix. We have also employed AutoDock, version 3.05 (Morris et al., 1998) for automated docking of coumarin in the various P450 models. This technique has been compared with other similar algorithms such as FlexX and GOLD (de Graaf et al., 2005) and appears to perform satisfactorily in comparison with the other methods.
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3. Results and discussion Fig. 2 shows how coumarin may fit within the putative active site of human CYP1A1 for metabolism at the experimentally observed position. It should be recognized that, in man, CYP1A1 is largely an extrahepatic enzyme, in contrast with the situation in experimental rodent species. The coumarin molecule is able to become orientated for 3,4-epoxidation by interactions with complementary amino acid residues which place the 3,4 bond directly above the haem moiety. It would appear to be a combination of hydrogen bond and p–p stacking contacts with key amino acids within the haem environment which position the coumarin substrate for 3,4epoxidation. Some of these have been probed using site-directed mutagenesis in CYP1A subfamily enzymes, and are known to affect the binding of other typical substrates. Table 1 shows the positions of some of these residues in an alignment between the substrate recognition sites (SRS regions) of CYP1A1, CYP1A2, CYP2A6 and CYP3A4, which has been based on the CYP2C5 crystallographic template used to generate homology models of these enzymes. From this table, it can be appreciated that there are certain similarities and also differences between the four matched regions of these enzymes. In fact, such comparisons between the relevant substrate recognition sites help to explain both the substrate
selectivity and altered regioselectivity of these P450s, particularly that of coumarin in this instance. For example, the presence of a phenylalanine residue at alignment position 205 (SRS2) tends to be associated with the binding of aromatic rings of typical substrates, and this residue is present in all of the listed enzymes except CYP3A4. Consequently, there tends to be a p–p stacking interaction between coumarin and the phenylalanine at position 205 in CYP1A1, CYP1A2, CYP2A6 and CYP2E1. However, in the case of CYP3A4, coumarin appears to be able to bind with an alternative phenylalanine residue. Apart from this aromatic p–p stacking information, at least one hydrogen bond-forming amino acid residue is apparently able to interact with coumarin in these P450s, and this tends to determine the site of metabolism in the substrate. For example, the asparagine residue at alignment position 290 in CYP2A6 can form a hydrogen bond with coumarin which orientates the molecule for 7-hydroxylation, as the relevant hydrogen atom in coumarin lies directly above the haem iron in the modelled enzyme–substrate complex (Lewis and Lake, 2002). Consequently, homology modelling of the relevant P450s can play a useful role in rationalizing the regioselectivity of these enzymes towards certain substrates, such as coumarin. Fig. 3 shows how coumarin may become orientated for 7-hydroxylation within the putative
Fig. 2. The putative active site of human CYP1A1 showing a possible orientation of coumarin for 3,4-epoxidation. Key amino acid residues are labelled according to their alignment position (see Table 1) with the CYP2C5 crystallographic template, and hydrogen bonds are shown in dashed lines between coumarin and the protein.
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Table 1 Similarities and differences between SRS regions in human P450 sequences SRS 1
Alignment position
CYP 1A1 1A2 2A6 2E1 3A4
100 L L Q L R
SRS 2
Alignment position
CYP 1A1 1A2 2A6 2E1 3A4
200 N K M L N
SRS 4
Alignment position
CYP 1A1 1A2 2A6 2E1 3A4
L N L A I
SRS 5
Alignment position
CYP 1A1 1A2 2A6 2E1 3A4
I S G I F
SRS 6
Alignment position
CYP 1A1 1A2 2A6 2E1 3A4
M L V L L
# T T T A F
Y Y A P P
L N M F T
D S D T P
T T S S K
L I L L F
L L W H V
N H G E K
# N E I N L
# F F F F L
G V Q H R
E E F L F
V T T L D
V A S S F
210 G S T T L
# G G I F F
* A A G A A
* G G G G G
F F T T Y
D D E E E
* T T T T T
V V V T T
L F V L I
360 P P P P –
F F M S M
T T S N R
# I I L L L
P P A P E
H H R H R
P P P P L
G G V I G
L L G G G
F F F F I
# L L I V A
I I K I S
470 Y Y H H L
S T F H F
# T T F F L
N D K R M
110 Q Q Y R S
T T D – P
N T L N K
290 D D N D I
# I I L A G
F S V F G
M M A G L
G G G D K
S S G G A
M L V I I
# S T V I S
# F F F F I
S S S N A
300 T T S S S
K K T C Q
–: denotes gap in the alignment with CYP2C5. *: denotes conserved residues in SRS4. #: potential substrate contact residue (as encountered in CYP2C5 substrate-bound crystal structure). SRS: substrate recognition site, a term coined by Gotoh (1992) in the analysis of CYP2 family sequences. Residues which have been probed using site-directed mutagenesis are emboldened (the ones in CYP2A6 correspond to those which have been probed for the other orthologues, such as CYP2A4 and CYP2A5). References: Lewis et al. (2003a), Domanksi et al. (2001), Hadjokas et al. (2002) and Domanski and Halpert (2001).
