p450 - publications

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1. Drug Metab Dispos. 2012 Feb 16. [Epub ahead of print]

Identification of the Residue in Human CYP3A4 that is Covalently Modified by
Bergamottin and the Reactive Intermediate that Contributes to the Grapefruit
Juice Effect.

Lin HL, Kenaan C, Hollenberg PF.

University of Michigan.

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
[(14)C]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 apo-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.
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 (272)LQLMIDSQNSK(282)
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.

PMID: 22344702 [PubMed - as supplied by publisher]