Why Is Quinidine an Inhibitor of Cytochrome P450 2D6?
2005; Elsevier BV; Volume: 280; Issue: 46 Linguagem: Inglês
10.1074/jbc.m505974200
ISSN1083-351X
AutoresLesley A. McLaughlin, Mark J. I. Paine, Carol A. Kemp, Jean‐Didier Maréchal, Jack U. Flanagan, C. J. Ward, Michael J. Sutcliffe, Gordon C. K. Roberts, C. Roland Wolf,
Tópico(s)Cancer therapeutics and mechanisms
ResumoWe have previously shown that Phe120, Glu216, and Asp301 in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolizing enzyme (Paine, M. J., McLaughlin, L. A., Flanagan, J. U., Kemp, C. A., Sutcliffe, M. J., Roberts, G. C., and Wolf, C. R. (2003) J. Biol. Chem. 278, 4021–4027 and Flanagan, J. U., Maréchal, J.-D., Ward, R., Kemp, C. A., McLaughlin, L. A., Sutcliffe, M. J., Roberts, G. C., Paine, M. J., and Wolf, C. R. (2004) Biochem. J. 380, 353–360). We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge at either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (Kd) for quinidine binding to the wild-type enzyme and the E216D and D301E mutants were 0.25–0.50 μm. The amide substitution of Glu216 or Asp301 resulted in 30–64-fold increases in the Kd for quinidine. The double mutant E216Q/D301Q showed the largest decrease in quinidine affinity, with a Kd of 65 μm. Alanine substitution of Phe120, Phe481,or Phe483 had only a minor effect on the inhibition of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolize quinidine. E216F produced O-demethylated quinidine, and F120A and E216Q/D301Q produced both O-demethylated quinidine and 3-hydroxyquinidine metabolites. Homology modeling and molecular docking were used to predict the modes of quinidine binding to the wild-type and mutant enzymes; these were able to rationalize the experimental observations. We have previously shown that Phe120, Glu216, and Asp301 in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolizing enzyme (Paine, M. J., McLaughlin, L. A., Flanagan, J. U., Kemp, C. A., Sutcliffe, M. J., Roberts, G. C., and Wolf, C. R. (2003) J. Biol. Chem. 278, 4021–4027 and Flanagan, J. U., Maréchal, J.-D., Ward, R., Kemp, C. A., McLaughlin, L. A., Sutcliffe, M. J., Roberts, G. C., Paine, M. J., and Wolf, C. R. (2004) Biochem. J. 380, 353–360). We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge at either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (Kd) for quinidine binding to the wild-type enzyme and the E216D and D301E mutants were 0.25–0.50 μm. The amide substitution of Glu216 or Asp301 resulted in 30–64-fold increases in the Kd for quinidine. The double mutant E216Q/D301Q showed the largest decrease in quinidine affinity, with a Kd of 65 μm. Alanine substitution of Phe120, Phe481,or Phe483 had only a minor effect on the inhibition of bufuralol 1′-hydroxylation and dextromethorphan O-demethylation and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolize quinidine. E216F produced O-demethylated quinidine, and F120A and E216Q/D301Q produced both O-demethylated quinidine and 3-hydroxyquinidine metabolites. Homology modeling and molecular docking were used to predict the modes of quinidine binding to the wild-type and mutant enzymes; these were able to rationalize the experimental observations. Human cytochrome P450 2D6 (CYP2D6) 4The abbreviations used are: CYP2D6, cytochrome P450 2D6; HPLC, high performance liquid chromatography.4The abbreviations used are: CYP2D6, cytochrome P450 2D6; HPLC, high performance liquid chromatography. plays a central role in drug metabolism, metabolizing >30% of the most commonly prescribed drugs (1.Jones B.C. Tyman C.A. Smith D.A. Xenobiotica. 