Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active-site residues in quinidine binding

Lesley A. McLaughlin, Mark J.I. Paine, Carol A. Kemp, Jean Didier Maréchal, Jack U. Flanagan, Clive J. Ward, Michael J. Sutcliffe, Gordon C.K. Roberts, C. Roland Wolf

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Abstract

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.

Original languageEnglish
Pages (from-to)38617-38624
Number of pages8
JournalJournal of Biological Chemistry
Volume280
Issue number46
DOIs
Publication statusPublished - Sep 2005

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Cytochrome P-450 CYP2D6
Quinidine
Catalytic Domain
Dextromethorphan
Enzymes
Hydroxylation
Substitution reactions
Metabolites
Amides
Alanine
Mutation

Cite this

McLaughlin, Lesley A. ; Paine, Mark J.I. ; Kemp, Carol A. ; Maréchal, Jean Didier ; Flanagan, Jack U. ; Ward, Clive J. ; Sutcliffe, Michael J. ; Roberts, Gordon C.K. ; Wolf, C. Roland. / Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active-site residues in quinidine binding. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 46. pp. 38617-38624.
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title = "Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active-site residues in quinidine binding",
abstract = "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., Mar{\'e}chal, 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.",
author = "McLaughlin, {Lesley A.} and Paine, {Mark J.I.} and Kemp, {Carol A.} and Mar{\'e}chal, {Jean Didier} and Flanagan, {Jack U.} and Ward, {Clive J.} and Sutcliffe, {Michael J.} and Roberts, {Gordon C.K.} and Wolf, {C. Roland}",
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McLaughlin, LA, Paine, MJI, Kemp, CA, Maréchal, JD, Flanagan, JU, Ward, CJ, Sutcliffe, MJ, Roberts, GCK & Wolf, CR 2005, 'Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active-site residues in quinidine binding', Journal of Biological Chemistry, vol. 280, no. 46, pp. 38617-38624. https://doi.org/10.1074/jbc.M505974200

Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active-site residues in quinidine binding. / McLaughlin, Lesley A.; Paine, Mark J.I.; Kemp, Carol A.; Maréchal, Jean Didier; Flanagan, Jack U.; Ward, Clive J.; Sutcliffe, Michael J.; Roberts, Gordon C.K.; Wolf, C. Roland.

In: Journal of Biological Chemistry, Vol. 280, No. 46, 09.2005, p. 38617-38624.

Research output: Contribution to journalArticle

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T1 - Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active-site residues in quinidine binding

AU - McLaughlin, Lesley A.

AU - Paine, Mark J.I.

AU - Kemp, Carol A.

AU - Maréchal, Jean Didier

AU - Flanagan, Jack U.

AU - Ward, Clive J.

AU - Sutcliffe, Michael J.

AU - Roberts, Gordon C.K.

AU - Wolf, C. Roland

PY - 2005/9

Y1 - 2005/9

N2 - 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.

AB - 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.

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