DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 DMD FastThis articleForward. has not Publishedbeen copyedited on and AugustDMD formatted. #22376# 25, The 2008 final version as doi:10.1124/dmd.108.022376 may differ from this version. The Molecular Basis of CYP2D6 mediated N-dealkylation.
- Balance between metabolic clearance routes and enzyme
inhibition
Britta Bonn, Collen M. Masimirembwa, Yasmin Aristei, and Ismael Zamora
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Discovery DMPK & Bioanalytical Chemisty, AstraZeneca R&D Mölndal, Sweden (B.B., C.M.M.) dmd.aspetjournals.org Department of Chemistry, Medicinal Chemistry, University of Gothenburg, Gothenburg, Sweden.
(B.B.)
African Institute of Biomedical Science & Technology (AiBST), Harare, Zimbabwe. (C.M.M) at ASPET Journals on September 24, 2021
Department of Chemistry, Laboratory for Chemometrics and Chemoinformatics, University of Perugia,
Perugia, Italy. (Y.A.)
Lead Molecular Design, S.L., San Cugat del Vallés, Spain. (I.Z.)
Institution Municipal d´Investigació Medica (IMIM), Universitat Pompeu Fabra, Barcelona, Spain. (I.Z)
Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics. 1 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. RUNNING TITLE PAGE
Molecular Basis of CYP2D6 mediated N-dealkylation
Corresponding author: Britta Bonn. Discovery DMPK & Bioanalytical Chemistry, AstraZeneca
R&D Mölndal, SE-431 81 Mölndal, Sweden. Tel.: +46 31 706 4262; fax: +46 31 776 3787; e-mail:
Number of text pages: 29 Downloaded from
Number of tables: 3
Number of figures: 2 dmd.aspetjournals.org Number of references: 47
Number of words in abstract: 160
Number of words introduction: 788 at ASPET Journals on September 24, 2021 Number of words in discussion: 1479
Abbreviations : CYP – Cytochrome P450, Clint – intrinsic clearance, SOM – Site of Metabolism,
DXM – dextromethorphan, DXO – dextrorphan, MEM – 3-methoxymorphinan, AMMC – 4- aminomethyl-7-methoxycoumarine, GRID-MIFs – GRID derived molecular interaction fields, FLAP – fingerprints for proteins and ligands, MRM – multiple reaction monitoring.
2 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version.
ABSTRACT
N-Dealkylation is a commonly observed metabolic reaction for drugs containing secondary and tertiary amines. On searching the literature, it is obvious that this reaction is far less common among cytochrome P450 2D6 catalyzed reactions compared to other CYPs. The CYP2D6 pharmacophore and characteristic features in the active site cavity suggest a favored substrate orientation that prevents
N-dealkylation from occurring. In this study, the literature was searched for N-dealkylated and Downloaded from non-N-dealkylated CYP2D6 substrates. The hypothesis suggested and confirmed was that
N-dealkylation occurs by this enzyme when the preferred site of metabolism is blocked towards other dmd.aspetjournals.org oxidative metabolic pathways. An interesting observation was also that addition of stable groups at preferred sites of metabolism generally improved the metabolic stability but also resulted in retained or increased inhibition of the enzyme. In addition, the effect of pH on N- and O-dealkylation of at ASPET Journals on September 24, 2021 dextromethorphan was shown to be consistent with the hypothesis that an ionized amino function favored substrate dockings resulting in O-dealkylation.
3 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. Cytochrome P450 2D6 (CYP2D6) is a polymorphic member of the cytochrome P450 superfamily important for the metabolism of a variety of xenobiotics (Guengerich, 2003). In drug discovery research it is of great interest to identify whether a compound is a substrate or not, and considerable effort has been put into the development of models to identify substrates and/or inhibitors of CYP2D6. Several pharmacophore models have been published identifying common features in CYP2D6 substrates and inhibitors (Koymans et al., 1992; de Groot et al., 1997; Lewis et al., 1997; de Groot et al., 1999). The pharmacophore comprises basic nitrogen atom 5-7Å or 10Å from the site of oxidation, a flat hydrophobic region near the site of oxidation and a negative molecular electrostatic potential (MEP) Downloaded from coplanar with this hydrophobic region (see examples in Fig 1). The intramolecular distances of 5 and
7Å were combined by Koymans et al. (Koymans et al., 1992) in a model assuming a carboxylate group dmd.aspetjournals.org as an anchor point in the protein with one of the oxygen atoms interacting with the “5Å substrates” and the other with the “7Å substrates”. Since several substrates with approximately 10Å between the nitrogen atom and the site of oxidation were also known, refined models were constructed including this at ASPET Journals on September 24, 2021 intramolecular distance (Lewis et al., 1997). Even though the early pharmacophore models are rather crude they have been successful in predicting the influence of CYP2D6 in the metabolism of xenobiotics as well as predicting possible sites of metabolism (e.g. 75 % predictability (de Groot et al.,
1999)).
Structural information of CYP2D6 from homology models (de Groot et al., 1996; De Rienzo et al.,
2000) and a crystal structure of ligand-free CYP2D6 (Rowland et al., 2006) revealed two acidic amino acids, Glu216 and Asp301, and two phenylalanine residues, Phe120 and Phe483, in the active site cavity. This was compatible with the previous pharmacophore models identifying the basic nitrogen and a planar hydrophobic area as characteristic features in CYP2D6 substrates. Site directed mutagenesis studies also confirmed the importance of the acidic amino acids for substrate specificity and catalytic activity (Mackman et al., 1996; Guengerich et al., 2003; Paine et al., 2003). Similar experiments also suggest that the hydrophobic and steric characters of the phenylalanine residues influence the
4 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. regiospecific metabolism in a substrate dependent manner (Flanagan et al., 2004; Lussenburg et al.,
2005). Despite the well-characterized CYP2D6 substrate/inhibitor pharmacophores prediction of the regio-specificity of CYP2D6-mediated metabolism is a complex matter with many different interactions to account for. However, in the present study the main focus has been on the electrostatic interactions and the influence of the acidic amino acids.
