Time-dependent inactivation of P450 3A4 by raloxifene: identification of Cys239 as the site of apoprotein alkylation.
Time-dependent inactivation of cytochrome P450s is typically a result of substrate bioactivation to form reactive species that subsequently alkylate the heme group, apoprotein, or both. The chemical identity of many reactive intermediates is generally proposed based on the products of trapping reactions with nucleophilic agents as only a few P450-drug adducts have been directly characterized. We describe the use of mass spectrometry to show that a single equivalent of raloxifene is bound to the intact P450 apoprotein. Furthermore, mass analysis of peptides following digestion with proteinase K revealed that the covalently bound drug is localized to residue Cys239. A mass shift of 471 Da to the intact protein and peptide, relative to control samples, indicated that time-dependent inactivation of P450 3A4 occurred through the raloxifene diquinone methide intermediately prior to nucleophilic attack of the sulfur of Cys239. 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 residue Cys239 exclusively, time-dependent inactivation of P450 3A4 by raloxifene was prevented. The change in 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 inactivation occurred through the bioactivation of raloxifene to the diquinone methide and subsequent alkylation of Cys239.
Baer
Brian R
BR
Amgen, Department of Pharmacokinetics and Drug Metabolism, 1201 Amgen Court West, Seattle, Washington 98119, USA.
Wienkers
Larry C
LC
Rock
Dan A
DA
D000477:Alkylating Agents / Q000737:chemistry / Q000494:pharmacology; D000478:Alkylation / Q000187:drug effects; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D019286:Endopeptidase K / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000737:chemistry / Q000494:pharmacology; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D007460:Iodoacetamide / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008874:Midazolam / Q000737:chemistry / Q000494:pharmacology; D015394:Molecular Structure; D011804:Quinolines / Q000737:chemistry / Q000378:metabolism; D020849:Raloxifene Hydrochloride / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors
D000477:Alkylating Agents; D001059:Apoproteins; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D004965:Estrogen Antagonists; D011804:Quinolines; D011994:Recombinant Proteins; C065956:7-benzyloxyquinoline; D020849:Raloxifene Hydrochloride; C029358:N-(1-pyrenyl)iodoacetamide; D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D019286:Endopeptidase K; D005978:Glutathione; D003545:Cysteine; D008874:Midazolam; D007460:Iodoacetamide