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Research ArticleArticle

Immobilized Cytochrome P450 for Monitoring of P450-P450 Interactions and Metabolism

Chris D. Bostick, Katherine M. Hickey, Lance A. Wollenberg, Darcy R. Flora, Timothy S. Tracy and Peter M. Gannett
Drug Metabolism and Disposition May 2016, 44 (5) 741-749; DOI: https://doi.org/10.1124/dmd.115.067637
Chris D. Bostick
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.)
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Katherine M. Hickey
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.)
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Lance A. Wollenberg
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.)
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Darcy R. Flora
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.)
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Timothy S. Tracy
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.)
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Peter M. Gannett
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.)
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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. KD values obtained via surface plasmon resonance show strong binding, in the nanomolar range, for the above pairs, with CYP2C9-CYP2D6 yielding the lowest KD, 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.

Footnotes

    • Received October 2, 2015.
    • Accepted March 9, 2016.
  • All authors are co-first authors.

  • This research was supported by West Virginia University via the National Science Foundation through the Experimental Program to Stimulate Competitive Research [Grant EPS-1003907]; the National Institutes of Health National Institute of General Medical Sciences [Grant R01-GM086891]; and the National Science Foundation Integrative Graduate Education and Research Traineeship Program [Grant DGE-1144676].

  • dx.doi.org/10.1124/dmd.115.067637.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

  • Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 44 (5)
Drug Metabolism and Disposition
Vol. 44, Issue 5
1 May 2016
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Research ArticleArticle

P450 Immobilization to Probe P450-P450 Interactions

Chris D. Bostick, Katherine M. Hickey, Lance A. Wollenberg, Darcy R. Flora, Timothy S. Tracy and Peter M. Gannett
Drug Metabolism and Disposition May 1, 2016, 44 (5) 741-749; DOI: https://doi.org/10.1124/dmd.115.067637

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Research ArticleArticle

P450 Immobilization to Probe P450-P450 Interactions

Chris D. Bostick, Katherine M. Hickey, Lance A. Wollenberg, Darcy R. Flora, Timothy S. Tracy and Peter M. Gannett
Drug Metabolism and Disposition May 1, 2016, 44 (5) 741-749; DOI: https://doi.org/10.1124/dmd.115.067637
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