title: Mechanism-based inactivation of cytochrome P450 2B1 by 8-methoxypsoralen and several other furanocoumarins.
abstract: Of several furanocoumarins [5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), 5-hydroxypsoralen (5-OH-P), 8-hydroxypsoralen (8-OH-P), 4',5'-dihydro-8-MOP (DH-8-MOP), and psoralen (P)] tested as mechanism-based inactivators (MBIs) of purified reconstituted cytochrome P450 (P450) 2B1, 8-MOP was found to be the most potent (KI, kinact, and partition ratio of 2.9 microM, 0.34 min-1, and 1.3, respectively). The inactivation was not prevented by reactive oxygen species scavengers or nucleophilic trapping agents and proceeded with a decrease in P450 spectral content. Liquid chromatography (LC) separation of the reconstituted enzyme mixture, followed by liquid scintillation counting, indicated that [14C]-8-MOP binding was specific to the apoprotein of P450 2B1 with a binding stoichiometry of 0.7:1. The major metabolites formed by P450 2B1 from the furanocoumarins that were MBIs were characterized by LC electrospray ionization tandem mass spectrometry (ESI-MS/MS) as dihydro diols. Results from H218O incorporation experiments supported initial oxidation of 8-MOP and P to an epoxide which can react with some nucleophilic active site residue and inactivate the enzyme or partition to a dihydro diol metabolite by hydrolytic ring opening. On the other hand, 5-MOP was converted to an epoxide or gamma-keto enal intermediate prior to inactivation or dihydro diol formation. Comparison of the ESI mass spectra of P450 2B1 and furanocoumarin exposed P450 2B1, indicated a mass difference consistent with the covalent addition of a furanoepoxide to P450 2B1.
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle 98195, USA.
authors.forename: L L
authors.initials: LL
authors.lastname: Koenigs
authors.forename: W F
authors.initials: WF
authors.lastname: Trager
mesh_terms: D000078223:5-Methoxypsoralen; D000818:Animals; D001665:Binding Sites; D002853:Chromatography, Liquid; D003374:Coumarins / Q000378:metabolism / Q000494:pharmacology*; D019362:Cytochrome P-450 CYP2B1 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005363:Ficusin / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D008730:Methoxsalen / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D009249:NADP / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D003374:Coumarins; D004791:Enzyme Inhibitors; D000078223:5-Methoxypsoralen; D009249:NADP; D019362:Cytochrome P-450 CYP2B1; D005363:Ficusin; D008730:Methoxsalen