Biochemistry 2002, 41, 11025-11034
11025
Diversity in the Oxidation of Substrates by Cytochrome P450 2D6: Lack of an Obligatory Role of Aspartate 301-Substrate Electrostatic Bonding† F. Peter Guengerich,*,‡ Grover P. Miller,‡,§ Imad H. Hanna,‡,| Martha V. Martin,‡ Serge Le´ger,⊥ Cameron Black,⊥ Nathalie Chauret,⊥ Jose´ M. Silva,⊥ Laird A. Trimble,⊥ James A. Yergey,⊥ and Deborah A. Nicoll-Griffith⊥ Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt UniVersity School of Medicine, NashVille, Tennessee 37232-0146, and Department of Medicinal Chemistry, Merck Frosst Centre for Therapeutic Research, P.O. Box 1005, Pointe Claire DorVal, Quebec H9H 4P8, Canada ReceiVed May 7, 2002 ABSTRACT: Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human debrisoquine hydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs containing a basic nitrogen. Residue Asp301 has been characterized as being involved in electrostatic interactions with substrates on the basis of homology modeling and site-directed mutagenesis experiments [Ellis, S. W., Hayhurst, G. P., Smith, G., Lightfoot, T., Wong, M. M. S., Simula, A. P., Ackland, M. J., Sternberg, M. J. E., Lennard, M. S., Tucker, G. T., and Wolf, C. R. (1995) J. Biol. Chem. 270, 29055-29058]. However, pharmacophore models based on the role of Asp301 in substrate binding are compromised by reports of catalytic activity toward substrates devoid of a basic nitrogen, which have generally been ignored. We characterized a high-affinity ligand for P450 2D6, also devoid of a basic nitrogen atom, spirosulfonamide [4-[3-(4-fluorophenyl)-2-oxo-1-oxaspiro[4.4]non-3-en-4-yl]benzenesulfonamide], with Ks 1.6 µM. Spirosulfonamide is a substrate for P450 2D6 (kcat 6.5 min-1 for the formation of a syn spiromethylene carbinol, Km 7 µM). Mutation of Asp301 to neutral residues (Asn, Ser, Gly) did not substantially affect the binding of spirosulfonamide (Ks 2.5-3.5 µM). However, the hydroxylation of spirosulfonamide was attenuated in these mutants to the same extent (90%) as for the classic nitrogenous substrate bufuralol, and the effect of the D301N substitution was manifested on kcat but not Km. Analogues of spirosulfonamide were also evaluated as ligands and substrates. Analogues in which the sulfonamide moiety was modified to an amide, thioamide, methyl sulfone, or hydrogen were ligands with Ks values of 1.7-32 µM. All were substrates, and the methyl sulfone analogue was oxidized to the syn spiromethylene carbinol analogue of the major spirosulfonamide product. The D301N mutation produced varying changes in the oxidation patterns of the spirosulfonamide analogues. The peptidometic ritonavir and the steroids progesterone and testosterone had been reported to be substrates for P450 2D6, but the affinities (Ks) were unknown; these were estimated to be 1.2, 1.5, and 15 µM, respectively (cf. 6 µM for the classic substrate bufuralol). The results are consistent with a role of Asp301 other than electrostatic interaction with a positively charged ligand. H-Bonding or electrostatic interactions probably enhance binding of some substrates, but our results show that it is not required for all substrates and explain why predictive models fail to recognize the proclivity for many substrates, especially those containing no basic nitrogen.
P450s1 are major enzymes involved in the oxidations of many organic chemicals (2, 3). Particular interest has been given to the mammalian P450 enzymes that dominate the † This research was supported in part by U.S. Public Health Service (USPHS) Grants R35 CA44353, R01 CA90426, and P30 ES00267. G.P.M. and I.H.H. were recipients of USPHS Postdoctoral Fellowships F32 GM19808 and F32 CA79162, respectively. * To whom correspondence should be addressed: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638B Robinson Research Building (Medical Research Building I), 23rd and Pierce Ave., Nashville, TN 37232-0146. Tel: 615-322-2261. Fax: 615-322-3141. E-mail: guengerich@ toxicology.mc.vanderbilt.edu. ‡ Vanderbilt University School of Medicine. § Present address: Department of Biochemistry and Molecular Biology, University of Arkansas Medical School, Little Rock, AR 72205. | Present address: Department of Drug Metabolism and Safety Assessment, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033. ⊥ Merck Frosst Centre for Therapeutic Research.
