Received for publication January 5, 2009.
Revised May 11, 2009.
Accepted for publication May 15, 2009.

CYP2D6-CYP2C9 Protein-Protein Interactions and Isoform-Selective Effects on Substrate Binding and Catalysis

Abstract

Abstract Cytochrome P450 (P450) protein-protein interactions have been observed with various in-vitro systems. Interestingly, these interactions appear to be isoform dependent, with some combinations producing no effect, 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 both changes in 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 co-incubation inhibited CYP2C9 mediated S-flurbiprofen metabolism in a protein concentration dependent manner. Vmax values were reduced by up to 50%, but no appreciable effect on Km was observed. Spectral binding studies revealed a 20-fold increase in the Ks of CYP2C9 towards S-flurbiprofen in the presence of CYP2D6. CYP2C9 co-incubation had no effect on CYP2D6-mediated dextromethorphan O-demethylation. The order of combination of the proteins (CYP2C9, CYP2D6 and cytochrome P450 reductase (CPR)) influenced the magnitude of catalysis inhibition as well as the ability of increased cytochrome P450 reductase to attenuate the change in activity. A simple scheme, 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.

Key words: CYP2C, CYP2D, cytochrome P450, protein-protein interactions