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J. Biol. Chem., Vol. 280, Issue 46, 38617-38624, November 18, 2005

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Why Is Quinidine an Inhibitor of Cytochrome P450 2D6?

THE ROLE OF KEY ACTIVE-SITE RESIDUES IN QUINIDINE BINDING^*

Lesley A. McLaughlin1, Mark J. I. Paine1, Carol A. Kemp, Jean-Didier Maréchal¶, Jack U. Flanagan, Clive J. Ward, Michael J. Sutcliffe¶, Gordon C. K. Roberts2, and C. Roland Wolf3

From the Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom and the Departments of Biochemistry and ^¶Chemistry, University of Leicester, Leicester LE1 7RH, United Kingdom

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 (K_d) 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 K_d for quinidine. The double mutant E216Q/D301Q showed the largest decrease in quinidine affinity, with a K_d 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.

Received for publication, June 1, 2005 , and in revised form, August 25, 2005.

^* This work was supported by the Drug Metabolism Consortium (AstraZeneca, Aventis, Boehringer Ingelheim, Celltech Chiroscience, GlaxoSmithKline, Hoffmann-La Roche, Johnston and Johnston Pharmaceuticals, Merck Sharp and Dohme, Novartis, Novo Nordisk, Pfizer, Pharmacia Corp., and Wyeth). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to the work.

² To whom correspondence may be addressed: Dept. of Biochemistry, University of Leicester, Henry Wellcome Bldg., P. O. Box 138, Lancaster Rd., Leicester LE1 9HN, UK. Tel.: 44-116-229-7100; Fax: 44-116-229-7053; E-mail: gcr{at}le.ac.uk. ³ To whom correspondence may be addressed: Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, UK. Tel.: 44-1382-632-621; Fax: 44-1382-668-278; E-mail: Roland.Wolf{at}cancer.org.uk.

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