1997; 27: 1025-1037Crossref PubMed Scopus (32) Google Scholar). The CYP2D6 gene is highly polymorphic, leading to wide interindividual and ethnic differences in CYP2D6-mediated drug metabolism (2.Mahgoub A. Idle J.R. Dring L.G. Lancaster R. Smith R.L. Lancet. 1977; 2: 584-586Abstract PubMed Scopus (1005) Google Scholar, 3.Eichelbaum M. Spannbrucker N. Steincke B. Dengler H.J. Eur. J. Clin. Pharmacol. 1979; 16: 183-187Crossref PubMed Scopus (598) Google Scholar, 4.Daly A.K. Brockmoller J. Broly F. Eichelbaum M. Evans W.E. Gonzalez F.J. Huang J.D. Idle J.R. Ingelman-Sundberg M. Ishizaki T. Jacqz-Aigrain E. Meyer U.A. Nebert D.W. Steen V.M. Wolf C.R. Zanger U.M. Pharmacogenetics. 1996; 6: 193-201Crossref PubMed Scopus (452) Google Scholar). Cytochrome P450-drug and drug-drug interactions involving CYP2D6 ligands are thus a prime consideration in the development of new drugs, emphasizing the importance of a detailed understanding of the factors that govern the substrate specificity of this enzyme. Quinidine is not metabolized by CYP2D6 and has long been established as a potent competitive inhibitor of the enzyme (5.von Bahr C. Spina E. Birgersson C. Ericsson O. Goransson M. Henthorn T. Sjoqvist F. Biochem. Pharmacol. 1985; 34: 2501-2505Crossref PubMed Scopus (93) Google Scholar, 6.Guengerich F.P. Miller G.P. Hanna I.H. Sato H. Martin M.V. J. Biol. Chem. 2002; 277: 33711-33719Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 7.Guengerich F.P. Muller-Enoch D. Blair I.A. Mol. Pharmacol. 1986; 30: 287-295PubMed Google Scholar, 8.Otton S.V. Brinn R.U. Gram L.F. Drug Metab. Dispos. 1988; 16: 15-17PubMed Google Scholar, 9.Branch R.A. Adedoyin A. Frye R.F. Wilson J.W. Romkes M. Clin. Pharmacol. Ther. 2000; 68: 401-411Crossref PubMed Scopus (96) Google Scholar). The fact that quinidine is an inhibitor rather than a substrate is intriguing because it produces a classical type I binding spectrum with CYP2D6 (10.Hayhurst G.P. Harlow J. Chowdry J. Gross E. Hilton E. Lennard M.S. Tucker G.T. Ellis S.W. Biochem. J. 2001; 355: 373-379Crossref PubMed Scopus (41) Google Scholar) that is usually associated with the binding of substrate molecules (11.Schenkman J.B. Sligar S.G. Cinti D.L. Pharmacol. Ther. 1981; 12: 43-71Crossref PubMed Scopus (168) Google Scholar). In addition, quinidine possesses a number of features normally associated with CYP2D6 substrates, including a basic nitrogen atom, a flat hydrophobic region, and a negative molecular electrostatic potential (12.Strobl G.R. von Kruedener S. Stockigt J. Guengerich F.P. Wolff T. J. Med. Chem. 1993; 36: 1136-1145Crossref PubMed Scopus (160) Google Scholar). Studies of the relationship between structure and inhibitory activity for quinidine and its (less potent) stereoisomer quinine have been reported (13.Hutzler J.M. Walker G.S. Wienkers L.C. Chem. Res. Toxicol. 2003; 16: 450-459Crossref PubMed Scopus (45) Google Scholar), but the protein-ligand interactions that are responsible for the fact that quinidine can bind tightly, but not in an orientation favorable for catalysis, have not hitherto been established. Recent models of the active site of CYP2D6 (e.g. Ref. 14.Kirton S.B. Kemp C.A. Tomkinson N.P. St. Gallay S. Sutcliffe M.J. Proteins. 