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Mechanism-Based Inactivation of Cytochrome P450 3A4 by L-754,394

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Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195, Department of Chemistry, Room 477 Fulmer Hall, Washington State University, Pullman, Washington 99164-4630, Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee, UK, and Department of Drug Metabolism, WP-26A-2044, Merck Research Laboratories, West Point, Pennsylvania 19486
Cite this: Biochemistry 2000, 39, 15, 4276–4287
Publication Date (Web):March 16, 2000
https://doi.org/10.1021/bi992412u
Copyright © 2000 American Chemical Society

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    Abstract

    Mechanism-based inactivation of human liver P450 3A4 by L-754,394, a Merck compound synthesized as a potential HIV protease inhibitor, was investigated using recombinant P450 3A4. Enzyme inactivation was characterized by a small partition ratio (3.4 or 4.3 ± 0.4), i.e., the total number of metabolic events undergone by the inhibitor divided by the number of enzyme inactivating events, lack of reversibility upon extensive dialysis, no decrease in the characteristic 450-nm species relative to control, and covalent modification of the apoprotein. The major and minor products formed during the inactivation of P450 3A4 were the monohydroxylated and the dihydrodiol metabolites of L-754,394, respectively. L-754,394 that had been adducted to P450 3A4 was hydrolyzed under the conditions used for SDS-PAGE, Ni2+ affinity chromatography, and proteolytic digestion. In addition, the modification was not stable to the acidic conditions of HPLC separation and CNBr digestion. The labile nature of the peptide adduct and the nonstoichiometric binding of the inactivating species to P450 3A4 precluded the direct identification of a covalently modified amino acid residue or the peptide to which it was attached. However, Tricine SDS-PAGE in combination with MALDI-TOF-MS and homology modeling, allowed I257-M317 to be tentatively identified as an active site peptide, while prior knowledge of the stability of N-, O-, and S-linked conjugates of activated furans implicates Glu307 as the active site amino acid that is labeled by L-754, 394.

     This work was supported by NIH Grant GM32165 (Bethesda, MD) and the Dorothy Danforth-Compton Graduate Student Fellowship (L.K.L.).

     Department of Medicinal Chemistry, University of Washington.

    §

     Present Address:  School of Pharmacy, Rm. S-926, Department of Pharmaceutical Chemistry, University of CA, San Francisco, San Francisco, CA 94143-0446.

     Department of Chemistry, Washington State University.

     Biomedical Research Centre.

    *

     To whom correspondence should be addressed:  Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195. Telephone:  (206) 543-9481. Fax:  (206) 685-3252. E-mail:  [email protected].

     Merck Research Laboratories.

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