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Research ArticleArticle

Identification of the Residue in Human CYP3A4 That Is Covalently Modified by Bergamottin and the Reactive Intermediate That Contributes to the Grapefruit Juice Effect

Hsia-lien Lin, Cesar Kenaan and Paul F. Hollenberg
Drug Metabolism and Disposition May 2012, 40 (5) 998-1006; DOI: https://doi.org/10.1124/dmd.112.044560
Hsia-lien Lin
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Cesar Kenaan
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Paul F. Hollenberg
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Abstract

Previous studies have demonstrated that bergamottin (BG), a component of grapefruit juice, is a mechanism-based inactivator of CYP3A4 and contributes, in part, to the grapefruit juice-drug interaction. Although the covalent binding of [14C]BG to the CYP3A4 apoprotein has been demonstrated by SDS-polyacrylamide gel electrophoresis, the identity of the modified amino acid residue and the reactive intermediate species of BG responsible for the inactivation have not been reported. In the present study, we show that inactivation of CYP3A4 by BG results in formation of a modified apoprotein-3A4 and a GSH conjugate, both exhibiting mass increases of 388 Da, which corresponds to the mass of 6′,7′-dihydroxybergamottin (DHBG), a metabolite of BG, plus one oxygen atom. To identify the adducted residue, BG-inactivated 3A4 was digested with trypsin, and the digests were then analyzed by liquid chromatography-tandem mass spectrometry (MS/MS). A mass shift of 388 Da was used for the SEQUEST database search, which revealed a mass increase of 388 Da for the peptide with the sequence 272LQLMIDSQNSK282, and MS/MS analysis of the adducted peptide demonstrated that Gln273 is the residue modified. Mutagenesis studies showed that the Gln273 to Val mutant was resistant to inactivation by BG and DHBG and did not generate two of the major metabolites of BG formed by 3A4 wild type. In conclusion, we have determined that the reactive intermediate, oxygenated DHBG, covalently binds to Gln273 and thereby contributes to the mechanism-based inactivation of CYP3A4 by BG.

Footnotes

  • This work was supported in part by the National Institutes of Health National Cancer Institute [Grant CA16954] (to P.F.H.).

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

    http://dx.doi.org/10.1124/dmd.112.044560.

  • ABBREVIATIONS:

    BG
    bergamottin
    DHBG
    6′,7′-dihydroxybergamottin
    WT
    wild type
    Q273V
    Gln273 to Val mutation of 3A4
    TFA
    trifluoroacetic acid
    Lys C
    lysyl endopeptidase
    CNBr
    cyanogen bromide
    PVDF
    polyvinylidene difluoride
    P450
    cytochrome P450
    NC
    nitrocellulose
    HPLC
    high-pressure liquid chromatography
    PAGE
    polyacrylamide gel electrophoresis
    ESI
    electrospray ionization
    LC-MS
    liquid chromatography-mass spectrometry
    LC-MS/MS
    liquid chromatography-tandem mass spectrometry
    apo
    apoprotein.

  • Received January 10, 2012.
  • Accepted February 16, 2012.
  • Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 40 (5)
Drug Metabolism and Disposition
Vol. 40, Issue 5
1 May 2012
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Research ArticleArticle

COVALENT MODIFICATION OF Gln273 IN 3A4 BY BERGAMOTTIN

Hsia-lien Lin, Cesar Kenaan and Paul F. Hollenberg
Drug Metabolism and Disposition May 1, 2012, 40 (5) 998-1006; DOI: https://doi.org/10.1124/dmd.112.044560

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Research ArticleArticle

COVALENT MODIFICATION OF Gln273 IN 3A4 BY BERGAMOTTIN

Hsia-lien Lin, Cesar Kenaan and Paul F. Hollenberg
Drug Metabolism and Disposition May 1, 2012, 40 (5) 998-1006; DOI: https://doi.org/10.1124/dmd.112.044560
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