2002; 49: 216-231Crossref PubMed Scopus (64) Google Scholar) suggest that two carboxylate groups (at Glu216 and Asp301) may play key roles in the recognition of substrates containing a basic nitrogen atom, and support for this has come from mutagenesis experiments (15.Ellis S.W. Hayhurst G.P. Smith G. Lightfoot T. Wong M.M. Simula A.P. Ackland M.J. Sternberg M.J. Lennard M.S. Tucker G.T. Wolf C.R. J. Biol. Chem. 1995; 270: 29055-29058Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 16.Guengerich F.P. Hanna I.H. Martin M.V. Gillam E.M. Biochemistry. 2003; 42: 1245-1253Crossref PubMed Scopus (64) Google Scholar, 17.Paine M.J. McLaughlin L.A. Flanagan J.U. Kemp C.A. Sutcliffe M.J. Roberts G.C. Wolf C.R. J. Biol. Chem. 2003; 278: 4021-4027Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). It has also been suggested that the aromatic residues Phe120, Phe481, and Phe483 may have roles in substrate binding through π-interactions with the planar hydrophobic regions common to many CYP2D6 substrates (10.Hayhurst G.P. Harlow J. Chowdry J. Gross E. Hilton E. Lennard M.S. Tucker G.T. Ellis S.W. Biochem. J. 2001; 355: 373-379Crossref PubMed Scopus (41) Google Scholar, 14.Kirton S.B. Kemp C.A. Tomkinson N.P. St. Gallay S. Sutcliffe M.J. Proteins. 2002; 49: 216-231Crossref PubMed Scopus (64) Google Scholar, 18.Smith G. Modi S. Pillai I. Lian L.Y. Sutcliffe M.J. Pritchard M.P. Friedberg T. Roberts G.C. Wolf C.R. Biochem. J. 1998; 331: 783-792Crossref PubMed Scopus (58) Google Scholar, 19.Venhorst J. ter Laak A.M. Commandeur J.N. Funae Y. Hiroi T. Vermeulen N.P. J. Med. Chem. 2003; 46: 74-86Crossref PubMed Scopus (99) Google Scholar). Here, we describe studies of a series of mutants of these five residues aimed at investigating their role in quinidine binding and in determining whether quinidine is a substrate or an inhibitor of this important drug-metabolizing enzyme. Materials—Terrific broth, chloramphenicol, dithiothreitol, glucose 6-phosphate, NADP+, phenylmethylsulfonyl fluoride, sodium dithionite, cytochrome c, and quinidine were purchased from Sigma (Poole, UK). Ampicillin was obtained from Beecham Research (Welwyn Garden City, UK), isopropyl β-d-thiogalactopyranoside and δ-aminolevulinic acid from Melford Laboratories (Ipswich, UK), and glucose-6-phosphate dehydrogenase (type VII) from Roche Applied Science (Lewes, UK). HPLC-grade solvents were from Rathburn Chemicals (Walkerburn, UK), and Agilent HPLC columns were from Crawford Scientific (Lanarkshire, Scotland, UK). DNA-modifying enzymes were obtained from Invitrogen (Paisley, UK) and Promega Corp. (Southampton, UK). Bufuralol, 1′-hydroxybufuralol, and (3S)-3′-hydroxyquinidine were purchased from Ultrafine Chemicals (Manchester, UK). Quinidine N-oxide was a kind gift from Merck Sharp and Dohme (Harlow, UK). All other chemicals were from BDH (Poole). Library efficient competent Escherichia coli strain JM109 was purchased from Promega Corp. Mutagenesis and Expression in E. coli—The Glu216 and Asp301 mutants of CYP2D6 used in this study were constructed and expressed in E. coli along with human cytochrome P450 reductase as described previously (17.Paine M.J. McLaughlin L.A. Flanagan J.U. Kemp C.A. Sutcliffe M.J. Roberts G.C. Wolf C.R. J. Biol. Chem. 2003; 278: 4021-4027Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). To obtain the remaining mutants, site-directed mutagenesis was performed following the single-stranded DNA template method (20.Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4540) Google Scholar) using pB81 as a template and the dut- ung- E. coli strain CJ236 along with an appropriate mutagenic oligonucleotide: F120A, 3′-ata gcg cgc cag agc cac ccc ttg gga-5′; F481A, 3′-cac cag gaa agc agc gac acc atg gtg-5′; and F483A, 3′-cac cag gaa agc agc gac acc atg gtg-5′. Note that the oligonucleotide sequences are reverse-complemented. Once the presence of the desired mutation was confirmed by automated DNA sequencing, the mutants were coexpressed with human cytochrome P450 reductase as described above. Varying quantities of cytochrome P420, reflecting different degrees of stability in the presence of dithionite, were observed for the different mutants. E216Q, D301E, D301Q, F481A, and F483A had P450:P420 peak area ratios of ∼9:1; E216N, E216Q/D301Q, and F120A had ratios of ∼1:1; and E216F, E216A, and D301N had ratios of ∼1:5. Quinidine Inhibition of Bufuralol 1′-Hydroxylation and Dextromethorphan O-Demethylation—Incubations were carried out in triplicate at 37 °C with shaking in 300 μl of 50 mm potassium phosphate (pH 7.4) containing E. coli membranes equivalent to 10 pmol of CYP2D6 (wild-type or mutant), quinidine (0, 1, 10, or 100 μm), an NADPH-generating system (comprising 5 mm glucose 6-phosphate, 1 unit of glucose-6-phosphate dehydrogenase, and 1 mm NADP+), and bufuralol or dextromethorphan at concentrations equivalent to the Km of each sample. The specific substrate concentrations used were as follows: CYP2D6, 1.1 μm bufuralol and 2.6 μm dextromethorphan; E216Q, 188 μm bufuralol and 51 μm dextromethorphan; E216D, 6 μm bufuralol and 13 μm dextromethorphan; E216F, 117 μm bufuralol and 30 μm dextromethorphan; E216A, 162 μm bufuralol and 63 μm dextromethorphan; E216K, 187 μm bufuralol and 312 μm dextromethorphan; D301E, 2 μm bufuralol and 11 μm dextromethorphan; D301Q, 142 μm bufuralol and 200 μm dextromethorphan; D301N, 160 μm bufuralol and 3598 μm dextromethorphan; E216Q/D301Q, 522 μm bufuralol and 438 μm dextromethorphan; F120A, 2.7 μm bufuralol and 1 μm dextromethorphan; F481A, 10 μm bufuralol and 11 μm dextromethorphan; F483A, 7.1 μm bufuralol and 9.5 μm dextromethorphan. After a 3-min preincubation at 37 °C, reactions were initiated by the addition of the NADPH-generating system and were allowed to proceed for 6 min before being stopped by the addition of 15 μl of 60% perchloric acid. 100-μl aliquots of the reaction supernatant were used for HPLC, separating the bufuralol and dextromethorphan metabolites as described previously (17.Paine M.J. McLaughlin L.A. Flanagan J.U. Kemp C.A. Sutcliffe M.J. Roberts G.C. Wolf C.R. J. Biol. Chem. 2003; 278: 4021-4027Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), using a Hewlett-Packard 1100 HPLC system and ChemStation software. As reported previously (17.Paine M.J. McLaughlin L.A. Flanagan J.U. Kemp C.A. Sutcliffe M.J. Roberts G.C. Wolf C.R. J. Biol. Chem. 2003; 278: 4021-4027Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 21.Flanagan J.U. Maréchal J.-D. Ward R. Kemp C.A. McLaughlin L.A. Sutcliffe M.J. Roberts G.C. Paine M.J. Wolf C.R. Biochem. J. 2004; 380: 353-360Crossref PubMed Google Scholar), the only significant decrease in activity for the mutants relative to the wild-type enzyme in the absence of inhibitor was for dextromethorphan O-demethylation by E216Q (∼4-fold) and E216K (∼10-fold). Quinidine Metabolism—To investigate quinidine metabolism, reaction mixtures consisted of 50 mm potassium phosphate (pH 7.4) containing 100 μm quinidine, E. coli membranes equivalent to 10 pmol of CYP2D6 (wild-type or mutant) and an NADPH-generating system (as described above) in a total volume of 200 μl. After a 3-min preincubation at 37 °C, reactions were initiated by the addition of the NADPH-generating system and incubated for an additional 15 min before being stopped with 100 μl of ice-cold methanol. Samples were left on ice for 10 min prior to centrifugation at 16,100 × g for 10 min. Metabolites were separated by HPLC using a Hypersil BDS-C18 column (5 μm, 250 × 4.6 mm) at a flow rate of 1 ml/min. Mobile phases of acetonitrile (solvent A) and sodium perchlorate/perchloric acid (14.05 g of sodium perchlorate and 1.6 ml of 60% perchloric acid dissolved in 5 liters of distilled H2O; solvent B) were mixed at a constant ratio of 15% solvent A to 85% solvent B (v/v) for the first 5 min, and then a linear gradient was applied over 4 min, ending at 31% solvent A to 69% solvent B (v/v), which was maintained for an additional 7 min. The retention times of (3S)-3′-hydroxyquinidine and quinidine N-oxide were established using authentic metabolite standards, with fluorescence detection at λex = 252 nm and λem = 302 nm. Identification of the Novel Quinidine Metabolite—Further analysis of the novel quinidine metabolite was undertaken by HPLC with mass spectrometric detection. 25 μl of the stopped incubation mixture was separated on a Luna C18 column (3 μm, 150 × 2 mm; Phenomenex, Cheshire, UK) with a linear gradient of 5 mm ammonium formate (pH 3.5) (solvent A) and acetonitrile (solvent B) delivered by a Waters 2795 separations module. The gradient ran from 5 to 30% solvent A over 20 min at a flow rate of 200 μl/min before returning to the starting conditions. The eluent was introduced into the source of a Quattro micro mass spectrometer (Micromass, Manchester, UK) and was ionized by electrospray ionization in the positive ion mode. The main parameters were as follows: capillary voltage, 3.3 kV; cone voltage, 30 V; source and desolvation temperatures, 100 and 300 °C, respectively; and cone and desolvation nitrogen gas flows, 90 and 300 liters/h-1, respectively. In collision-induced dissociation experiments, argon was used as the collision gas with a collision energy of 30 eV. Data were acquired and analyzed by MassLynx software. Quinidine Binding—Quinidine binding was measured by optical difference spectroscopy of E. coli membranes containing CYP2D6 and NADPH-cytochrome P450 reductase (EC 1.6.2.4) using a Cary 4000 UV-visible spectrophotometer. E. coli membranes containing wild-type or mutant CYP2D6 enzymes were diluted in 100 mm potassium phosphate buffer (pH 7.4) to a final concentration of 0.5 μm cytochrome P450 and split into two matched black-walled quartz cuvettes. After running a base line, 1-μl aliquots of quinidine dissolved in deionized water were added to the sample cuvette, and equal volumes of water were added to the reference cuvette. The samples were left for 2 min between additions to equilibrate, and the difference spectrum was then run between 360 and 460 nm. The final volume of additions was kept to 95% inhibition of both bufuralol 1′-hydroxylation and dextromethorphan O-demethylation. It is clear that the negative charges at Glu216 and Asp301 are important for the inhibitory effect of quinidine. The conservative substitutions E216D and D301E showed behavior similar to that of the wild-type enzyme, with >90% inhibition by 1 μm quinidine, whereas enzymes with non-conservative replacements were at least 50% active at 10 μm quinidine. The double mutant