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FEBS Letters 586 (2012) 1749–1753

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Hypothesis Rat CYP2D2, not 2D1, is functionally conserved with human CYP2D6 in endogenous formation ⇑ Nadja Grobe 1, Toni M. Kutchan , Meinhart H. Zenk 2

Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO 63132, USA

article info abstract

Article history: The assumption that CYP2D1 is the corresponding rat cytochrome to human CYP2D6 has been revis- Received 22 March 2012 ited using recombinant proteins in direct assays. CYP2D1 and 2D2 were incubated with Revised 4 May 2012 known CYP2D6 substrates, the three morphine precursors thebaine, codeine and (R)-reticuline. Accepted 12 May 2012 Mass spectrometric analysis showed that rat CYP2D2, not 2D1, catalyzed the 3-O-demethylation Available online 26 May 2012 reaction of thebaine and codeine. In addition, CYP2D2 incubated with (R)-reticuline generated four Edited by Miguel De la Rosa products corytuberine, pallidine, salutaridine and isoboldine while rat CYP2D1 was completely inac- tive. This intramolecular phenol-coupling reaction follows the same mechanism as observed for CYP2D6. Michaelis–Menten kinetic parameters revealed high catalytic efficiencies for rat CYP2D2. Keywords: CYP2D6 These findings suggest a critical evaluation of other commonly accepted, however untested, CYP2D1 CYP2D1 and CYP2D2 substrates. Mammalian morphine biosynthesis Ó 2012 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. LC–MS/MS analysis

1. Introduction rats deficient in CYP2D1 were found to convert the two morphine precursors, thebaine and codeine, less efficiently as microsomes Human CYP2D6 is involved in the oxidation of about 30% of from Sprague–Dawley rats expressing CYP2D1 [6,7]. clinically relevant drugs including the morphine precursor codeine Our interest in elucidating mammalian involved in [1,2]. The enzyme has been connected to a phenomenon known as mammalian morphine biosynthesis led us to test rat CYP2D1 in di- the debrisoquine/sparteine metabolism of drug oxidation which is rect enzyme assays similar to the CYP2D6 approach described pre- caused by aberrant metabolism patterns due to the absence or viously [8]. Surprisingly, rat CYP2D1 was not capable of catalyzing deficiency of functional allelic variants of CYP2D6 [3]. At least the 3-O-demethylation of thebaine and codeine nor the phenol- 112 allelic variants of CYP2D6 have been described since the dis- coupling reaction of (R)-reticuline. The rat genome was subse- covery of the genetic polymorphism resulting in a total of four quently searched and five isoforms were identified: CYP2D2, individual phenotypes: poor, intermediate, extensive and ultra- CYP2D3, CYP2D4, CYP2D5 and CYP2D18 among which CYP2D2 rapid metabolizers [4]. Considering the multitude of clinically was described as poorly expressed in the Dark-Agouti rat strain relevant compounds that are metabolized by human CYP2D6, drug next to CYP2D1 with similar catalytic activities as observed for dosages tailored towards individual phenotypes are currently dis- human CYP2D6 [9,10]. We obtained microsomes containing rat cussed [2]. CYP2D2 from a commercial source and describe herein its In the past, an in vitro model to test possible CYP2D6 substrates enzymatic characterization using the three morphine precursors utilized microsomal preparations of different rat strains that differ- (R)-reticuline, thebaine and codeine. entially express CYP2D1, the proposed rat orthologue to CYP2D6 [5]. Using this approach, liver microsomes obtained from Dark-Agouti 2. Materials and methods

2.1. Enzymes Abbreviations: CYp450s, cytochrome P450; MRM, multiple reaction monitoring; LCMS, liquid chromatography mass spectrometry CYP2D1 and CYP2D2 were purchased from BD Biosciences ⇑ Corresponding author. Fax: +1 314 587 1573. (Woburn, MA). For confirmation of amino acid sequences, the E-mail address: [email protected] (T.M. Kutchan). P450 enzymes were subjected to SDS–PAGE on a 10% (w/v) acryl- 1 Present address: Wright State University, Department of Pharmacology and amide gel. Protein bands at approximately 55–58 kDa were Toxicology, Boonshoft School of Medicine, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA. excised, eluted and sequenced using a 6520 QTOF with Chip Cube 2 M.H.Z. passed away prior to completion of this manuscript. (Agilent, Santa Clara, CA).