metabolism of drugs (4). Variability among individuals can have a major influence on the efficacy of drugs, and human P450s are an important target of efforts in pharmacogenomics (5). P450 2D6 was first identified as the polymorphic debrisoquine hydroxylase (6, 7). This enzyme is estimated to be involved in the metabolism of ∼30% of the drugs on the market today, more than any other P450 except P450 3A4 (4). Today the polymorphism of P450 2D6 is relatively well understood, at least in terms of the polymorphisms that exist (6-8), and efforts have been directed to better understand the biochemical basis of P450 2D6 activity. In early research 1 Abbreviations: P450, microsomal cytochrome P450 [also termed “heme-thiolate protein P450” (1)]; 1D, one dimensional; 2D, two dimensional; DMSO, dimethyl sulfoxide; HPLC, high-performance liquid chromatography; MS, mass spectrometry; HO, hydroxy; NOE, nuclear Overhauser effect; DFQ, double-filtered quantum; COSY, correlated spectroscopy; HMQC, heteronuclear multiple quantum correlation.
10.1021/bi020341k CCC: $22.00 © 2002 American Chemical Society Published on Web 08/15/2002
11026 Biochemistry, Vol. 41, No. 36, 2002
Guengerich et al.
Scheme 1: Spirosulfonamide and Oxidation Products
with purified P450 2D6 in this laboratory (9), the observation was made that most of the known substrates for P450 2D6 contained a basic nitrogen atom, which is located 5-7 Å away from the site of oxidation on the substrate (10, 11). This concept was developed with more detailed models of P450 2D6, in terms of both pharmacophore models and homology modeling and combinations of the two (12-14). Site-directed mutagenesis of Asp301 led to loss of catalytic activity in yeast recombinant P450 2D6 (15), and the result has been widely interpreted as evidence that this acidic amino acid serves as a point anionic charge to dock the basic nitrogen atom of the inhibitor (11) or substrate (16-20). For example, one often finds statements such as the following in the current literature on P450 2D6: “All of our data [on propranolol derivatives] are consistent with the generally accepted model for binding of CYP2D6 [P450 2D6] substrates via formation of an ion pair of the protonated amine with the carboxylate anion of Asp301 in the enzyme active site and subsequent oxidation at a distant site in the molecule” and “The presence of basic nitrogens is a common requirement of high-affinity substrates...” (21). One problem with models of the P450 2D6 structure (and pharmacophore models) was that they did not rationalize N-dealkylation reactions, such as those observed with deprenyl (22) and other drugs (23). One explanation for this dilemma was that the amine docks with Asp301 but is rapidly deprotonated to yield an unchanged substrate for 1-electron oxidation (22). Subsequently, models were expanded to accommodate this deficiency, with aromatic residues involved (24, 25). Experimental studies have provided only limited and rather nongeneralized support for roles of these suggested Phe residues (Phe481, Phe483) (25, 26). Further, reports have appeared of catalytic activity of P450 2D6 toward peptidometics devoid of a basic nitrogen (27) and even steroids (28-30). However, these reports have been largely ignored in consideration of P450 2D6 substrates (21). We identified a ligand, spirosulfonamide, devoid of a basic nitrogen but having high affinity for P450 2D6, raising further concerns about the reliability of P450 2D6 models based on a critical electrostatic interaction with Asp301. Spirosulfonamide was also a substrate for P450 2D6 (Scheme 1). Neutral Asp301 mutants (Asn, Ser, Gly) showed relatively
high affinity for spirosulfonamide (Kd 10-6 M). The compromised catalytic activity of these mutants for spirosulfonamide hydroxylation parallels that observed with the basic substrate bufuralol, and attenuated electrostatic interaction of substrate cannot be used as an explanation of the role of Asp301. P450 2D6 also bound and oxidized analogues of spirosulfonamide in which the sulfonamide group was replaced by other groups. We also estimated the affinity of P450 2D6 for other substrates (ritonavir, progesterone, and testosterone) and report binding affinities as good or better than for many classic ligands with basic nitrogen. EXPERIMENTAL PROCEDURES Chemicals. Spirosulfonamide (1), syn and anti HOspirosulfonamides, and keto spirosulfonamide were synthesized according to the procedures outlined in the Supporting Information (31). Other spirosulfonamide analogues, compounds 2-6, were