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. 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.Cytochrome P450s (P450s) are responsible for the metabolism of more than 75% of the drugs on the market (Guengerich, 2006). Multiple subfamilies of cytochrome P450s can activate and metabolize a wide spectrum of substrates by a two-electron transfer catalytic cycle, in which electrons are provided by either NADPH-cytochrome P450 reductase (CPR) or cytochrome b 5 (b5), depending on the stage of the cycle (Guengerich, 2001). A number of studies have investigated the role of CPR and b5 and the nature of P450, CPR, and b5 interactions. Although CPR is indispensable for metabolism, cytochrome b 5 either has no effect or significantly enhances metabolism (Shimada et al., 1994;Locuson et al., 2006). Yamazaki et al. (1997) demonstrated that b5 stimulated the metabolism of tolbutamide and (S)-warfarin by CYP2C10 but had no effect on bufuralol hydroxylation by CYP1A1 and CYP2D6. In addition, both apo-and holo-b5 enhanced metabolism by CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5 in reconstituted systems (Shimada et al., 1994), suggesting that the effect of these protein-protein interactions of the redox partner with P450 may involve enzyme conformational changes or other mechanisms besides just provision of electrons.In addition to interactions with CPR and b5, P450s can also interact with each other, resulting in c...

CYP2C8 Exists as a Dimer in Natural Membranes

“…Experimentally, both hetero-and homodimerization have been shown to affect P450 function. Enzyme kinetic analysis of pairs of P450s, including 2B4/1A4, 2D6/2C9, 2C9/3A4, and 2C9/2C19 has provided evidence that hetero-interactions between the P450s affect the activity of one or both of the P450s in the dimer (Cawley et al, 2001;Hazai and Kupfer, 2005;Subramanian et al, 2009Subramanian et al, , 2010Reed et al, 2010). Homo-oligomerization also has functional consequences.…”

“…Solubilized P450s also have been shown to form homooligomers containing up six or eight protein molecules in some cases, which were mediated by the signal anchor sequence (Von Wachenfeldt and Johnson, 1995). Interactions among P450s may have functional significance because coexpression of a second P450 with a P450 can either inhibit or increase the activity of the first P450 (Cawley et al, 2001;Hazai and Kupfer, 2005;Subramanian et al, 2009Subramanian et al, , 2010Reed et al, 2010). The functional significance of homooligomerization is not clear, but oligomerization of CYP3A4 has been shown to decrease reduction of the P450 by dithionite (Davydov et al, 2005) or the soluble flavin domain of P450BM-3 (Davydov et al, 2010).…”

CYP2C9-CYP3A4 Protein-Protein Interactions: Role of the Hydrophobic N Terminus

“…Considering that P450s and CPR, and P450s and b5 interact within the cell membrane, P450-P450 interactions also appear conceivable. Indeed, in vitro evidence of P450-P450 interactions have been observed in several studies, including interactions of human CYP2C9-CYP2C19, rabbit CYP1A2-CYP2B4, rabbit CYP1A2-CYP2E1, human CYP3A4-CYP1A1, human CYP3A4-CYP1A2, and human CYP2C9-CYP2D6 (Cawley et al, 1995;Yamazaki et al, 1997;Backes et al, 1998;Backes and Cawley, 1999;Kelley et al, 2005Kelley et al, , 2006Subramanian et al, 2009). Substrate-dependent activation or inhibition of one or both isoforms has been observed.…”

“…CYP1A1-CYP3A2 and CYP2E1-CYP2C2 complexes were identified using bimolecular fluorescence complementation spectroscopy and chemical cross-linking followed by immunoprecipitation, respectively (Alston et al, 1991;Ozalp et al, 2005). In addition, molecular modeling studies have also suggested the formation of heteromers (Backes and Kelley, 2003;, whereas an order of addition study provided indirect evidence for the formation of quasi-irreversibly formed CYP2C9 and CYP2D6 heteromers (Subramanian et al, 2009).Although the in vitro evidence for P450-P450 interactions is compelling, the in vivo evidence of and physiological significance for P450-P450 interactions remains to be established. It has been speculated that heteromerization of P450s may exist to ensure proper cellular targeting into the membrane, cytosol, and Golgi complexes (Szczesna-Skorupa and Kemper, 2008).…”

Interactions among Cytochromes P450 in Microsomal Membranes

“…This type of relationship is exhibited by CYP3A4/CYP1A2 (26), CYP2C19/CYP2C9 (27), CYP2D6/CYP2C9 (28), and CYP3A4/ CYP2C9 (29) pairs. The most extensively studied P450-P450 pair is that of rabbit CYP1A2 and CYP2B4 enzymes (3, 6, 13, 30 -34).…”