0014-5793/$36.00 Ó 2012 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. http://dx.doi.org/10.1016/j.febslet.2012.05.021 1750 N. Grobe et al. / FEBS Letters 586 (2012) 1749–1753

2.2. Enzyme assays gradient elution, using solvent A (0.2% acetic acid) and B (99.8%

CH3CN, 0.2% acetic acid) (all v/v). The gradient started with 100% Reactions were conducted in a total volume of 250 ll containing A for 5 min and was increased to 60% B over 20 min. Elution was 100 mM potassium phosphate buffer (pH 7.4), 15 lg L-a-dilauroyl- continued for 5 min at 60% B. A detailed description of the LC– sn-glycero-3-phosphocholine, 1 mM NADP+, 10 mM glucose 6- MS/MS analysis for reaction mixtures containing reticuline has phosphate, 1 unit/ml glucose-6-phosphate dehydrogenase (yeast), been published previously [8]. Incubations containing debrisoquine 1000 units/ml catalase (bovine liver), 10 lg bovine erythrocyte were analyzed using an Eclipse XDB-C18 HPLC column combined superoxide dismutase, and 0.25–50 lM alkaloidal . Reac- with a microfilter unit (Sigma, 1/16 OD tubing). The mobile phase tions were started by adding 5–30 pmol P450 enzyme (BD Biosci- total flow was set to 0.2 ml/min with binary gradient elution, using ences) and incubated for 10–120 min at 37 °C. Extraction of solvent A (0.1% formic acid) and B (CH3CN). The gradient started alkaloids was conducted as previously described [8]. Alkaloids were with 95% A for 2 min and was increased to 100% B over 8 min. analyzed by LC–MS/MS. Kinetic parameters were estimated by non- Elution was continued for 2 min at 100% B. Identification of analytes linear regression with GraphPad Prism. The debrisoquine oxidation was based on retention times, MRM transition ratios, and compar- assay was prepared according to Hiroi et al. 2002 [10]. Reactions ison with the expected values for standards. Quantitation was per- were conducted in a total volume of 250 ll containing 100 mM formed by constructing standard curves for each analyte and potassium phosphate buffer (pH 7.4), 0.8 mM NADPH, and 4 lM integrating the peak area of the quantifier MRM transition with debrisoquine or alkaloidal substrate. Reactions were started by add- Analyst 1.4.1 (Applied Biosystems, MDS SCIEX Instruments). ing 10 pmol P450 enzyme (BD Biosciences), incubated for 10– 120 min and stopped with 25 ll 10% trichloroacetic acid. After cen- 3. Results and discussion trifugation, the supernatant was analyzed by LC–MS/MS. Microsomal studies suggested rat P450 enzyme CYP2D1 as 2.3. LC–MS/MS analysis functionally conserved with human CYP2D6 and an enzymatic cat- alyst for the 3-O-demethylation reaction of thebaine and codeine Enzyme activities were analyzed with a 4000 QTRAP LC–TIS– [6,7]. Thus, CYP2D1 was obtained from a commercial source and MS–MS (Agilent) system using the multiple reaction monitoring tested in incubations containing these morphine precursors. Since (MRM) in positive ionization mode. Compound dependent parame- the 3-O-demethylation of thebaine, m/z 312, or codeine, m/z 300, ters (collision energy, declustering potential, quantifier MRM, qual- results in a mass loss of 14 mass units, specific multiple reaction ifier MRM, dwell time) are listed in Table 1. For the analysis of monitoring (MRM) for oripavine, m/z 298, and morphine, m/z incubations containing codeine and thebaine, separation of 286, was selected. Minor formation of m/z 298 was (10 ll) samples was achieved by using a Gemini C18 octadecylsi- observed in incubations with CYP2D1 and thebaine while codeine lane HPLC column (Phenomenex, 5 lm, 150 mm  2 mm) com- was not converted by CYP2D1 (Fig. 1). bined with a C18 guard column (Phenomenex, 4 mm  2 mm). However, MS/MS data of the generated product m/z 298 were The mobile phase total flow was set to 0.1 ml/min with binary not identical to the MS/MS obtained for the standard oripavine. It

Table 1 Compound dependent parameters for the LC–MS/MS method.

Analyte Collision energy (V) Declustering potential (V) Quantifier MRM transition Qualifier MRM transition Dwell time (ms) Reticuline 35 45 330 ? 192 330 ? 137 50 Corytuberine 40 50 328 ? 265 328 ? 282 50 Pallidine 40 50 328 ? 211 328 ? 237 50 Salutaridine 40 50 328 ? 211 328 ? 237 50 Isoboldine 40 50 328 ? 265 328 ? 237 50 Thebaine 35 40 312 ? 251 312 ? 281 50 Oripavine 35 40 298 ? 218 298 ? 249 50 Codeine 40 47 300 ? 215 286 ? 225 50 Morphine 45 45 286 ? 165 286 ? 201 50 Debrisoquine 35 70 176 ? 134 176 ? 117 100 Hydroxydebrisoquine 35 70 192 ? 130 192 ? 115 100

Fig. 1. Conversion of thebaine and codeine by rat CYP2D1. (A) MRM for the substrate thebaine, m/z 312, after incubation with CYP2D1. (B) MRM for the demethylated product, m/z 298, after incubation of thebaine with CYP2D1. (C) MRM for the substrate codeine, m/z 300, after incubation with CYP2D1. (D) MRM for the demethylated product, m/z 286, after incubation of codeine with CYP2D1. Illustrated is the sum of selected MRM. Incubations were carried out for 120 min at 37 °C. N. Grobe et al. / FEBS Letters 586 (2012) 1749–1753 1751

was therefore concluded that the observed mass loss of 14 was due to a possible CYP2D1-catalyzed demethylation of thebaine at the ring nitrogen. Indeed, further comparison and mass spectrometric characterization suggested the CYP2D1 product as N-demethylated thebaine (Fig. 2). To confirm that CYP2D1 was still active in incuba- tions lasting as long as 120 min, the rat P450 enzyme was incu- bated with the reference substrate, debrisoquine, as described previously [10]. Indeed, as shown in Fig. 3A and B debrisoquine was hydroxylated by CYP2D1 as previously described [9,10,15]. Using the same incubation conditions, it was again confirmed that CYP2D1 was not active with the morphine precursors. Fig. 2. Identification of product, m/z 298, formed from thebaine by CYP2D1. (A) MS/ Since CYP2D1 did not show similar catalytic activities to human MS of CYP2D1 product, m/z 298. (B) MS/MS of northebaine. CYP2D6 in incubations with the alkaloidal substrates, we searched

Fig. 3. Conversion of debrisoquine by rat CYP2D1 and CYP2D2. (A) MRM for the substrate debrisoquine, m/z 176, after incubation with CYP2D1. (B) MRM for the product hydroxydebrisoquine, m/z 192, after incubation of debrisoquine with CYP2D1. (C) MRM for the substrate debrisoquine, m/z 176, after incubation with CYP2D2. (D) MRM for the product hydroxydebrisoquine, m/z 192, after incubation of debrisoquine with CYP2D2. Illustrated is the sum of selected MRM. Incubations were carried out for 120 min at 37 °C.

Fig. 4. Conversion of thebaine and codeine by rat CYP2D2. (A) MRM for the substrate thebaine, m/z 312, after incubation with CYP2D2. (B) MRM for the demethylated product, m/z 298, after incubation of thebaine with CYP2D2. (C) MS/MS of m/z 298 formed from thebaine by CYP2D2. (D) MS/MS of oripavine. (E) MRM for the substrate codeine, m/z 300, after incubation of CYP2D2. (F) MRM for the demethylated product, m/z 286, after incubation of CYP2D2 with codeine. (G) MS/MS of m/z 286 formed from codeine by CYP2D2. (H) MS/MS of morphine. Illustrated is the sum of selected MRM. Incubations were carried out for 120 min at 37 °C. Note that substrates were completely converted to respective reaction products. 1752 N. Grobe et al. / FEBS Letters 586 (2012) 1749–1753