experimental drugs that were prepared at Merck-Frosst using synthetic procedures analogous to that used for spirosulfonamide. Ritonavir was a gift of Abbott Laboratories (North Chicago, IL). Bufuralol hydrochloride was a gift of Hoffman-LaRoche (Nutley, NJ). Deuterated NMR solvents were obtained from C/D/N Isotopes (Montreal, Quebec, Canada) and Cambridge Isotope Laboratories (Andover, MA). Other chemicals were obtained from commercial sources and were of reagent, analytical, or HPLC grade, as appropriate. Microsomal Fractions and Enzymes. Human liver samples were obtained from organ donors through Tennessee Donor Services (Nashville, TN), stored at -80 °C, and used to prepare microsomes (33). In some of the preliminary experiments with spirosulfonamide, microsomes prepared from insect cells infected with baculovirus vectors were used as sources of human P450s, NADPH-P450 reductase, and, in the case of P450 3A4, cytochrome b5 [Gentest Co., Woburn, MA (now BD Bioscience)]. The baculovirus microsome products used were those designated control, CYP2C8, CYP2C9-arg, CYP2C19, CYP2D6, and CYP3A4 by the supplier. The cDNA sequence of P450 2D6 (DB6) (34) was modified by polymerase chain reaction to incorporate a
P450 2D6 Oxidations C-terminal (His)5 peptide (35).2 The modified protein was expressed in Escherichia coli (MV1304 strain) in the presence of 1.0 mg of chloramphenicol L-1 (38). Expressed P450s were purified by Ni2+-immobilized metal affinity chromatography as described (35, 39). Rat NADPH-P450 reductase was expressed in E. coli (TOPP 3 strain) utilizing plasmid pOR263 (40) and was purified as described (39, 41). Biotransformations of Spirosulfonamide (1). Incubations and metabolite characterization by mass spectrometry and NMR are completely detailed in the Supporting Information. Briefly, hepatocyte and preparative microsomal incubations were conducted under standard conditions (42). Initial mass spectral characterization was conducted by capillary HPLC continuous-flow liquid secondary ion MS analysis on a JEOL HX-110A mass spectrometer (43). Isolation of the major metabolite (M1, subsequently identified as syn HO-spirosulfonamide) was performed using a combination of preparative HPLC and solid-phase extraction procedures similar to those described previously (43). Conventional 1H NMR spectra for the isolated metabolite were acquired on a Bruker AMX 500 spectrometer (Bruker Spectrospin, Fremont, CA) using a combination of homonuclear coupling, 1D NOE, and 2D DFQ-COSY and HMQC experiments (44). Biotransformations of Spiromethyl Sulfone (2). Preparative scale incubations and HPLC and mass spectrometry of the product were done as in the case of 1 (see Supporting Information). HPLC-NMR spectra of the methyl sulfone product were acquired on a Varian Inova 600 spectrometer (Varian NMR Systems, Palo Alto, CA) equipped with a Varian HPLC system (Varian Chromatography Systems, Walnut Creek, CA) using a COSY experiment and by comparison to the parent compound. Full details can be found in the Supporting Information. Determination of P450s InVolVed in Spirosulfonamide Oxidation. Preliminary studies involved incubations with microsomes of baculovirus-infected insect cells expressing specific P450s. The incubations were conducted under standard oxidative conditions using 50 pmol of P450 (as stated by the vendor), an NADPH-generating system, and 20 µM spirosulfonamide (dissolved in DMSO, to 1% final volume) in a final total volume of 250 µL. The incubations were conducted for 60 min at 37 °C and quenched by the addition of an equal volume of CH3CN. Analysis was conducted by HPLC under conditions similar to those described below. Binding of Spirosulfonamide and Analogues to P450 2D6. Heme perturbation spectra were recorded with purified P450 2D6 enzyme preparations (2-5 µM, in 0.10 M potassium phosphate buffer, pH 7.4) using stocks of spirosulfonamide dissolved in CH3CN (45, 46). Absorbance spectra were recorded on Aminco DW2a/OLIS and Cary 14/OLIS instruments (On-Line Instrument Systems, Bogart, GA). Other work has indicated that neither DMSO nor CH3CN hinder P450 2D6 reactions when added at concentrations up to 1.0% (v/v) (47). The analogues were also dissolved in CH3CN and added to P450 2D6 to generate type I perturbation spectra. 2 The cDNA we expressed in previous work (34) had been obtained with a change (to Met) at codon 374 that appears to be the result of a cloning artifact (36, 37). The change M374V was made, and the resulting sequence is that generally agreed to be the most common allele (6).