Considering the common structure of CYP2D6 substrates with a basic nitrogen 5-7Å from the site of oxidation interacting with the acidic amino acid Glu216, it would be unlikely that N-dealkylation Downloaded from reactions occur. The main reason is that the electrostatic interaction between the basic nitrogen in the substrate and the carboxylic acid group of Glu216 on top of the active site cavity would favor an dmd.aspetjournals.org orientation of the substrate with the nitrogen directed away from the heme. Nevertheless N-dealkylation does occur in CYP2D6 but is far less common than in e.g. CYP3A4 (Coutts et al., 1994). In a previous study the influence and importance of the positively charged nitrogen on the orientation of the substrates at ASPET Journals on September 24, 2021 in the active site cavity was explored (Upthagrove and Nelson, 2001). It was shown that addition of substituents that increase the basicity of the compound also increased the affinity for the enzyme.
In the present study, this was investigated by comparing the N-dealkylation of dextromethorphan
(DXM) at different pH-values to see how different ratios of unprotonated/protonated forms of the ligand affect the formation of the N-dealkylated metabolite. Besides the effect of the degree of ionization, which may be affected by the micro-environment in the enzyme active site cavity, another working hypothesis was that N-dealkylation in CYP2D6 occurs e.g. when aromatic hydroxylation or
O-demethylation reactions are not possible. That is, when the preferred site of metabolism is prevented from participating in an oxidative reaction, the substrate will be N-dealkylated to a more significant extent. To study this hypothesis the literature was searched in order to select CYP2D6 substrates that were reported to be N-dealkylated and substrates that were not. The preferred site of metabolism for these compounds was then identified by aligning the compounds to CYP2D6 probe substrates, using the
5 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. program FLAP (Fingerprints for Ligands And Proteins) (Baroni et al., 2007). The site of metabolism
(SOM) prediction program MetaSite (Zamora et al., 2003) was also used and compared with the results from FLAP. In addition, some compounds were selected for experimental determinations of the major metabolites formed, the rate of formation of N-dealkylated product, as well as the inhibition potential in recombinant CYP2D6. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 24, 2021
6 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. MATERIALS AND METHODS
Materials
The source of recombinant enzymes was bacterial membranes containing human cytochrome P450
CYP2D6 co-expressed with NADPH-cytochrome P450 reductase supplied from Cypex Ltd. Scotland,
UK. The test compounds were purchased from Sigma Chemicals Co. St. Louis, USA
(dextromethorphan hydrobromide, (S,S)-sertraline hydrochloride, maprotiline hydrochloride, citalopram hydrobromide, lidocaine hydrochloride, alprenolol hydrochloride, clomipramine hydrochloride, imipramine hydrochloride, nisoxetine hydrochloride, (R)-tomoxetine hydrochloride, cis-tramado Downloaded from hydrochloride, cis-O-desmethyltramadol), ICN Biomedicals Inc. Ohio, USA (thioridazine hydrochloride, dextrorphan tartrate), Toronto Research Chemicals, North York, Canada (fluoxetine dmd.aspetjournals.org hydrochloride), Bionet Research, Cornwall, UK (Bionet-BB 6N-710, Bionet-BB 4N-726, Bionet-BB
6N-708), Aldrich Chemical Company, Milwaukee (N,N-dimethyl-5-methoxytryptamine) and Fluka
Chemie AG, Buchs, Switzerland (N,N-dimethyltryptamine). NADPH was purchased from Sigma at ASPET Journals on September 24, 2021 Chemicals Co, St. Louis and 4-aminomethyl-7-methoxycoumarine (AMMC) from BD Gentest inc.
Woburn, MA, USA. All other chemicals were of analytical grade and highest quality available.
Computer hardware and software
The calculations were performed in a Linux environment on a HP workstation xw6200 and a
Pentium IV. The softwares used were GRID version 22.2.2, MetaSite version 2.7.5, MOKA version 1.0,
Mizer version 1.0 and a command line version of FLAP beta version from Molecular Discovery
(http://www.moldiscovery.com) and ISIS draw version 2.4 (MDL® Information Systems, Inc. http://www.mdl.com/).
Data set
From a literature search on CYP2D6 substrates compounds for which the published data clearly showed the metabolites formed via CYP2D6 catalysis were chosen. Since the basic nitrogen was of
7 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. interest as a pharmacophoric feature in this study compounds with a predicted pKa below 8 were filtered away, ending up with a set of CYP2D6 substrates being protonated at physiological pH. In order to get a comparable environment around the basic nitrogen and thereby simplify the analysis an additional filtering of the data set was done to only include methyl- and ethylamines. The final data set of 43 compounds was divided into two groups: 1) substrates with N-dealkylation as a major metabolic pathway in CYP2D6 and 2) substrates undergoing other metabolic reactions (e.g. aromatic hydroxylation, O-dealkylation) as primary routes in CYP2D6.
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From this data set 20 compounds were selected for experimental determination of metabolism and inhibition potential using recombinant CYP2D6 enzyme. As mentioned above the focus of this study dmd.aspetjournals.org was on major metabolites formed by CYP2D6. For data on compounds found in the literature the major metabolite formed by CYP2D6 was rationalized from the available references. For the compounds classified as not being N-dealkylated by this enzyme some were still reported to be N-dealkylated but at ASPET Journals on September 24, 2021 only to minor extent and were therefore not considered to be of importance in this study. For the experimental data set, metabolites with an abundance <20 % of all metabolites formed were considered minor and >20 % were considered to be major metabolites. This estimation was based on the LC-MS response areas. For these estimations it was assumed that the ionization efficiencies for the metabolites were similar to that of the parent drug, which is not always the case. However, since no authentic standards were available for the metabolites the LC-MS response areas were used for a rough quantification and distribution of metabolites.
For the computational part all structures were drawn in ISIS and exported as sdf-files. Since the basic
CYP2D6 substrates would be protonated at physiological pH and the fact that the positive charge is suggested to be of importance for affinity of the substrate to the enzyme (Upthagrove and Nelson, 2001) the compounds were protonated prior to further analysis. This was done with the program MOKA
(Milletti et al., 2007), which protonates the molecules at a selected pH based on predicted pKa-values.