Fig. 5. Test for phenol-coupling activities of CYP2D1 or CYP2D2. (A) MRM for m/z 330 after incubation of CYP2D1 with (R)-reticuline. (B) MRM for m/z 328 after incubation of CYP2D1 with (R)-reticuline. (C) MRM for m/z 330 after incubation of CYP2D2 with (R)-reticuline. (D) MRM for m/z 328 after incubation of CYP2D2 with (R)-reticuline (pallidine 8.92 min, corytuberine 10.25 min, salutaridine 12.58 min and isoboldine 13.44 min). Illustrated is the sum of selected MRM for all four products. Incubations were carried out for 120 min at 37 °C.

Table 2 Catalytic parameters for rat CYP2D2.

À1 À1 Substrate Product Km, lM kcat, pmol/min/pmol P450 kcat//Km,mM s (R)-reticuline Corytuberine 1.0 ± 0.18 0.03 ± 0.001 0.42 Pallidine 0.8 ± 0.10 0.86 ± 0.03 17.9 Salutaridine 0.7 ± 0.12 2.26 ± 0.11 53.9 Isoboldine 0.6 ± 0.10 0.69 ± 0.03 19.3 Thebaine Oripavine 42 ± 9.0 63 ± 7.6 25.0 Codeine Morphine 29 ± 4.6 14 ± 1.1 8.0

Table 3 Relative capacity of salutaridine synthase, CYP2D6, CYP3A4 and CYP2D2 to catalyze the oxidative phenol-coupling of (R)- and (S)-reticuline.

Enzyme (À)-corytuberine/(+)-corytuberine (+)-pallidine/(À)-pallidine salutaridine/sinoacutine (+)-isoboldine/(À)-isoboldine (%) (%) (%) (%) Salutaridine synthase nda/nda nda/nda 100/nda nda/nda [13,14] CYP2D6 [8] 4/2 23/17 7/5 66/76 CYP3A4 [8] 8/12 22/17 34/35 36/36 CYP2D2 [this work] 1/4 20/34 74/56 5/4

a nd: not detected.

the rat genome for other possible candidates capable of catalyzing towards this third morphine precursor were compared. Since the the 3-O-demethylation reaction of thebaine or codeine. Five iso- mechanism of the phenol-coupling reaction of (R)-reticuline, m/z forms were identified CYP2D2, CYP2D3, CYP2D4, CYP2D5 and 330, leads to a total loss of 2 mass units, the MRM for m/z 328 was CYP2D18 among which rat CYP2D2, next to CYP2D1, was described monitored. Fig. 5 illustrates that while CYP2D1 was inactive, as poorly expressed in the Dark-Agouti rat strain [9]. Moreover, CYP2D2 formed the four expected products corytuberine, pallidine, studies with cDNA-expressed rat CYP isoforms elucidated that salutaridine and isoboldine. Using (R)-reticuline as substrate, an CYP2D2, CYP2D4 and CYP2D18, but not CYP2D1, catalyzed the optimal pH of 7.4 was determined for CYP2D2. The relative distribu- hydroxylation of tyramine to dopamine with similar kinetic char- tion of the four phenol-coupled products was the same after 10 and acteristics for CYP2D2 compared with human CYP2D6 [11]. While 120 min of incubation as well as at any pH between 5 and 9. progesterone hydroxylation properties of CYP2D4 and CYP2D6 Michaelis–Menten kinetic parameters revealed that, similar to were alike [10,12], CYP2D2 and CYP2D6 resembled each other in human CYP2D6, rat CYP2D2 shows a higher Km value for thebaine their catalytic activities for the hydroxylation of bufuralol, debr- and codeine (29–46 lM) in comparison with (R)-reticuline (0.6– isoquine and propranolol, which are common CYP2D substrates 1.0 lM) (Table 2). However, catalytic efficiencies for all three sub-

