CYP2D6-CYP2C9 protein-protein interactions and isoform-selective effects on substrate binding and catalysis.
Cytochrome P450 (P450) protein-protein interactions have been observed with various in vitro systems. It is interesting to note that these interactions seem to be isoform-dependent, with some combinations producing no effect and others producing increased or decreased catalytic activity. With some exceptions, most of the work to date has involved P450s from rabbit, rat, and other animal species, with few studies including human P450s. In the studies presented herein, the interactions of two key drug-metabolizing enzymes, CYP2C9 and CYP2D6, were analyzed in a purified, reconstituted enzyme system for changes in both substrate-binding affinity and rates of catalysis. In addition, an extensive study was conducted as to the "order of mixing" for the reconstituted enzyme system and the impact on the observations. CYP2D6 coincubation inhibited CYP2C9-mediated (S)-flurbiprofen metabolism in a protein concentration-dependent manner. V(max) values were reduced by up to 50%, but no appreciable effect on K(m) was observed. Spectral binding studies revealed a 20-fold increase in the K(S) of CYP2C9 toward (S)-flurbiprofen in the presence of CYP2D6. CYP2C9 coincubation had no effect on CYP2D6-mediated dextromethorphan O-demethylation. The order of combination of the proteins (CYP2C9, CYP2D6, and cytochrome P450 reductase) influenced the magnitude of catalysis inhibition as well as the ability of increased cytochrome P450 reductase to attenuate the change in activity. A simple model, congruent with current results and those of others, is proposed to explain oligomer formation. In summary, CYP2C9-CYP2D6 interactions can alter catalytic activity and, thus, influence in vitro-in vivo correlation predictions.
Subramanian
Murali
M
Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, 308 Harvard Street SE, Minneapolis, MN 55126, USA. tracy017@umn.edu
Low
Michael
M
Locuson
Charles W
CW
Tracy
Timothy S
TS
D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D001665:Binding Sites; D055162:Biocatalysis; D020134:Catalytic Domain; D065729:Cytochrome P-450 CYP2C9; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism*; D003640:Dealkylation; D003915:Dextromethorphan / Q000378:metabolism; D003917:Dextrorphan / Q000378:metabolism; D005480:Flurbiprofen / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D008954:Models, Biological; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D013057:Spectrum Analysis; D013379:Substrate Specificity
D003917:Dextrorphan; C065522:4'-hydroxyflurbiprofen; D005480:Flurbiprofen; D003915:Dextromethorphan; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D019389:Cytochrome P-450 CYP2D6; D009251:NADPH-Ferrihemoprotein Reductase