title: Inhibition of bupropion metabolism by selegiline: mechanism-based inactivation of human CYP2B6 and characterization of glutathione and peptide adducts.
abstract: Selegiline, the R-enantiomer of deprenyl, is used in the treatment of Parkinson's disease. Bupropion, an antidepressant, often used to treat patients in conjunction with selegiline, is metabolized primarily by CYP2B6. The effect of selegiline on the enzymatic activity of human cytochrome CYP2B6 in a reconstituted system and its effect on the metabolism of bupropion were examined. Selegiline was found to be a mechanism-based inactivator of the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation (7-EFC) activity of CYP2B6 as well as bupropion metabolism. The inactivations were time-, concentration-, and NADPH-dependent and were characterized by K(I) values of 0.14 and 0.6 μM, k(inact) values of 0.022 and 0.029 min⁻¹, and t(½) values of 31.5 and 24 min, respectively. In standard inhibition assays, selegiline increased the K(m) of CYP2B6 for bupropion from 10 to 92 μM and decreased the k(cat) by ∼50%. The reduced carbon-monoxide difference spectrum revealed over a 50% loss in the cytochrome P450 spectrum in the inactivated sample, with no loss in heme, and there was ∼70% loss in enzyme activity. Trapping of the reactive metabolite using GSH led to the identification of a GSH-selegiline conjugate with a m/z 528 that could be explained by hydroxylation of selegiline followed by the addition of glutathione to the propargyl moiety after oxygenation to form the ketene intermediate. Liquid chromatography-tandem mass spectrometry analysis of the labeled protein following digestion with trypsin revealed the peptide ⁶⁴DVFTVHLGPR⁷³ as the peptide modified by the reactive metabolite of selegiline and the site of adduct formation is Asp64.
authors.affiliation: Department of Pharmacology, The University of Michigan, Ann Arbor, MI, USA.
authors.forename: Chitra
authors.initials: C
authors.lastname: Sridar
authors.forename: Cesar
authors.initials: C
authors.lastname: Kenaan
authors.forename: Paul F
authors.initials: PF
authors.lastname: Hollenberg
mesh_terms: D000595:Amino Acid Sequence; D018687:Antidepressive Agents, Second-Generation / Q000378:metabolism; D000978:Antiparkinson Agents / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016642:Bupropion / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D065702:Cytochrome P-450 CYP2B6; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004347:Drug Interactions; D004926:Escherichia coli / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D012642:Selegiline / Q000494:pharmacology*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D018687:Antidepressive Agents, Second-Generation; D000978:Antiparkinson Agents; D065607:Cytochrome P-450 Enzyme Inhibitors; D010455:Peptides; D016642:Bupropion; D012642:Selegiline; D006418:Heme; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating; D005978:Glutathione