8 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. The 2D-3D conversion was made with the program Mizer, generating one conformer minimized with the MM3 force field.
pH-dependent N-dealkylation of DXM
DXM was incubated with recombinant CYP2D6 and buffers at different pH-values. Since it is difficult to establish the pH of the microenvironment in the active site cavity it was assumed that this pH resemble that of the surrounding buffer, which is also used to determine the protonation of DXM. The buffers used were potassium phosphate at pH 6.0, 6.6 and 7.4 and Tris-HCl at pH 8.0 and 8.8. Stock Downloaded from solutions of DXM were prepared in 50% acetonitrile at 50 times the concentration in the incubation.
The incubations were performed in triplicates at two different concentrations based on the Km-values for dmd.aspetjournals.org formation of the two metabolites dextrorphan (DXO) and 3-methoxymorphinan (MEM) in human liver microsomes. The compound (3 µM and 200 µM) was mixed with recombinant CYP2D6 (50 pmol/ml) and buffer (0.1 M). After 10 min preincubation, the reaction was initiated by the addition of NADPH at ASPET Journals on September 24, 2021 (1 mM) and incubated for 7, 15, 20 and 30 min. Zero time-point samples were taken just before the addition of NADPH. All incubations were performed in glass vials in a water bath at 37 °C. The reaction was quenched with two parts of ice-cooled acetonitrile with 0.8% formic acid, the samples were centrifuged at 2737g for 20 min at 4 °C and the supernatant diluted 1:2 with water prior to analysis. For analysis of DXM at high concentration, the samples were diluted 1:400 in water. Standard curves were prepared in the incubation matrix for DXM, DXO and MEM (30; 10; 3.3; 0.37; 0.12; 0.04;
0.014 and 0.0046 µM). The standard samples were precipitated with acetonitrile and diluted in water in the same way as the incubation samples.
Clint and metabolite identification in recombinant CYPs
Since literature data can be difficult to evaluate and compare due to different experimental conditions such as enzyme source, substrate concentrations etc., compounds were selected for experimental determination of metabolism in CYP2D6. Twenty compounds were incubated with recombinant
9 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. CYP2D6 in order to determine major metabolites, formation of N-dealkylated metabolites and disappearance of parent compound (Clint). For determination of Clint-values all compounds were incubated at 1 µM and additional incubations were done at 10 µM for metabolite identification. For a proper comparison of Clint the concentration used should be based at the Km-value of each substrate.
However, since the Km –value for several of the test compounds were unknown the concentration at
1 µM was chosen for all the Clint determinations. This concentration is commonly used in early screening on metabolic stability on drug candidates and is assumed to give a comparable measurement of Clint over a set of compounds for which the Km is unknown. The compounds were mixed with Downloaded from recombinant CYP2D6 (50 pmol/ml) and potassium phosphate buffer (0.1 M, pH 7.4). After 10 min preincubation the reaction was initiated by addition of NADPH (1 mM) and incubated for 7, 15, 20 and dmd.aspetjournals.org 30 min. Zero samples were taken just before addition of NADPH. All incubations were performed in microtiter plates at 37 °C. The reaction was quenched with two parts ice-cooled acetonitrile with 0.8% formic acid, the samples were centrifuged at 2737g for 20 min at 4 °C and the supernatant diluted 1:2 at ASPET Journals on September 24, 2021 with water prior to analysis.
For the interpretation of the metabolic stability data the following classification was used: compounds with Clint-values below 0.7 µl/min/pmol P450 were considered to be low clearance compounds in
CYP2D6 while Clint -values above 3 µl/min/pmol P450 were classified as high clearance. These cut off values were estimated from values used in metabolic stability screens in human liver microsomes (low
Clint <10 µL/min/mg and high Clint >45 µl/min/mg, corresponding to hepatic clearance predictions of
<30% of liver blood flow and >70% of liver blood flow in vivo). In product sheets on pooled human liver microsomes from BD Gentest it was declared that the amount CYP2D6 was 15 pmol/ml HLM (BD
Gentest inc. Woburn, MA, USA). This value was used to get the corresponding values for Clint in recombinant CYP2D6.
IC50-determinations
10 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. For determination of inhibition potential of CYP2D6 full IC50-curves were obtained for each compound using a fluorescence end-point assay (Crespi et al., 1997). A serial dilution of the test compounds was done in 50% acetonitrile (50, 16.667, 5.556, 1.852, 0.617, 0.206, 0.069, and 0.023 µM) and added to a master mix containing enzyme (20 pmol/ml), potassium phosphate buffer (0.1 M, pH 7.4) and the fluorescent probe substrate AMMC (15 µM). Also included were blank samples without test compound and substrate and incubations only containing the substrate, representing 100% activity.
The incubation mixtures were preincubated for 10 min and initiated by addition of NADPH (0.4 mM), except for blank samples. All incubations were performed in microtiter plates at 37 °C. After 30 min of Downloaded from incubation the reactions were quenched with 80% acetonitrile and 20% Trisbase. The fluorescence was measured at 390/460 nm using a SpectraMax Gemini XS (Molecular Devices Corporation, Sunnyvale, dmd.aspetjournals.org CA, USA). The IC50-values were determined by nonlinear least-squares regression analysis using
XLfit4 version 4.1.1 (idbs, Guildford, UK).
at ASPET Journals on September 24, 2021 In this assay an IC50-value below 10 µM was considered to be an indication of potential CYP inhibition.
Bioanalytical equipment and analysis
Analysis and quantification of DXM and the two metabolites DXO (O-demethylation) and MEM (N- demethylation) from the incubation of DXM at different pH were done using LC-MS. The HPLC system was an Agilent 1100 Series (Hewlett Packard GmbH, Walbronn, Germany) coupled to a HTC
PAL auto sampler (CTC Analytics AG, Zwingen, Germany). The analytical column used for chromatographic separation was a HyPurity C18 column (50 × 2.2 mm, 5µm) and the mobile phases consisted of water and acetonitrile acidified with 0.1% formic acid. The gradient used was 5-90% acetonitrile in 4.5 min at a flow rate of 0.75 mL/min. The HPLC system was connected to a Sciex API
4000 quadrupole mass spectrometer with ESI interface (Applied Biosystems Ontario, Canada). The analytes were recorded with multiple reaction monitoring (MRM) in positive mode with Q1/Q3 masses
11 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. 272/171 for DXM, 258/171 for DXO and 258/157 for MEM. The Analyst 1.4.1 software (Applied
Biosystems) was used for analysis and storage of data. All samples were analyzed regarding disappearance of DXM and formation of the two metabolites DXO and MEM. Authentic standards were used for quantification.