active site of CYP2A6 as outlined above, whereas Fig. 4 indicates that the positioning of coumarin in CYP1A2 is somewhat different from the situation in CYP2A6 due to the changed spacial arrangement of hydrogen bond forming residues. The putative active site of human CYP1A1 is closely related to that of CYP1A2, although some changes in certain residue positions exist, probably explaining the slightly different, but overlapping, substrate preferences of the two CYP1A subfamily enzymes. However, the route of coumarin metabolism is the same for both CYP1A1 and CYP1A2, as summarized in Table 2 which also provides information for CYP2A6, CYP2E1 and CYP3A4-mediated metabolism
of this substrate. As far as CYP3A4 is concerned, coumarin metabolism is directed towards either 3-hydroxylation or 3,4-epoxidation (Born et al., 2002; Zhuo et al., 1999), whereas the position in CYP2E1 mirrors that of the CYP1A-mediated situation where coumarin forms the 3,4-epoxide (see Table 2 and Fig. 1). In a previous study, we showed that the differences in coumarin metabolism in the rat and mouse may be due to a variation in the active site residues of rat CYP2A1 and mouse CYP2A4 and CYP2A5 (Lewis and Lake, 2002). For the two mouse orthologues, CYP2A4 and CYP2A5, it is likely that only a single amino acid residue change (L209F) is sufficient to alter the substrate
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Fig. 3. The putative active site of CYP2A6 showing an orientation of coumarin for 7-hydroxylation. Key amino acid residues are labelled according to their alignment position (see Table 1) with respect to the CYP2C5 crystallographic template. Hydrogen bonds are shown as dashed lines between coumarin and the protein.
selectivity of CYP2A4 from towards testosterone (as a 15a-hydroxylase) to that of coumarin (as a 7-hydroxylase) as encountered in CYP2A5 (Lindberg and Negishi, 1989). Consequently, it is interesting to note that the corresponding residue position in human CYP2A6 to Phe-209 in the mouse orthologue CYP2A5 is also a phenylalanine, and this is shown in Table 1 at alignment position 205 with respect to the CYP2C5 structural template. In fact, phenylalanine is present at this position for all of the human P450s discussed in this work apart from CYP3A4 where the corresponding residue at this point is leucine. However, if a phenylalanine is required for p–p stacking with the aromatic ring system of coumarin, this is afforded by one such residue at alignment position 102 in the CYP3A4 SRS1, as shown in Table 1. Therefore, it would appear that the presence of an aromatic amino acid in the active site region is probably essential for coumarin metabolism by the P450 concerned, although the actual position of coumarin oxygenation depends on other factors and this is probably caused by the disposition of a key hydrogen bond donor residue in the active site. Of the five human P450 enzymes under consideration, only CYP2A6 metabolizes coumarin at the 7-position and it is interesting to observe that the aspargine residue at position 290 (Table 1) may form a hydrogen bond with the substrate and thus orientate it for hydroxylation at the 7-position.
This becomes an aspartate in the CYP1A1, CYP1A2 and CYP2E1 sequences, and all of these enzymes are known to exhibit coumarin-3,4-epoxidase activity. In the case of CYP3A4, which mediates in 3-hydroxylation of coumarin (Born et al., 2002), there is a nonconservative change to isoleucine at this point (i.e. at the residue corresponding to position 290 in the alignment) and it appears that the carbonyl oxygen of coumarin can enter into hydrogen-bonded interactions with an active site serine at alignment position 113 (see Table 1) which corresponds to Ser-119 in CYP3A4. This residue has been the subject of site-directed mutagenesis experiments in CYP3A4, albeit with reference to other substrates of this enzyme (reviewed in Domanski and Halpert, 2001). However, the evidence suggests that Ser-119 would lie relatively close to the haem group as the hydrogen bond acceptor atoms on CYP3A4 sub˚ ngstroms from the site of metabstrates lie only a few A olism. Therefore, it is likely that coumarin binds via a hydrogen-bonded interaction with Ser-119 in CYP3A4, which serves to orientate the substrate for 3-hydroxylation. Fig. 5 shows how such an orientation may be achieved for coumarin binding to the putative active site of CYP3A4, where there is also the possibility of p–p stacking to a complementary aromatic amino acid residue, which can thus augment the hydrogen bond interaction between the substrate and Ser-119.
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261
Fig. 4. The putative active site of human CYP1A2 showing the substrate oritentated for 3,4-epoxidation. Key amino acid residues are labelled according to their alignment position (see Table 1) with the CYP2C5 crystallographic template. Hydrogen bonds are shown as dashed lines between coumarin and the protein.
