Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active-site residues in quinidine binding.
We have previously shown that Phe(120), Glu(216), and Asp(301) 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 microm. The amide substitution of Glu(216) or Asp(301) 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 microm. Alanine substitution of Phe(120), Phe(481),or Phe(483) 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.
McLaughlin
Lesley A
LA
Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.
Paine
Mark J I
MJ
Kemp
Carol A
CA
Maréchal
Jean-Didier
JD
Flanagan
Jack U
JU
Ward
Clive J
CJ
Sutcliffe
Michael J
MJ
Roberts
Gordon C K
GC
Wolf
C Roland
CR
D000409:Alanine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry* / Q000235:genetics; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D010090:Oxidoreductases, O-Demethylating / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011802:Quinidine / Q000737:chemistry / Q000494:pharmacology*; D013053:Spectrophotometry; D013379:Substrate Specificity
D065690:Cytochrome P-450 CYP2D6 Inhibitors; D001224:Aspartic Acid; D018698:Glutamic Acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010090:Oxidoreductases, O-Demethylating; C052861:dextromethorphan O-demethylase; D019389:Cytochrome P-450 CYP2D6; C043872:bufuralol 1'-hydroxylase; D011802:Quinidine; D000409:Alanine