The analysis of the experimental data set of CYP2D6 substrates regarding disappearance of parent compound and formation of metabolites was done on a Waters Micromass LCT premiere TOF mass spectrometer with an ESI interface (Wythenshawe, UK). For chromatographic separation this was Downloaded from connected to a Waters Aqcuity UPLC system (Milford, MA, US). The analytical column used was an
Acquity UPLC C18 column (2.1 x 50 mm, 1.7 µm). The mobile phases consisted of water and dmd.aspetjournals.org acetonitrile acidified with 0.2% formic acid and the gradient used was 5-90% acetonitrile in 5 min with a flow rate of 0.75 mL/min. A generic MS method was used for all compounds with a capillary voltage of 3000 V and the cone voltage set to 35 V. Detailed metabolite identification was performed with at ASPET Journals on September 24, 2021 LC-MS/MS using a Waters Q-TOF Premiere (Wythenshawe, UK) instrument. The MS/MS-analysis was done on the metabolites identified in the LCT premiere TOF spectrometer. The collision energy was ramped from 15 to 40 eV and the cone voltage was set to 30 V. For chromatographic separation the same system and settings were used as above. The MassLynx v. 1.4 (Waters, Milford, MA, USA) software was used for analysis and storage of data.
Computational analysis
FLAP
For identification of the site of metabolism (SOM) based on the CYP2D6 pharmacophore all compounds were aligned to DXM (Fig 1b), a rigid molecule representing a 7Å CYP2D6 substrate, and to the larger substrate tropisterone (Fig 1c) to cover the 10Å substrates. The alignments were done using the program FLAP (Baroni et al., 2007). This program uses GRID derived molecular interaction fields
(GRID-MIFs) (Goodford, 1985) to create 4-point pharmacophores for the substrates. For these
12 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. calculations a hydrophobic probe (DRY), a hydrogen bond donor (N1) and a hydrogen bond acceptor
(O) probe were used to describe the substrates. For each ligand, a maximum of 25 conformers were generated using random search by rotating a maximum of 10 rotable bonds. The calculated 4-point pharmacophores for each conformer was used to determine pair wise similarity with the 4-point pharmacophore for DXM or tropisterone resulting in a best conformer overlap for each compound.
After confirming that the basic nitrogen atoms in DXM/tropisterone and the test compound were aligned, the parts of the test molecule that were overlapping with the known SOM in the templates were identified to be the preferred SOM. In the cases where solutions were obtained with both DXM and Downloaded from tropisterone as templates, the solution with the best alignment of the molecules and the shortest distance between the basic nitrogen atoms was selected. dmd.aspetjournals.org
For the interpretation of FLAP results the following rules were used: 1) the FLAP predicted SOM is within two atoms from the group overlapping with the SOM in the template molecule, 2) N-dealkylation at ASPET Journals on September 24, 2021 is suggested to be the metabolic reaction if the SOM in the template molecule is overlapping with, or within one atom away from a halogen atom, 3) N-dealkylation is suggested to be the metabolic reaction if the SOM in the template molecule is overlapping with other atoms unable to undergo oxidative metabolism e.g. exact overlap with an hydroxyl group.
MetaSite
The identification of SOM with the FLAP methodology was compared with the results from the previously known SOM prediction software MetaSite (Zamora et al., 2003). This methodology considers structural complementarity between the active site of the enzyme and the ligand as well as reactivity of the ligand. To compare the enzyme and ligand, two sets of descriptors are calculated. For the enzyme these are based on flexible GRID-MIFs. In order to generate the descriptor set of the ligand each atom of the molecule is classified as a GRID probe and the distances between them are calculated.
The resulting fingerprint of the ligand is compared with the description of the enzyme active site and the
13 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. most optimal orientations are obtained. Based on this, all atoms in the molecule are ranked according to their accessibility to the heme. In addition, a reactivity factor based on fragment recognition is added. In summary, the site of metabolism is described by a probability index, which is the product of the similarity between ligand and protein (i.e. accessibility of one atom to the heme) and the reactivity.
For this analysis, the compounds were loaded into MetaSite as single conformers and protonated at the nitrogen atom according to the predicted pKa of the compounds. When analyzing the results the top two averaged ranked solutions with reactivity factor enabled were considered. Downloaded from
dmd.aspetjournals.org at ASPET Journals on September 24, 2021
14 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. RESULTS
pH-dependent N-dealkylation of DXM
One aim of this study was to examine how formation of the two major metabolites of DXM, DXO and
MEM, was influenced by the pH of the incubation mixture. The pKa of DXM is 9.12 (Gilligan and Li
Wan Po, 1991), and for the pH range used in the experiments (pH 6.0 to pH 8.8) this would theoretically correspond to a variation of the amount of ionized form of the molecule from 99.9 to 66.6%.The amount of metabolites formed after incubation of DXM was plotted against the time of incubation and the rate of formation was caclulated from the slope of the curve. This analysis was done for the different Downloaded from incubation conditions to see how the rate of formation varied with pH (Fig 2a and Fig 2b). Based on the
Km-values for the two reactions the incubations were done at DXM concentrations of 200 µM and dmd.aspetjournals.org 3 µM, respectively. In the incubations at 3 µM only minute amounts of MEM (formed via N- demethylation) were formed, therefore the results obtained from this concentration were not included in further analysis. The O-demethylation of DXM to DXO had a maximal rate at pH 7.4 and at higher pH- at ASPET Journals on September 24, 2021 values the reaction rate decreased (Fig 2a). The N-demethylation (formation of MEM) also peaked at pH
7.4 but the rate did not decrease as much at increasing values (Fig 2b). Since the change in pH probably affected the protein and its catalytical activity the comparison done is difficult to evaluate. For a more proper evaluation of the influence of pH on the N- and O-demethylation of DXM, the ratio between the rates of formation of the two metabolites was calculated (DXO/MEM). To use this ratio would diminish the effect on the protein assuming that the decreased catalytic activity at high and low pH was the same for both reactions. The O-demethylation/N-demethylation ratio clearly decreased at higher pH (Fig 2c) indicating that more N-demethylation occurred when the pH increased and DXM was present to a higher extent as the free base.
