title: Formation of raloxifene homo-dimer in CYP3A4, evidence for multi-substrate binding in a single catalytically competent P450 active site.
abstract: Raloxifene is a polyaromatic compound which has been reported to form radicals when incubated with horseradish peroxidase resulting in formation of a homo-dimer product. Polyaromatic phenols have also been reported to undergo oxidation by P450 enzymes to form reactive intermediates, presumably through the formation of phenoxy radical species. Recently, we observed that a raloxifene homo-dimer was formed in vitro when incubated with CYP3A4. In response to this finding, a series of experiments were designed to determine whether the observed raloxifene homo-dimer was formed via solution phase chemistry similar to that previously documented with horseradish peroxidase or if generation of the homo-dimer occurred within the P450 active site. To this end, a series of experiments were carried out to determine the structure of the CYP3A4 generated raloxifene homo-dimer using analytical techniques including: high resolution MS, NMR and H/D exchange. In addition, a variety of in vitro techniques were applied to characterize the mechanism responsible for formation of the raloxifene homo-dimer. Collectively, the results of these experiments suggest that unlike the homo-dimer formed by peroxidase enzymes, raloxifene homo-dimer formation mediated by CYP3A4 is a consequence of two raloxifene molecules binding simultaneously within the active site of a catalytically competent P450 enzyme.
authors.affiliation: Amgen Inc., Department of Phamacokinetics and Drug Metabolism, 1201 Amgen Court West, Seattle, WA 98119, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Davis
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Greene
authors.forename: Sean
authors.initials: S
authors.lastname: Han
authors.forename: Dan A
authors.initials: DA
authors.lastname: Rock
authors.forename: Larry C
authors.initials: LC
authors.lastname: Wienkers
mesh_terms: D020134:Catalytic Domain; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry* / Q000378:metabolism*; D041961:Deuterium Exchange Measurement; D019281:Dimerization; D055664:Electrochemical Techniques; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D020849:Raloxifene Hydrochloride / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D020849:Raloxifene Hydrochloride; D006735:Horseradish Peroxidase; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human