[10]. Thus, recombinant CYP2D2 was obtained from a commercial strates became similar due to lower maximum rates (kcat) for the source and tested first with debrisoquine to confirm activity. Sim- conversion of (R)-reticuline. Overall catalytic efficiencies for theba- ilar to rat CYP2D1, CYP2D2 converted debrisoquine to hydroxy- ine, codeine and (R)-reticuline were at least an order of magnitude debrisoquine in incubations as long as 120 min (Fig. 3C and D). higher than for human CYP2D6 (Table 2 and [8]). Moreover, as shown in Fig. 4 CYP2D2 was highly active with both Including this work, four enzymes are known to catalyze the morphine precursors, thebaine and codeine, using the same incu- intramolecular formation of the critical C12–C13 bridge in salutari- bation conditions. The reaction products at m/z 298 and m/z 286 dine during morphine biosynthesis [8,13,14]. Table 3 compares the showed identical MS/MS spectra upon comparison to oripavine relative capacity of these four cytochrome P450s to catalyze the and morphine standards, respectively (Fig. 4). formation of phenol-coupled products from reticuline. While plant After confirming 3-O-demethylating activity for CYP2D2 with salutaridine synthase only generates salutaridine and does not thebaine and codeine, the phenol coupling reaction of (R)-reticuline accept the (S)-enantiomer as substrate, the mammalian P450 was examined and enzymatic activities of rat CYP2D1 and CYP2D2 enzymes appear to be more promiscuous. Among all, rat CYP2D2 N. Grobe et al. / FEBS Letters 586 (2012) 1749–1753 1753 is the most efficient mammalian P450 enzyme to produce the crit- [7] Mikus, G., Somogyi, A.A., Bochner, F. and Eichelbaum, M. (1991) Codeine O- ical morphine precursor. demethylation: rat strain differences and the effects of inhibitors. Biochem. Pharmacol. 41, 757–762. In conclusion, new enzymatic activities were found for rat [8] Grobe, N., Zhang, B., Fisinger, U., Kutchan, T.M., Zenk, M.H. and Guengerich, F.P. CYP2D2 that have been incorrectly proposed for CYP2D1. A similar (2009) Mammalian cytochrome P450 enzymes catalyze the phenol-coupling correction of CYP2D1/CYP2D2 has previously been made for the step in endogenous morphine biosynthesis. J. Biol. Chem. 284, 24425–24431. [9] Yamamoto, Y., Tasaki, T., Nakamura, A., Iwata, H., Kazusaka, A., Gonzalez, F.J. drug debrisoquine [9,15]. These findings become particularly impor- and Fujita, S. (1998) Molecular basis of the Dark Agouti rat drug oxidation tant for studies relying on results based on comparison of enzyme polymorphism: importance of CYP2D1 and CYP2D2. Pharmacogenetics 8, activities between different rat strains [6,7,16–20]. Thus, the metab- 73–82. [10] Hiroi, T., Chow, T., Imaoka, S. and Funae, Y. (2002) Catalytic specificity of olism of some drugs, such as metoprolol, dextromethophan or 3,4- CYP2D isoforms in rat and human. Drug Metab Dispos. 30, 970–976. methylenedioxymethamphetamine, might need further evaluation. [11] Bromek, E., Haduch, A. and Daniel, W.A. (2010) The ability of cytochrome P450 2D isoforms to synthesize dopamine in the brain: an in vitro study. Eur. J. Pharmacol. 626, 171–178. Acknowledgments [12] Hiroi, T., Kishimoto, W., Chow, T., Imaoka, S., Igarashi, T. and Funae, Y. (2001) Progesterone oxidation by cytochrome P450 2D isoforms in the brain. The authors would like to thank Dr. Leslie Hicks and Dr. Sophie Endocrinology 142, 3901–3908. Alvarez, Donald Danforth Plant Science Center, for sequencing the [13] Gerardy, R. and Zenk, M.H. (1993) Formation of salutaridine from (R)- reticuline by a membrane-bound cytochrome P-450 enzyme from Papaver commercially available CYP2D1 and CYP2D2. We would like to somniferum. 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