FLAP alignment and MetaSite analysis of literature compounds
15 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. Table 1 summarizes the reported SOM, the preferred SOM based on the FLAP alignment and the predicted SOM using MetaSite for a literature data set of 23 compounds. Also included in the table are the predicted pKa-values from MOKA. The majority of the compounds gave the best alignments with
DXM (7Å substrate) but eletriptan, brofaromine, buflomedil, carteolol, tolperisone, bufuralol, ibogaine, propafenone and bunitrolol were better aligned with tropisterone (10Å substrate). For five compounds
(dexfenfluramine, hydromorphone, morphine, chlorpheniramine and eletriptan) in the literature data set
N-dealkylation was reported as one of the major metabolic pathways in CYP2D6. For four of these compounds (dexfenfluramine, hydromorphone, morphine and chlorpheniramine) the part identified as Downloaded from the preferred site of metabolism by the FLAP alignments were blocked towards oxidative metabolism according to the FLAP prediction criteria (see Materials and Methods section). The fifth N-dealkylated dmd.aspetjournals.org compound, eletriptan, did not follow the working hypothesis since the molecule did not contain any groups unreactive towards oxidation. On the other hand, the electron withdrawing sulfon would make the aromatic moiety in eletriptan less prone to undergo oxidative metabolism. For this group of at ASPET Journals on September 24, 2021 compounds MetaSite predicted N-dealkylation in one of five cases (Table 3). It is though worth mentioning that MetaSite predicted SOM for morphine and eletriptan to be at a carbon atom in the alfa- position to the nitrogen but not the carbon in the N-methyl group. A hydroxylation at the MetaSite predicted carbon atom would eventually result in a cleavage of the nitrogen-carbon bond. However this will not result in the N-demethylated metabolite reported in the literature and is not considered as a correct prediction.
In the larger set comprising the compounds reported to be metabolized by CYP2D6 via other metabolic routes such as aromatic hydroxylation and/or O-demethylation the FLAP alignment did not identify any stable groups at the preferred site of metabolism. In 14 of 18 cases, the FLAP-predicted site of metabolism was correct compared with what was reported in the literature. One of the four wrongly predicted compounds, bisoprolol, was not successfully aligned to any of the template structures. The
16 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. prediction rate for MetaSite was 16 of 18 correctly predicted compounds for this set. A summary of the predictions rates is presented in Table 3.
It was difficult to find a large number of methyl- /ethyl aminyl compounds metabolized through N- dealkylation as major metabolic route by CYP2D6, however the compounds identified support the working hypothesis stated in this study.
Metabolite identification, Clint- and IC50-determinations of CYP2D6 substrates Downloaded from Twenty compounds were selected for experimental determinations of major metabolites, intrinsic clearance (Clint) and inhibition potential in recombinant CYP2D6. The selection was done with the aim
to represent both N-dealkylated and non N-dealkylated substrates. Another goal was to find some dmd.aspetjournals.org compounds with similar core structures in order to compare the effect of stable groups in the molecule.
The compounds were incubated with recombinant CYP2D6 and the Clint was determined from disappearance of the parent compound. These incubations were also analyzed with LC-MS/MS in order at ASPET Journals on September 24, 2021 to rationalize the structure of major metabolites. The inhibition potential of CYP2D6, the IC50-value, was determined with an end-point assay using a fluorescent probe substrate. Most of the compounds used were racemates but due to the availability (S,S)-sertraline and (R)-tomoxetine were used as pure stereoisomers. Tramadol and O-desmethyltramadol were used as the cis-racemates.
Predicted pKa-values, major metabolites formed in CYP2D6, calculated Clint-values and IC50-values for the 20 compounds are reported in Table 2. Due to difficulties in determining specific regioselectivity for hydroxylation of some compounds larger and more unspecific areas were suggested as the major
SOM. However, from the results it is still possible to discriminate if the metabolism occurred in the proximity of the nitrogen or not, which actually was the aim of this study. Metabolite identification data from LC-MS/MS analysis of the parent compounds and major metabolites are available as
Supplementary Information.
17 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version.
The compounds were divided into two groups, those with N-dealkylation as one major metabolic route and compounds with other metabolic reactions responsible for the biotransformation by CYP2D6.
The group with N-dealkylation as a major metabolic pathway consisted of eight compounds (nisoxetine, fluoxetine, Bionet-BB 6N-710, Bionet-BB 6N-708, lidocaine, (S,S)-sertraline, maprotiline, and citalopram). The major metabolite of citalopram was the N-oxide and not the N-dealkylated compound.
Despite this observation citalopram was, however, included in this group due to the fact that the oxidation takes place on the basic nitrogen. Of these eight compounds FLAP alignment identified stable Downloaded from groups at the preferred site of metabolism for fluoxetine, Bionet-BB 6N-708, (S,S)-sertraline and citalopram (Table 2). The other compounds did not contain any stable groups in the molecule but on the dmd.aspetjournals.org other hand Bionet-BB 6N-710 and lidocaine were both stable according to their Clint-values. Even though metabolites were found, the compounds were not metabolized by CYP2D6 to any greater extent.
The high IC50-value for lidocaine also indicates poor affinity for the isoenzyme. Comparing the at ASPET Journals on September 24, 2021 predicted pKa of the compounds, lidocaine also had the lowest pKa-value (pKa 7.99), which would further support that N-dealkylation occurs due to a larger fraction present as the free base (~20%). The metabolism of nisoxetine and maprotiline could not be explained by our working hypothesis.
Interestingly the IC50-values were in general within the lower range for this group (all except for citalopram had IC50-values below 10 µM with most of them below 5 µM), indicating a high inhibition potential. The high inhibition potential could be an indication that the compounds adopt a binding mode in CYP2D6 based on the “traditional” pharmacophore, which does not lead to efficient formation of any metabolite due to blocking groups at the predicted SOM.
