Carcinogenesis vol.17 no.6 pp. 1357-1360, 1996

The metabolism of tamoxifen by human cytochromes P450 isrationalized by molecular modelling of the enzyme-substrateinteractions: potential importance to its proposedanti-carcinogenic/carcinogenic actions

Helen Wiseman and David F.V.Lewis1

Department of Nutrition and Dietetics, School of Life, Basic Medical andHealth Sciences, King's College London, Campden Hill Road, London W87AH and 'Molecular Toxicology Group. School of Biological Sciences,University of Surrey. Guildford, Surrey GU2 5XH, UK

'To whom correspondence should be addressed

A molecular modelling study on the human cytochromeP450-mediated metabolism of tamoxifen is reported. Usingthree-dimensional models of human P450s constructedfrom the bacterial crystal structure template, P450bm3

(CYP102), the likely mode of substrate binding is demon-strated, which is consistent with the known positions ofmetabolism in tamoxifen. In particular, the CYP102-derived structures of CYP3A4, CYP2D6 and CYP2C9 areable to rationalize the routes of tamoxifen metabolismreported in human subjects. The implications for potentialtoxicity of tamoxifen in man is discussed in the light ofthese findings.

Introduction

There is current interest in the use of the anti-oestrogeniccompound tamoxifen for the treatment of post-menopausalbreast cancer and the prevention of breast cancer, osteoporosisand heart disease (1). Although toxic effects have beendemonstrated in rodent species, it is thought that these maynot have relevance to human subjects, due to significantspecies differences in metabolism. Most phase I metabolismis mediated by one or more of the cytochromes P450 (CYP*)and the relevant human P450 isoforms involved in the metabol-ism of tamoxifen have recently been reported (2), indicatingthat the major pathways of tamoxifen metabolism are mediatedvia CYP3A and CYP2C isoforms, which together constitute~50% of the human hepatic P450 complement; the averagepercentages of various P450s in human hepatic samples (3)are shown in Table I.

As molecular models of the major human P450s haverecently been published (4-6), based on the new bacterialP450 crystal structure, P450bm3 (CYP102), it is of interest toinvestigate whether the tamoxifen molecule is able to fit therelevant putative enzyme active sites in such a way as toexplain the known experimental findings on P450-mediatedmetabolism. It is important to develop models which mayassist in the prediction of xenobiotic metabolism (7) andthe structural characteristics of specific P450 substrates andinhibitors can be useful guidelines in such investigations (8).In particular, the involvement of enzymes from the CYP2Cand CYP2D subfamilies, which exhibit genetic polymorphismin man (9), may represent significant limitations on the clinicaluse of novel pharmaceutical products in sections of certainethnogeographical groups, whereas other P450 families, especi-

•Abbreviations: CYP, cytochrome P450; PM, 'poor metabolizer'; EM,'extensive metabolizer'.

© Oxford University Press

ally CYP1 (10), have been shown to play a major role inthe metabolic activation of procarcinogens and mutagens,including chemicals to which man is likely to be exposed.

The metabolism of tamoxifen has been studied in smallrodents (11) and in human liver (2,12-15). Although knownto exhibit genotoxicity, mutagenicity and carcinogenicity inanimal and other test systems, the possible link betweentamoxifen therapy and human cancers is controversial, despitean apparent correlation between tamoxifen usage and endomet-rial cancer (16). There is clear structural similarity betweentamoxifen and the potent carcinogen diethylstilbestrol, but itis likely that the relatively high clearance of tamoxifen,resulting from addition of the /V-dimethylaminoethoxy sidechain, will diminish the potential toxic risk to man, especiallyas the important Af-dimethylation step is catalysed by CYP3A4,which is the major component (see Table I) of the humanhepatic P450 complement (3). Enzymes of the CYP2C subfam-ily also constitute a relatively high percentage of human P450in the liver and it would appear that of these, 2C9 mediates4-hydroxylation of tamoxifen (2), although 2D6 may also beinvolved in this pathway, despite conflicting evidence (2,15,17).The likely role of P450 enzymes from the CYP2C and CYP2Dsubfamilies in phase I metabolism of tamoxifen could haveimportant implications for prescribing this anti-estrogen topatients who exhibit 'poor metabolizer' (PM) status withrespect to 2D6 or 2C19 genetic polymorphisms. Furthermore,production of the 4-hydroxy metabolite represents the route tothe active anti-estrogenic agent and it has been shown that4-hydroxy tamoxifen is able to bind at an interaction site closeto the surface of the human oestrogen receptor (18). Apparently,the 'extensive metabolizer' (EM) phenotype for CYP2D6-mediated debrisoquine metabolism has been associated withlung cancer incidence in several human ethnogeographicalpopulations (19) and it is possible that CYP2D6-catalysedactivation of the nicotine pyrolysate NNK could represent alink between tobacco smoking, lung cancer and the 2D6EM phenotype (20). However, in the case of tamoxifen the4-hydroxylation step, which is thought to be catalysed by bothCYP2C9 and, to a lesser extent, CYP2D6, can be regarded asa potentially detoxifying process and it is not clear whetherthe metabolic activation of tamoxifen (i.e. epoxidation of thealkene moiety) is relevant to man. Recent evidence suggeststhat the activation of tamoxifen in Homo sapiens may involve4-hydroxylation and/or a-hydroxylation of the ethyl group, asthese can both give rise to DNA adducts (21), whereastransacts isomerization of the 4-hydroxy metabolite couldrepresent an important step in the pathway to activation (17).