E216Q/D301Q, with complete removal of the charge but not the polarity, was found to be strikingly insensitive to inhibition by quinidine, retaining 80% of its bufuralol 1′-hydroxylase activity and 85% of its dextromethorphan O-demethylase activity in the presence of 100 μm quinidine. By contrast, alanine substitution of the aromatic side chain of Phe120, Phe481, or Phe483 had only a minor effect on the inhibition of catalytic activity by quinidine. The effects of the mutations were generally similar for quinidine inhibition of both bufuralol 1′-hydroxylase and dextromethorphan O-demethylase activities, although for most of the mutants, quinidine was found to be a somewhat better inhibitor with respect to dextromethorphan compared with bufuralol. These observations suggest that the negative charges at Glu216 and Asp301, but not the aromatic rings of the three phenylalanine residues, are important for the binding of quinidine; this is broadly consistent with the effects of mutation of these residues on the Km values of substrates containing a basic nitrogen atom (10.Hayhurst G.P. Harlow J. Chowdry J. Gross E. Hilton E. Lennard M.S. Tucker G.T. Ellis S.W. Biochem. J. 2001; 355: 373-379Crossref PubMed Scopus (41) Google Scholar, 16.Guengerich F.P. Hanna I.H. Martin M.V. Gillam E.M. Biochemistry. 2003; 42: 1245-1253Crossref PubMed Scopus (64) Google Scholar, 17.Paine M.J. McLaughlin L.A. Flanagan J.U. Kemp C.A. Sutcliffe M.J. Roberts G.C. Wolf C.R. J. Biol. Chem. 2003; 278: 4021-4027Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 18.Smith G. Modi S. Pillai I. Lian L.Y. Sutcliffe M.J. Pritchard M.P. Friedberg T. Roberts G.C. Wolf C.R. Biochem. J. 1998; 331: 783-792Crossref PubMed Scopus (58) Google Scholar, 21.Flanagan J.U. Maréchal J.-D. Ward R. Kemp C.A. McLaughlin L.A. Sutcliffe M.J. Roberts G.C. Paine M.J. Wolf C.R. Biochem. J. 2004; 380: 353-360Crossref PubMed Google Scholar, 32.Keizers P.H. Lussenburg B.M. de Graaf C. Mentink L.M. Vermeulen N.P. Commandeur J.N. Biochem. Pharmacol. 2004; 68: 2263-2271Crossref PubMed Scopus (46) Google Scholar). Quinidine Binding to the Glu216 and Asp301 Mutants—The effects of the mutations on quinidine binding were determined directly by measuring optical difference spectra upon addition of quinidine to bacterial membranes expressing cytochrome P450. Wild-type CYP2D6 showed a type I binding spectrum upon quinidine addition, with λmax and λmin of ∼420 and ∼390 nm, respectively (Fig. 2A), characteristic of the change from a low to high spin state of the ferric iron that is usually associated with the binding of substrate molecules (11.Schenkman J.B. Sligar S.G. Cinti D.L. Pharmacol. Ther. 1981; 12: 43-71Crossref PubMed Scopus (168) Google Scholar). None of the mutants showed evidence of a type II spectrum, characteristic of direct coordination to the heme iron. The majority showed type I difference spectra (Fig. 2A) or variations thereof (Fig. 2C), but three showed a different form of spectrum with an increase in absorbance at shorter wavelengths (Fig. 2B), suggesting either a change in the heme environment or light scattering from membrane or protein aggregation. 5The difference spectra reported here for quinidine binding to the E216Q and E216A mutants appear to be different from those reported by Guengerich et al. (16.Guengerich F.P. Hanna I.H. Martin M.V. Gillam E.M. Biochemistry. 2003; 42: 1245-1253Crossref PubMed Scopus (64) Google
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