The last group, containing the compounds not N-dealkylated by CYP2D6 to any major extent, included the highest Clint-values. Only 3/12 compounds were classified as low Clint compared to the first group where 5/8 were low Clint and neither of the compounds classified as high clearance drugs in
CYP2D6. This indicates that the compounds in the second group were more efficiently metabolized by
18 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. CYP2D6 compared to those metabolized through N-dealkylation. The IC50-values ranged between
<1 µM up to approximately 40 µM. Thus, many of these compounds can adopt productive binding modes that lead to metabolites that do not cause any major inhibition. To rationalize these differences in
Clint-values and inhibition potential, a general trend observed for these compounds was that when introducing stable groups in the molecule the metabolic stability was improved while the inhibition potential was increased. This effect can be highlighted with the example of Bionet-BB 4N-726 and
Bionet-BB 6N-708 (Table 2), two compounds with the same core structure but with different substituents. Bionet-BB 4N-726 had a Clint-value of 4.3 µl/min/pmol P450 and an IC50-value of Downloaded from
3.33 µM. Bionet-BB 6N-708, in which stable groups are introduced, was stable in the Clint assay
(<0.2 µl/min/pmol P450) but the IC50-value was almost ten times lower (0.38 µM) than that of Bionet- dmd.aspetjournals.org BB 4N-726.
As for the literature data set, the majority of the compounds in the experimental data set were at ASPET Journals on September 24, 2021 successfully aligned with DXM, but fluoxetine, lidocaine and alprenolol were better aligned with tropisterone. For compounds not metabolized by N-dealkylation as a major pathway, the success rate for predicting the SOM using the FLAP alignment was 9/12. Of the three wrongly predicted compounds O- desmethyltramadol was not successfully aligned with either of the two template molecules. For this group MetaSite predicted correct sites of metabolism in 10 of 12 cases. On the other hand MetaSite was not successful in predicting compounds that were N-dealkylated (1 of 8). A summary of the prediction rates is presented in Table 3.
19 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. DISCUSSION
Prediction of regio-specific metabolism of CYP2D6 substrates is complex. Different determinants such as aromatic stacking and hydrophobic interaction with Phe120 and Phe483 (Flanagan et al., 2004;
Lussenburg et al., 2005) as well as the hypothesis that different oxygenating species are responsible for different reactions (Keizers et al., 2005; Newcomb and Chandrasena, 2005) have been previously reported. This study, however, focuses on another determinant, the influence of the electrostatic interaction between the acidic amino acids in the active site cavity and the basic nitrogen in the Downloaded from substrate.
dmd.aspetjournals.org In this study we have explored how structural factors influence the occurrence of the less common
N-dealkylation reactions by CYP2D6. N-Dealkylation is a common metabolic pathway for drugs containing secondary and tertiary amines and in many cases CYP3A4, CYP1A2 or CYP2C19 are at ASPET Journals on September 24, 2021 involved in the catalysis (Coutts et al., 1994). This reaction also occurs in CYP2D6 but it is far less common than in the above-mentioned isoforms. In order to focus on how CYP2D6 catalyzes
N-dealkylation reactions this study only considered the in vitro situation using recombinant enzyme.
From the results it could be seen that the few compounds that were identified to or reported to be metabolized through N-dealkylation had a low Clint-value and were poorly cleared from the system. In the in vivo situation N-dealkylation via CYP2D6 is probably of minor importance and CYP3A4 with less substrate specificity will dominate the metabolism at high concentrations of the substrate.
Early pharmacophore models and the crystal structure of CYP2D6 have pointed out one major characteristic of CYP2D6 substrates, a basic nitrogen present in the molecule 5-7 or 10 Å from the site of oxidation, which can interact with an acidic amino acid residue on top of the active site. At physiological pH the positive charge of the basic nitrogen would favor an orientation of the substrate with the nitrogen away from the heme due to electrostatic interactions with the acidic amino acid in the
20 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. active site cavity. Even though a major fraction of the amines will be protonated there is still a small neutral fraction whose orientation would not be restrained by the electrostatic interactions in the cavity.
Thus, increasing the fraction of neutral molecules would theoretically increase the probability for
N-dealkylation to occur. The results from incubation of DXM with CYP2D6 at different pH values clearly support this hypothesis. The ratio between the rate of O-demethylation and N-demethylation shows a pH dependence with an increase in N-demethylation at higher pH-values. The influence of electrostatic interactions in CYP2D6 has previously been studied by Upthagrove et al. where it was shown that an increase of the basicity of CYP2D6 substrates by introducing different substituents in the Downloaded from molecule, increased the affinity for the enzyme (Upthagrove and Nelson, 2001). These studies show a relationship between protonation of substrate with affinity and orientation of CYP2D6 substrates in the dmd.aspetjournals.org active site cavity of the enzyme. In a study by Miller et al. the issues of protonation of substrates in binding and catalysis were studied in CYP2D6 (Miller et al., 2001). From these results it was suggested that the basicity of the amine was decreased upon binding resulting in enhanced availability of the at ASPET Journals on September 24, 2021 unprotonated form.
Searching the literature for compounds that are N-dealkylated by CYP2D6 and compounds not N- dealkylated gave the opportunity to investigate another working hypothesis that N-dealkylation occurs in CYP2D6 if the preferred SOM is blocked in one way or another. Since the amounts of CYP2D6 substrates that are N-dealkylated are rather limited in the literature it is difficult to do a comparison with statistical significance. Nevertheless, this study gives the opportunity to evaluate the general trends observed. There are however difficulties in drawing conclusions based only on literature data since experimental conditions often differ with different enzyme sources, different concentrations etc. In this study, 20 compounds were selected for experimental determination of the metabolism by CYP2D6 in order to obtain comparable data. The Km-values for the metabolic reactions studied vary over a wide range, however the experiments performed to study and compare the formation of metabolites and disappearance of parent compound were run at a concentration of 1 µM in all cases. In incubations at
21 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. higher concentrations, N-dealkylated metabolites could be detected for almost all compounds. This indicates that at some point, with an increased substrate concentration, there will be some molecules oriented with the nitrogen towards the heme even if this is not the most favorable orientation.
Analyzing the results, several of the substrates that are N-dealkylated have a stable blocking group in the molecule. On the other hand all of these compounds have low Clint-values and are thus poorly metabolized by CYP2D6. This could be explained by the above-mentioned discussion that only the non- charged fraction of the molecules is N-dealkylated. When the preferred SOM is blocked, other Downloaded from metabolic reactions like O-demethylation and hydroxylation will not occur and mainly N-dealkylated metabolites are observed. Regarding the metabolic properties of the compounds, adding blocking groups dmd.aspetjournals.org at sites of metabolism seemed to increase the metabolic stability but also resulted in an increased inhibition potential. This has important implications in solving metabolic issues, in that minor changes in the molecular structure in order to improve metabolic stability might increase the enzyme inhibition at ASPET Journals on September 24, 2021 potential of the compound and result in undesirable drug-drug interactions. This phenomenon of improved metabolic stability but retained or increased inhibition potential as a consequence of the addition of metabolically stable groups in the molecules, has also been observed by Ahlström et al. in the case of CYP2C9 (Ahlstrom et al., 2007).
