title: Mechanism-based inactivation of cytochrome P450 3A4 by L-754,394.
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, Ni(2+) 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.
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195, USA.
authors.forename: L K
authors.initials: LK
authors.lastname: Lightning
authors.forename: J P
authors.initials: JP
authors.lastname: Jones
authors.forename: T
authors.initials: T
authors.lastname: Friedberg
authors.forename: M P
authors.initials: MP
authors.lastname: Pritchard
authors.forename: M
authors.initials: M
authors.lastname: Shou
authors.forename: T H
authors.initials: TH
authors.lastname: Rushmore
authors.forename: W F
authors.initials: WF
authors.lastname: Trager
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003198:Computer Simulation; D003488:Cyanogen Bromide / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D003956:Dialysis; D004926:Escherichia coli / Q000166:cytology; D017320:HIV Protease Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D006900:Hydroxylation; D007189:Indans / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D009249:NADP / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010879:Piperazines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D017320:HIV Protease Inhibitors; D007189:Indans; C094644:L 754394; D010446:Peptide Fragments; D010879:Piperazines; D011993:Recombinant Fusion Proteins; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; D014357:Trypsin; D003488:Cyanogen Bromide