Materials and methods

The construction of three-dimensional models of mammalian cytochromesP450 based on the CYP102 template has been described previously (4,5). Thehuman cytochromes P450 reported in this work were produced from theCYP102 crystal structure (Brookhaven Protein Databank, reference code 2hpd)using a novel amino acid sequence alignment, which included considerationof site-directed mutagenesis and other experimental evidence (4). It has been

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H.Wiseman and D.F.V.Lewis

Table I. Average percentage of P450s in human liver

CYP subfamily Percentage

3A2C1A2E2A2D2B

302013742

Fig. 1. A possible mode of interaction between tamoxifen and the putativeactive site region of CYP3A4. Specific amino acid residues likely to beinvolved in substrate binding, orientation and metabolism (Thr268) arelabelled. The substrate is orientated for N-demethylation via contacts withAsn74 and Phe72.

Fig. 2. A possible mode of interaction between tamoxifen and the putativeactive site of CYP2C9. Amino acid residues likely to be associated withsubstrate binding and oxygenation (Thr268 ) are labelled. In this positionthe substrate is orientated for 4-hydroxylation via contacts, primarily, withSer331 and Leu332.

shown that a model of the human isoform CYP2A6 is able to rationalizecoumarin metabolism in man, in addition to explaining substrate specificitiesin the rat and mouse CYP2A orthologues (5). The putative active sites ofthese and other mammalian P450s modelled via the same procedure areconsistent with the information obtained from protein engineering studiesregarding the effect of certain amino acid residues on P450 substrate specificityand regioselectivity (4,5).

Alignment with the CYP 102 sequence exhibits higher overall homologywith mammalian microsomal P450s than those obtained using the CYP 101template (22), especially when the results from site-directed mutagenesisexperiments, molecular probe analysis and other experimental data are takeninto account. For example, the sequence identity between CYP102 andCYP3A4 is 27.1%, whereas for CYP10I and CYP3A4 this is significantly

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Fig. 3. A possible mode of interaction between tamoxifen and the putativeactive site of CYP2D6. Specific amino acid residues which may beassociated with substrate binding and metabolism (Thr268) are labelled.Tamoxifen is orientated for oxygenation in the 4-position via contacts withGlul84, Glu78, Phe87 and Val328.

lower at 16.7%. CYP102 can be regarded as the preferred template formicrosomal P45Os (23) due to the fact that there is a commonality in redoxpartner, as all utilize an NADPH-dependent oxidoreductase and, therefore,CYP 102 has been classified as belonging to the eukaryotic class of P450s(24). Moreover, in contrast to either CYP101 or CYP108, there are relativelyfew residue insertions required for the generation of microsomal P450structures via alignment with CYP102, which considerably facilitates themodelling process. Following the necessary residue changes, as required bythe alignment (4), geometry optimzation of the raw structures gave minimumenergy values of —1000 kcal/mol for the human P450 models investigated(4,5). The minimized tamoxifen structure was then docked interactively intothe putative active sites of the relevant P450s thought to be involved in itsmetabolism, using the Sybyl molecular modelling software system (TriposAssociates, St Louis, MO).