Table 2 Metabolism of coumarin by human P450s CYP
Route of metabolism
References
1A1 1A2 2A6 2E1 3A4
3,4-Epoxidation 3,4-Epoxidation 7-Hydroxylation 3,4-Epoxidation 3-Hydroxylation and 3,4-epoxidation
Born et al. (2002) and Zhuo et al. (1999) Born et al. (2002) and Zhuo et al. (1999) Shimada et al. (1996), Pelkonen et al. (2000), Zhuo et al. (1999) and Lake (1999) Born et al. (2002) and Zhuo et al. (1999) Born et al. (2002) and Zhuo et al. (1999)
5
4 3
6
Coumarin structure showing sites of metabolism (arrowed) mediated by human P450s listed above. 2
7 8
O 1
O
Calculations based on substrate lipophilicity (using the log P value, where P is the octanol/water partition coefficient) and typical average energies for hydrogenbonded and p–p stacking interactions, generally provide reasonably satisfactory estimates for the overall enzyme–substrate binding energy, which agree well with experimentally determined values (Lewis, 2003). Table 3 provides a summary of these calculations based on an average value of 2 kcal mol1 for a typical hydrogen bond and 0.9 kcal mol1 for an aromatic p–p
stacking interaction between two six-membered aromatic rings. In the case of coumarin, there is no contribution from loss in bond rotational freedom due to the rigid planar structure of the molecule. Therefore, any differences in enzyme–substrate binding energy should be associated with the number of hydrogen-bonded and p–p stacking interactions, as the desolvation component will also likely be the same in each case, if one assumes that the hydrophobicity of the active sites are similar. Consequently, it is feasible to make reasonable
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Fig. 5. The putative site of CYP3A4 with coumarin positioned for 3-hydroxylation. Key amino acid residues are labelled according to their alignment position (see Table 1) with respect to the CYP2C5 crystallographic template. Hydrogen bonds are shown as dashed lines between coumarin and the protein.
Table 3 Estimation of binding energies for coumarin-P450 interactions CYP
DGdesol
DGhb
DGp–p
DGcalc bind
AutoDock
Km (lM)
DGexpt bind
1A1 1A2 2A6 2E1 3A4
1.9717 1.9717 1.9717 1.9717 1.9717
2.0 4.0 4.0 2.0 4.0
1.8 0.9 1.8 0.9 0
5.7717 6.8717 7.7717 6.8717 5.9717
5.28 5.81 5.70 5.61 5.98
51 19 6 12 54
6.0887 6.6969 7.4070 6.9800 6.0535
The agreement between experimental and calculated values for binding affinity (DGbind) shows an R2 of 0.95, for the five points. CYP, cytochrome P450 enzyme; DGcalc bind ¼ DGdesol þ DGhb þ DGp–p ; DGdesol = RT ln P, where P is the octanol/water partition coefficient; DGhb, average hydrogen bond energy of 2 kcal mol1 per hydrogen bond; DGp–p, average p–p stacking interaction energy of 0.9 kcal mol1 per p–p stack; DGexpt bind ¼ RT ln K m , where Km is the Michaelis constant (lM) taken from the literature (Zhuo et al., 1999); R, gas constant (1.9872 cal mol1 K1); T, absolute temperature (taken as 310 K); AutoDock, calculated binding energy (kcal mol1) between the coumarin substrate and each CYP using the AutoDock software (Morris et al., 1998).
estimates of overall binding affinity based on the likely contributions from the major contributing factors of desolvation, hydrogen bonding and p–p stacking according to the relationship: DGbind ¼ DGdesolv þ DGhb þ DGp–p where these terms have been described previously (Lewis, 2003) with the binding free energy (DGbind) being obtained from the Km value (Bauer et al., 2001). The results shown in Table 3, albeit based on a limited dataset, indicate that there is a very good agreement (R2 = 0.95) between calculated and experimental binding energies based on the above expression. This finding indicates that the overall method of estimating binding energies in this way is likely to be fairly sound, and may be employed for other P450 substrates in predicting their Km values provided that the likely interactions
within the enzyme active sites are either known, or can be inferred from molecular modelling investigations. The calculated binding energies obtained via AutoDock are listed in Table 3 and, apart from that of CYP3A4, are all somewhat lower than the experimentally determined values. It is thought that a possible explanation for this may lie in certain key interactions not being fully taken into account, particularly p–p stacking between nearby aromatic ring systems. The discrepancies for CYP1A1, CYP1A1 and CYP2E1 are approximately 1 kcal mol1, whereas that of CYP2A6 is nearer 2 kcal mol1 which suggests that one of the hydrogen bond interactions may not have been identified by the automated docking program. Taking these considerations into account, the agreement between AutoDock and the experimental data is quite satisfactory, and of a similar magnitude to that estimated
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from first principles via the additive method described above.
4. Conclusions The metabolism of coumarin by several human P450s can be rationalized in terms of the likely interactions between the substrate and enzyme active site in each case. In addition, it is possible to derive estimated values for the binding interaction energy which agree well with experimental findings. Automated docking of coumarin using the AutoDock software also produced satisfactory results although a discrepancy of about 1 kcal mol1 in the calculated binding energy was observed in most cases. It is anticipated, therefore, that a similar approach may be feasible for other P450–substrate interactions, and that there could be wider applicability of these techniques to other areas of ligand–receptor or enzyme– substrate binding interactions.
Acknowledgements The financial support of GlaxoSmithKline Research and Development Limited, Merck, Sharp & Dohme Limited, British Technology Group Limited and the University of Surrey, is gratefully acknowledged by one of us (DFVL). Yuko Ito would like to thank the Japanese government for the award of a visiting scientist scholarship as part of a PhD programme.
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