The program FLAP was used to identify the preferred SOM according to the CYP2D6 pharmacophore. To cover the range of CYP2D6 substrates DXM, a “7Å substrate”, and tropisterone, a
“10Å substrate”, were used as templates. The prediction success rates were encouraging and when summarizing the results from the literature data set and the experimental data set, SOM was successfully predicted in 77% of the compounds not metabolized by N-dealkylation. Of these 30 compounds, two were not successfully aligned (bisoprolol and O-desmethyltramadol) with either template, thus not fitting the CYP2D6 pharmacophore. This method is a ligand-based approach and a comparison of the prediction rate with MetaSite, an approach that considers both protein-ligand complementarity and
22 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. reactivity, was done. For the compounds not metabolized by N-dealkylation MetaSite predicted the correct site of metabolism in 87 % of the cases. This is somewhat better than the result obtained from
FLAP alignments, but still this simple ligand based approach works well for CYP2D6. Afzelius et al. compared different computational approaches for SOM predictions in CYP2C9 and CYP3A4 and showed that MetaSite is superior, but that also the pure ligand-based approaches perform well (Afzelius et al.). It was argued that due to the large and flexible active site cavity of CYP3A4, reactivity is thought to be the driving force in metabolism of the substrates of this isoform. The study also indicated that
CYP2C9 is reactivity driven, despite of its smaller active site cavity resulting in more specific Downloaded from interactions. In the case of CYP2D6, steric hindrance is also believed to be of importance for selectivity, thus indicating that structure based methods would be successful for SOM predictions. Nevertheless, dmd.aspetjournals.org with the well-characterized pharmacophoric features of CYP2D6 substrates it is not surprising that a pharmacophore based alignment method is successful in predicting the site of metabolism of this isoform. To identify compounds that are N-dealkylated by CYP2D6 the FLAP alignment can be a useful at ASPET Journals on September 24, 2021 method. For all compounds that were N-dealkylated and contained stable groups in the molecule FLAP identified the stable group at the preferred site of metabolism, however there were some false negatives where N-dealkylation occurs even though no blocking group was present. MetaSite did not perform as well in identifying N-dealkylation as site of metabolism and the FLAP alignment could therefore be a good complement to this methodology.
In conclusion, the hypothesis that N-dealkylation occurs in CYP2D6 when the preferred site of metabolism is blocked seems to be correct in most cases studied here. It is also evident that the fraction of unprotonated molecules affects the extent to which N-dealkylation will occur. In addition, an important finding is that blocking of the sites of metabolism can retain, or increase problems with enzyme inhibition. This study also includes an investigation of a novel computational tool, FLAP, for site of metabolism predictions. Firstly, using this tool to spot stable groups at preferred SOMs to identify N-dealkylation reactions by CYP2D6 was shown to be successful and could be a valuable
23 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. complement to other available SOM prediction tools such as MetaSite. Secondly, this fairly simple approach to predict SOM for CYP2D6 substrates by alignment to a template molecule worked satisfactorily, which highlights the importance and influence of the CYP2D6 pharmacophore.
Nevertheless, further work needs to be done to explore ways of disrupting the CYP2D6 pharmacophore to address both metabolic stability issues (SOM) and enzyme inhibition issues while retaining the pharmacological activity of compound classes.
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24 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. ACKNOWLEDGEMENT
We acknowledge Dr Emre Isin for excellent help in interpretation of mass spectral data, Prof. Neal
Castagnoli for insightful discussions with respect to pH dependence studies and Prof. Kristina Luthman and Dr Richard Thompson for valuable discussions and critical reading of the manuscript.
Downloaded from dmd.aspetjournals.org at ASPET Journals on September 24, 2021
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31 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. LEGENDS FOR FIGURES
Fig 1. CYP2D6 probe substrates a) debrisoquine b) DXM and c) tropisterone with the proposed major sites of metabolism in CYP2D6 indicated with arrows.
Fig 2. The effect of pH on the formation of N- and O-demethylated metabolites of DXM in CYP2D6. a)
Rate of formation of DXO (O-demethylation) after incubation of 200 µM DXM. b) Rate of formation of Downloaded from
MEM (N-demethylation) after incubation of 200 µM DXM. c) Ratio between O- and N-demethylation
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32 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. TABLES
TABLE 1
FLAP predictions, MetaSite predictions and reported site of metabolism in CYP2D6 for the literature
data set
pKaa FLAPb MetaSitec Reported site of Ref.
metabolismd
Downloaded from
N-dealkylated by 2D6
F F F F F F dmd.aspetjournals.org F F F (Gross et Dexfenfluramine 9.59 al., 1996) NH NH NH
N N (Benetton et at ASPET Journals on September 24, 2021 f H H H N Hydromorphone 8.52 H H H al., 2004) OH O OH O O O OH O O
N N N (Projean et f H H H Morphine 8.35 H H H al.) OH O H O OH O H O OH O H O
Cl Cl Cl (Fried et al., Chlorpheniramine 9.05 N N N 2002) N N N
(Evans et e,f O O O Eletriptan 9.29 S N S N S N O O O al., 2003) N N N H H H
Not N-dealkylated by 2D6
33 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version.
O O O (Haritos et Doxepin 8.93 al.) N N N
(Lin et al., MDMA 9.65 NH NH NH O O O 1997) O O O
S S S (Yoshii et Chlorpromazine 9.24 N Cl N Cl N Cl al., 2000) N N N
(Sorensen Downloaded from Perhexiline 10.32 et al., 2003) N N N H H H N-Butyl
(Bach et al., dmd.aspetjournals.org 10.09 N N N 1999) amphetamine H H H
(Bach et al., Ethylamphetamine 9.99 N N N H H H 1999) at ASPET Journals on September 24, 2021
4-Methoxy-ethyl (Bach et al., 9.99 N N O H N H O H O 1999) amphetamine
H H H N N N (Feifel et e O Brofaromine 9.55 O O Br Br Br al., 1993)
O O O
(Kanmacher e O O O O O O Buflomedil 9.48 N N N et al., 2000) O O O O O O
N N N (Nakamura Mequitazine 9.36 H H H N N N et al., 1998)
S S S
34 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version.
