title: Immobilized Cytochrome P450 for Monitoring of P450-P450 Interactions and Metabolism.
abstract: Cytochrome P450 (P450) protein-protein interactions have been shown to alter their catalytic activity. Furthermore, these interactions are isoform specific and can elicit activation, inhibition, or no effect on enzymatic activity. Studies show that these effects are also dependent on the protein partner cytochrome P450 reductase (CPR) and the order of protein addition to purified reconstituted enzyme systems. In this study, we use controlled immobilization of P450s to a gold surface to gain a better understanding of P450-P450 interactions between three key drug-metabolizing isoforms (CYP2C9, CYP3A4, and CYP2D6). Molecular modeling was used to assess the favorability of homomeric/heteromeric P450 complex formation. P450 complex formation in vitro was analyzed in real time utilizing surface plasmon resonance. Finally, the effects of P450 complex formation were investigated utilizing our immobilized platform and reconstituted enzyme systems. Molecular modeling shows favorable binding of CYP2C9-CPR, CYP2C9-CYP2D6, CYP2C9-CYP2C9, and CYP2C9-CYP3A4, in rank order.KDvalues obtained via surface plasmon resonance show strong binding, in the nanomolar range, for the above pairs, with CYP2C9-CYP2D6 yielding the lowestKD, followed by CYP2C9-CYP2C9, CYP2C9-CPR, and CYP2C9-CYP3A4. Metabolic incubations show that immobilized CYP2C9 metabolism was activated by homomeric complex formation. CYP2C9 metabolism was not affected by the presence of CYP3A4 with saturating CPR concentrations. CYP2C9 metabolism was activated by CYP2D6 at saturating CPR concentrations in solution but was inhibited when CYP2C9 was immobilized. The order of addition of proteins (CYP2C9, CYP2D6, CYP3A4, and CPR) influenced the magnitude of inhibition for CYP3A4 and CYP2D6. These results indicate isoform-specific P450 interactions and effects on P450-mediated metabolism.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, West Virginia (C.D.B., K.M.H.); Array BioPharma, Boulder, Colorado (L.A.W.); Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota (D.R.F.); College of Pharmacy, University of Kentucky, Lexington, Kentucky (T.S.T.); and Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University, Ft. Lauderdale, Florida (P.M.G.).
authors.forename: Chris D
authors.initials: CD
authors.lastname: Bostick
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, West Virginia (C.D.B., K.M.H.); Array BioPharma, Boulder, Colorado (L.A.W.); Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota (D.R.F.); College of Pharmacy, University of Kentucky, Lexington, Kentucky (T.S.T.); and Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University, Ft. Lauderdale, Florida (P.M.G.).
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Hickey
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, West Virginia (C.D.B., K.M.H.); Array BioPharma, Boulder, Colorado (L.A.W.); Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota (D.R.F.); College of Pharmacy, University of Kentucky, Lexington, Kentucky (T.S.T.); and Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University, Ft. Lauderdale, Florida (P.M.G.).
authors.forename: Lance A
authors.initials: LA
authors.lastname: Wollenberg
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, West Virginia (C.D.B., K.M.H.); Array BioPharma, Boulder, Colorado (L.A.W.); Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota (D.R.F.); College of Pharmacy, University of Kentucky, Lexington, Kentucky (T.S.T.); and Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University, Ft. Lauderdale, Florida (P.M.G.).
authors.forename: Darcy R
authors.initials: DR
authors.lastname: Flora
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, West Virginia (C.D.B., K.M.H.); Array BioPharma, Boulder, Colorado (L.A.W.); Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota (D.R.F.); College of Pharmacy, University of Kentucky, Lexington, Kentucky (T.S.T.); and Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University, Ft. Lauderdale, Florida (P.M.G.).
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Tracy
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, West Virginia (C.D.B., K.M.H.); Array BioPharma, Boulder, Colorado (L.A.W.); Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota (D.R.F.); College of Pharmacy, University of Kentucky, Lexington, Kentucky (T.S.T.); and Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University, Ft. Lauderdale, Florida (P.M.G.) pgannett@nova.edu.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Gannett
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D008958:Models, Molecular; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000378:metabolism*
chemical_list: D004364:Pharmaceutical Preparations; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase