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Research ArticleArticle

Cytochrome P450 Architecture and Cysteine Nucleophile Placement Impact Raloxifene-Mediated Mechanism-Based Inactivation

Brooke M. VandenBrink, John A. Davis, Josh T. Pearson, Robert S. Foti, Larry C. Wienkers and Dan A. Rock
Molecular Pharmacology November 2012, 82 (5) 835-842; DOI: https://doi.org/10.1124/mol.112.080739
Brooke M. VandenBrink
Biochemistry and Biophysics Group, Departments of Pharmacokinetics and Drug Metabolism, Amgen, Inc., Seattle, Washington
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John A. Davis
Biochemistry and Biophysics Group, Departments of Pharmacokinetics and Drug Metabolism, Amgen, Inc., Seattle, Washington
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Josh T. Pearson
Biochemistry and Biophysics Group, Departments of Pharmacokinetics and Drug Metabolism, Amgen, Inc., Seattle, Washington
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Robert S. Foti
Biochemistry and Biophysics Group, Departments of Pharmacokinetics and Drug Metabolism, Amgen, Inc., Seattle, Washington
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Larry C. Wienkers
Biochemistry and Biophysics Group, Departments of Pharmacokinetics and Drug Metabolism, Amgen, Inc., Seattle, Washington
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Dan A. Rock
Biochemistry and Biophysics Group, Departments of Pharmacokinetics and Drug Metabolism, Amgen, Inc., Seattle, Washington
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Abstract

The propensity for cytochrome P450 (P450) enzymes to bioactivate xenobiotics is governed by the inherent chemistry of the xenobiotic itself and the active site architecture of the P450 enzyme(s). Accessible nucleophiles in the active site or egress channels of the P450 enzyme have the potential of sequestering reactive metabolites through covalent modification, thereby limiting their exposure to other proteins. Raloxifene, a drug known to undergo CYP3A-mediated reactive metabolite formation and time-dependent inhibition in vitro, was used to explore the potential for bioactivation and enzyme inactivation of additional P450 enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A5). Every P450 tested except CYP2E1 was capable of raloxifene bioactivation, based on glutathione adduct formation. However, raloxifene-mediated time-dependent inhibition only occurred in CYP2C8 and CYP3A4. Comparable inactivation kinetics were achieved with KI and kinact values of 0.26 μM and 0.10 min−1 and 0.81 μM and 0.20 min−1 for CYP2C8 and CYP3A4, respectively. Proteolytic digests of CYP2C8 and CYP3A4 Supersomes revealed adducts to Cys225 and Cys239 for CYP2C8 and CYP3A4, respectively. For each P450 enzyme, proposed substrate/metabolite access channels were mapped and active site cysteines were identified, which revealed that only CYP2C8 and CYP3A4 possess accessible cysteine residues near the active site cavities, a result consistent with the observed kinetics. The combined data suggest that the extent of bioactivation across P450 enzymes does not correlate with P450 inactivation. In addition, multiple factors contribute to the ability of reactive metabolites to form apo-adducts with P450 enzymes.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    http://dx.doi.org/10.1124/mol.112.080739.

  • ABBREVIATIONS:

    P450
    cytochrome P450
    GSH
    glutathione
    MS
    mass spectrometry
    TDI
    time-dependent inhibition.

  • Received June 21, 2012.
  • Accepted August 2, 2012.
  • Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 82 (5)
Molecular Pharmacology
Vol. 82, Issue 5
1 Nov 2012
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Research ArticleArticle

Raloxifene-Selective P450 Inactivation

Brooke M. VandenBrink, John A. Davis, Josh T. Pearson, Robert S. Foti, Larry C. Wienkers and Dan A. Rock
Molecular Pharmacology November 1, 2012, 82 (5) 835-842; DOI: https://doi.org/10.1124/mol.112.080739

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Research ArticleArticle

Raloxifene-Selective P450 Inactivation

Brooke M. VandenBrink, John A. Davis, Josh T. Pearson, Robert S. Foti, Larry C. Wienkers and Dan A. Rock
Molecular Pharmacology November 1, 2012, 82 (5) 835-842; DOI: https://doi.org/10.1124/mol.112.080739
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