Biochemistry, Vol. 41, No. 36, 2002 11027 Values of A390-A420 were plotted vs the ligand concentration to estimated Ks, a spectrally estimated dissociation constant (45), using Graphpad Prism software (Graphpad, San Diego, CA). Hyperbolic fitting was used with high Ks values, and a quadratic expression was used when Ks was near the P450 concentration or the program DynaFit was applied (see Supporting Information) (48). Some of the compounds tested as ligands did not elicit perturbation spectra, and an alternate approach was used. Bufuralol elicits type I binding spectra (Ks ) 6.2 ( µM). Two P450 samples were balanced against each other as before in the Cary 14/OLIS spectrophotometer, and then (()bufuralol was added to the sample cuvette to 8 µM, a value near the Ks, to induce a type I difference spectrum. Subsequently, varying amounts of CH3CN solutions of ritonavir, progesterone, or testosterone were added to the cuvette to attenuate the difference spectrum. The competition between the “second” ligand and bufuralol was assumed to be competitive, and plots of A390-A420 vs the concentration of the second ligand were fit to the model
bufuralol + P450 2D6 a bufuralol‚P450 2D6 second ligand + P450 2D6 a second ligand‚P450 2D6
Ks Kd
using the software program DynaFit (48) operating on an Apple Macintosh G4 computer, with Ks ) 6.2 µM (determined experimentally). The system yielded fits for Kd and for ∆A390-420,max, along with error estimates. Oxidation of Spirosulfonamide and Analogues by P450 2D6. Standard oxidation reactions were conducted in 1.0 mL final volumes of 0.10 M potassium phosphate buffer (pH 7.4) containing either 0.05 or 0.10 nmol of P450 2D6, a 3-fold molar excess of NADPH-P450 reductase, and freshly sonicated L-R-dilauroyl-sn-glycero-3-phosphocholine (Sigma Chemical Co., St. Louis, MO) (30 µg). [In cases where human liver microsomes were used instead of purified P450 2D6, 0.75 mg of microsomal protein was substituted for the above components, in the same buffer, in the absence or presence of 2.0 µM quinidine (Aldrich Chemical Co., Milwaukee, WI), in triplicate.] Spirosulfonamide and its analogues were dissolved in CH3CN to prepare stock solutions [final concentration e1% (v/v) in the reactions]. The mixtures were incubated for 3 min at 37 °C, and the reactions were started by the addition of an NADPHgenerating system (33). Incubations were carried out for 5 min at 37 °C. Reactions were stopped by the addition of 2.0 mL of CH2Cl2 and chilled on ice, followed by mixing using a vortex device. The layers were separated by centrifugation [(3 × 104)g, 10 min]. A 1.6 mL aliquot of each CH2Cl2 (lower) layer was removed with a glass pipet, transferred to a 2.0 mL Reacti-vial (Pierce Chemical Co., Rockford, IL), and concentrated to dryness under an N2 stream. The residues were dissolved in 100 µL of CH3CN, and 50 µL aliquots were analyzed by HPLC using a 6.2 × 80 mm Zorbax octylsilane (C8) HPLC column (3 µm; Mac-Mod, Chadds Ford, PA). Solvent A was 1.0 mM HClO4 and solvent B was CH3CN-H2O (9:1 v/v). The elution program consisted of a linear gradient increasing from 5% to 75% solvent B (v/v) over the following 20 min (flow rate 2.5 mL min-1,
11028 Biochemistry, Vol. 41, No. 36, 2002 A245). The alcohols (syn and anti HO-spirosulfonamide) and ketone (keto spirosulfonamide) were separated with baseline resolution (vide infra).
Guengerich et al. Scheme 2: Analogues of Spirosulfonamide and Other P450 2D6 Ligandsa
RESULTS Characterization of P450-Catalyzed Oxidations of Spirosulfonamide in LiVer Microsomes and BaculoVirus Recombinant P450 2D6. Preliminary studies indicated that spirosulfonamide, devoid of a basic nitrogen atom, might be preferentially oxidized by P450 2D6. When spirosulfonamide was incubated with baculovirus-generated microsomes containing individual human P450s (50 pmol of P450, 20 µM spirosulfonamide, 37 °C, 60 min), the decreases in residual spirosulfonamide with each P450 were as follows: 2D6, 84%; 3A4, 43%; 2C8, 20%; 2C9,