Results and discussion

Figure 1 shows the proposed mode of interaction betweentamoxifen and the putative active site region of CYP3A4. Itappears that hydrogen bonding between Asn74 and the oxygenatom of the ethanolamino side chain, together with n-Kstacking between the phenyl ring of Phe72 and one of thearomatic rings will orientate tamoxifen for A/-demethylation.One of the iV-methyl groups is relatively close to the hemeiron and, although the distances involved are slightly differentfrom those encountered with typical CYP3A4 substrates (25),this orientation certainly explains the known CYP3A4-medi-ated metabolism of tamoxifen in terms of key interactionpoints found with many CYP3A4 substrates. There is also aclose hydrophobic contact between one of the putative activesite aliphatic amino acid residues and the ethyl group oftamoxifen, which gives further support to this model of theenzyme-substrate interaction. However, one of the aromaticrings is rather close to Thr73, which would not be regardedas favourable, although it is possible that the substrate maymove closer to the heme following this initial binding inter-action and this could then involve JI-JI stacking betweenthis aromatic ring and Phe72, a significantly more favoured

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Tamoxifen metabolism by human cytochromes P4S0

Table II. Interatomic distances between key atoms in tamoxifen and within the putative active sites of human P450s

P450 model Interatomic distance8 Description6

CYP3A4

CYP2C9

CYP2D6

O....N-Me5.673 AN-Me...Fe-O3.767 AO....4-OH9.471 A4-OH...Fe-O2.894 AN+....4-OH12.234 A4-OH...Fe-O2.238 A

Distance between the side chain oxygen atom, which forms a hydrogen bond with Asn74, and the yV-methyl group

Distance between the N-methyl group (site of 3A4 metabolism) and the iron-oxygen of the heme moiety

Distance between the side chain oxygen atom, which forms a hydrogen bond with Ser331, and the 4-hydroxyl group

Distance between the 4-hydroxyl (site of 2C9 metabolism) and the iron-oxygen of the heme moiety

Distance between the protonated nitrogen atom on the side chain, which ion pairs with Glul84, and the 4-hydroxylgroupDistance between the 4-hydroxyl (site of 2D6 metabolism?) and the iron-oxygen of the heme moiety

aThe distances between key interacting atoms of the tamoxifen substrates and sites of metabolism are within the ranges encountered with other substrates ofthese enzymes, with the exception of 2D6, although a known 2D6 substrate, tropisetron, shows a similar distance value.'The interactions between the tamoxifen molecule and each enzyme active site amino acid residues and heme groups are shown in Figures 1-3.

interaction in energy terms. Furthermore, such a movement ofthe substrate would bring the /V-methyl group nearer to theheme iron, thus facilitating oxygenation and N-demethylation.As the active site of CYP3A4 is relatively large, it is possiblethat there may be alternative docking interactions for tamoxifen,especially as one side of the heme pocket appears to containa number of aromatic amino acid residues that would beexpected to be capable of binding tamoxifen via its phenylring moieties. However, the orientation shown in Figure 1 isconsistent with the typical structural requirements for CYP3A4substrates in terms of interactions with Asn74, Phe72 and oneof the active site hydrophobic residues, together with thedistances between interacting groups and the site of meta-bolism.

The possibility of 4-hydroxylation of tamoxifen being medi-ated by CYP2C9 was postulated on the grounds of structuralmodelling within the enzyme active site, prior to publicationof the relevant human P450 studies of White and co-workers(2). The reason for this speculation was the distance betweenthe hydrogen bond acceptor atom (i.e. the oxygen of theethanolamine side chain) and the site of metabolism at the4-position. Moreover, modelling of tamoxifen within the putat-ive active site of CYP2C9 indicates that p-hydroxylation islikely on the grounds of orientation of the substrate withrespect to Ser331, a known site of interaction for 2C-specificcompounds (4). Furthermore, p-hydroxylation of the aromaticrings is a common feature of CYP2C-mediated metabolism, e.g.phenytoin and mephenytoin. Confirmation of this hypothesis bythe experimental work of White et al. (2) clearly buildsconfidence in the use of enzyme modelling as a means ofpredicting the route of P450-catalysed metabolism. The likelyinteraction between tamoxifen and the CYP2C9 model isshown in Figure 2, where it can be seen that hydrogen bondingfrom Ser331 to the oxygen of the side chain orientates thesubstrate for hydroxylation at the 4-position on the relevantphenyl ring. There are other interactions between the substrateand putative active site residues which augment this mode ofbinding, such as hydrophobic contacts between the N-dimethylgroup on the substrate and the side chain of Leu332, andbetween the ethyl group on tamoxifen and Val87, togetherwith n-Tt stacking between one of the aromatic rings on thesubstrate and Phe88. It has been demonstrated via site-directedmutagenesis experiments that Ser331 is an important determin-

ant of substrate regioselectivity in the CYP2C subfamily(26,27).

