文摘
Time-dependent inactivation of cytochrome P450s is typically a result of substrate bioactivation toform reactive species that subsequently alkylate the heme group, apoprotein, or both. The chemical identityof many reactive intermediates is generally proposed based on the products of trapping reactions withnucleophilic agents as only a few P450-drug adducts have been directly characterized. We describe theuse of mass spectrometry to show that a single equivalent of raloxifene is bound to the intact P450apoprotein. Furthermore, mass analysis of peptides following digestion with proteinase K revealed thatthe covalently bound drug is localized to residue Cys239. A mass shift of 471 Da to the intact proteinand peptide, relative to control samples, indicated that time-dependent inactivation of P450 3A4 occurredthrough the raloxifene diquinone methide intermediately prior to nucleophilic attack of the sulfur ofCys239. Association between raloxifene adduction to P450 3A4 apoprotein and the observed time-dependent inactivation was further investigated with the use of cysteine-specific modifying reagents.When P450 3A4 was treated with iodoacetamide or N-(1-pyrene)iodoacetamide, which alkylated residueCys239 exclusively, time-dependent inactivation of P450 3A4 by raloxifene was prevented. The changein protein mass of 471 Da combined with the protection from inactivation that occurred through pre-alkylation of Cys239 provided conclusive evidence that raloxifene-mediated P450 3A4 inactivationoccurred through the bioactivation of raloxifene to the diquinone methide and subsequent alkylation ofCys239.