NH NH NH OH OH OH (Kudo et Carteolole 9.34 O O O al., 1997)
N O N O N O H H H
(Dalmadi et e Tolperisone 9.27 ON ON ON al., 2003)
(Narimatsu e OH OH OH Bufuralol 8.92 H H H O N O N O N et al., 2002)
(Obach et Downloaded from e N N N Ibogaine 8.81 O O O N N N al., 1998) H H H
OH OH OH N N N H H H (Kroemer et dmd.aspetjournals.org Propafenonee 9.13 O O O O O O al., 1989)
N H N H N H (Fischer et O O O Tropisterone 9.16 NH NH NH at ASPET Journals on September 24, 2021 O O O H H H al., 1994)
(Narimatsu e N N N Bunitrolol 9.34 N N N H H H O OH O OH O OH et al., 1994)
OH OH H H O N O N (Horikiri et Bisoprolol 9.24 Cpd not aligned O O al.) O O a Predicted pKa values from MOKA b Encircled are the parts of the molecules aligned with site of metabolism in DXM/tropisterone from the FLAP alignment c Encircled are the parts of the molecules comprising the top 2 ranked sites of metabolism from MetaSite d Encircled is the site of metabolism in CYP2D6 reported in the literature e FLAP alignment with tropisterone
35 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. f Protonation of the nitrogen results in a stereogenic center. The MetaSite predictions comprise both the R and S stereoisomers Downloaded from dmd.aspetjournals.org at ASPET Journals on September 24, 2021
36 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. TABLE 2
Predicted pKa-values, experimentally determined IC50-values, Clint-values and major metabolites in
CYP2D6 together with FLAP and MetaSite predictions for the experimental data set.
pKaa IC50 Clintb FLAPc MetaSited Major
metabolitee (µM) (µl/min/pmol
P450)
Downloaded from
N-dealkylation major metabolic route in CYP2D6
O O O dmd.aspetjournals.org Nisoxetine 9.46 1.25 1.4 O O O
NH NH NH
O O f O F F at ASPET Journals on September 24, 2021 Fluoxetine 9.46 8.56 0.43 F F F F F NH F NH F NH
O O O Bionet-BB 6N-710g 9.29 1.21 <0.2i
N N N
F F F F F Cl F Cl F F Cl F O O O Bionet-BB 6N-708g 9.29 0.38 <0.2i N N N
f i O O O Lidocaine 7.99 >50 <0.2 N N N H H H N N N
NH NH NH (S,S)-Sertraline 9.95 3.33 0.25
Cl Cl Cl Cl Cl Cl
37 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version.
N N N Maprotiline 10.21 4.11 2.4 H H H
H H H
N N N O O N N O N Citalopram 9.00 14.0 2.0
F F F
Major metabolite in incubation with CYP2D6 other than N-dealkylation Downloaded from
h N N N Clomipramine 9.24 1.89 2.7 Cl Cl Cl
N N N
dmd.aspetjournals.org cis-Tramadolh 9.29 >50 0.5 N N N O O O OH OH OH
OH OH at ASPET Journals on September 24, 2021 OH g,h Dextrorphan 9.16 13 0.29 H H H N N N
H H N H N,N-Dimethyl-5- N N 8.98 18.2 0.93 h N O N methoxytryptamine N O O
j (R)-tomoxetine 9.46 1.24 >8 O O O
NH NH NH
O O g j F F O Bionet-BB 4N-726 9.29 3.33 >8 F N N N
Imipramine 9.24 4.3 7.2 N N N
N N N
38 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version.
H N,N- H N H N N 8.98 39.8 1.12 N Dimethyltryptamine N N
cis-O - N 9.29 28 0.35 N N not aligned OH OH Desmethyltramadol OH OH
O O O Dextromethorphang 9.16 3.2 6 k H H H (9.12 ) N N N
S S Downloaded from S
N S N S Thioridazineg 9.16 0.27 1.4 N S N N N
dmd.aspetjournals.org
OH OH OH H H H O O N O N Alprenololf 9.24 22.3 >8j N
a Predicted pKa values from MOKA at ASPET Journals on September 24, 2021 b Clint calculated after incubation with 1 µM of the compound with recombinant CYP2D6 c Encircled is the part of the molecule aligned with site of metabolism in DXM/tropisterone from the FLAP alignment d Encircled is the parts of the molecule comprising the top 2 ranked site of metabolism from MetaSite e Encircled is the site of metabolism for major metabolites after incubation with CYP2D6 f FLAP alignment with tropisterone g Protonation of the nitrogen results in a stereogenic center. The MetaSite predictions comprise both the R and the S stereoisomers. h N-dealkylated metabolite present but considered to have minor contribution i no curves obtained due to stable compound j no measurable amount of compound left after 7 minutes of incubation k experimentally determined pKa-value (Gilligan and Li Wan Po, 1991)
39 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited andDMD formatted. #22376# The final version may differ from this version. TABLE 3
Summary of FLAP and MetaSite predictions of SOMa
N-dealkylation by CYP2D6 No N-dealkylation by CYP2D6
FLAP predicted MetaSite correctly FLAP correctly MetaSite correctly
blocking group at predicted SOMa predicted SOMa predicted SOMa
preferred SOMa (%b) (%b) (%b) (%b)
Downloaded from
Literature 80 20 78 89 data set dmd.aspetjournals.org
Experimental 50c 13c 75 83 data set
Total 62 15 77 87 at ASPET Journals on September 24, 2021
a SOM = site of metabolism b Prediction rate calculated by dividing the number of correctly predicted compounds with the total number of compounds in the analysis. c N-dealkylated metabolite one of major metabolites after incubation of 1µM with recombinant CYP2D6.
40 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 24, 2021 DMD Fast Forward. Published on August 25, 2008 as DOI: 10.1124/dmd.108.022376 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 24, 2021