In addition to the involvement of. CYP2C9 in 4-hydroxylation of tamoxifen, there is also some evidence forthe CYP2D6 isozyme catalysing the same reaction (2). Thedistance between the protonatable nitrogen atom in tamoxifenand the para position on the adjacent aromatic ring is > 12 A,which is 5 A greater than the upper limit for CYP2D6 substratespecificity according to the currently accepted template model(28). Consequently, there has to be some refinement of thisCYP2D6 model to account for these findings. In the structureof CYP2D6 generated from CYP102 (4,29,30) it is possiblethat tamoxifen can occupy the putative active site with anorientation that presents the para position on the appropriatearomatic ring for oxygenation, as shown in Figure 3. Inorder to rationalize the known CYP2D6-mediated route ofmetabolism the tamoxifen molecule has to be orientateddifferently from that of other typical substrates of this isoform.However, the CYP2D6 substrate tropisetron does exhibit someanalogy with tamoxifen in terms of the distance of theprotonated nitrogen from the site of metabolism (30). Thisinvolves ion pairing with Glu 184, instead of the usual Asp260(Asp301 in CYP2D6), together with somewhat different inter-actions with other active site residues. Although there is todate no evidence from site-directed mutagenesis for the possiblerole of Glu 184 in CYP2D6 substrate binding, this position isclose to that shown by Lindberg and Negishi (31) to be criticalfor substrate specificity in the mouse CYP2A orthologues.Molecular modelling of CYP2A subfamily isozymes indicatesthat both positions 181 and 184 are involved in substratebinding interactions and orientation for metabolism in theexperimental observations (5), whereas residue 180 (212 inCYP2D6) corresponds to an allelic variant (Gly-»Glu) in 2D6,which is associated with lowered debrisoquine metabolism.Consequently, there is some evidence that residues on theF helix, situated above the heme binding site, may be associatedwith substrate binding interactions in the CYP2 family and,furthermore, position 182 in the model (4) corresponds toThrl85 in CYP101, which has been shown by site-directedmutagenesis to be important for substrate regiospecificity(32,33). Table II summarizes the relevant interatomic distancesin tamoxifen and with respect to its interaction with theputative active sites of CYP2D6, CYP2C9 and CYP3A4. It

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H.Wiseman and D.F.V.Lewis

is not known which P450 isoform is responsible for a-hydroxylation of the ethyl group, but preliminary modellingstudies (data not shown) indicate that tamoxifen is able to fitthe putative active site of CYP2B6, where interactions withcomplementary amino acid side chains could orientate themolecule for oxygenation at the a-position on the ethyl moiety.However, this is a minor pathway in man and CYP2B6constitutes a very low percentage of human hepatic P450.

Although one might expect that 4-hydroxylation of tamoxi-fen represents a detoxifying pathway, as conjungation reactionswith the phenolic group would be possible, there is recentevidence (21) which suggests that this step is actually apotentially activating route of metabolism. If this is the case,then the CYP2C9- and CYP2D6-mediated pathways couldhave some relevance to a possible carcinogenic risk fromtamoxifen use in human populations, particularly with respectto genetic polymorphism in either of these human P450isoforms. (Although the association between genetic defectsin CYP2D6 and CYP2C19 and lowered metabolism have beenestablished, there is evidence to suggest that allelic variantsof 2C9 may also give rise to PM status). It remains to be seenwhether any linkage exists between cancer incidence associatedwith tamoxifen use and PM or EM phenotypes of CYP2D6or CYP2C19. However, the detoxifying role of CYP3A4, themajor human hepatic isoform, in the metabolism of tamoxifenprobably explains the fact that, in contrast to the situation inexperimental rodents, the potential carcinogenic risk is likelyto be relatively low.

Ackowledgement

The financial support of Glaxo Research and Development Ltd is gratefullyacknowledged by DFVL.

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Received on October 25, 1995; revised on February 12, 1996; accepted onFebruary 27, 1996

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