title: Identification of cytochrome P450 3A4 modification site with reactive metabolite using linear ion trap-Fourier transform mass spectrometry.
abstract: Covalent binding of reactive metabolites to cytochrome P450s (P450s) often causes their mechanism-based inactivation (MBI), resulting in drug-drug interactions or toxicity. The detection and identification of the P450 sites to which reactive metabolites bind would elucidate MBI mechanisms. We describe a proteomic approach using nano-LC/linear ion trap-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to characterize the binding of a reactive metabolite of raloxifene, which is a known P450 3A4 inhibitor, to the P450 3A4 isozyme. LTQ-FT analyses revealed that the metabolic reaction of raloxifene in a reconstituted P450 3A4 system formed a reactive metabolite adduct to P450 3A4 apoprotein, accompanied by a mass shift of 471 Da relative to intact P450 3A4 apoprotein. The reaction mixtures were digested with trypsin, and then the tryptic digests were analyzed by nano-LC-MS/MS. This technique revealed that VWGFYDGQQPVLAITDPDMIK (position 71-91) was a tryptic peptide modified by the reactive metabolite derived from raloxifene. The site of adduction with the reactive metabolite was further postulated to be the nucleophilic OH group of Tyr-75 of P450 3A4. A proteomic approach using LTQ-FT can yield direct information on the P450 3A4 modification site without radiolabeled compounds. In addition, this information can elucidate mechanisms involved in the covalent binding of reactive metabolites and the inactivation of P450 3A4.
authors.affiliation: Drug Metabolism and Pharmacokinetics Research Laboratories, R&D Division, Daiichi-Sankyo Co., Ltd., Tokyo, Japan. yukinaga.hideo.ds@daiichisankyo.co.jp
authors.forename: Hideo
authors.initials: H
authors.lastname: Yukinaga
authors.forename: Tomonori
authors.initials: T
authors.lastname: Takami
authors.forename: Sho-Hei
authors.initials: SH
authors.lastname: Shioyama
authors.forename: Zenzaburo
authors.initials: Z
authors.lastname: Tozuka
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Masumoto
authors.forename: Osamu
authors.initials: O
authors.lastname: Okazaki
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Sudo
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D004965:Estrogen Antagonists / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000379:methods; D020836:Protein Structure, Quaternary; D040901:Proteomics; D020849:Raloxifene Hydrochloride / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D004965:Estrogen Antagonists; D020849:Raloxifene Hydrochloride; D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human