Dehydrogenation of Raloxifene by Cytochrome P450 3A4
Creators: name: Moore, Chad Douglas
Publisher: name: University of Utah
Year: 2012
Subjects: Raloxifene
Dehydrogenation
Cytochrome P450 3A4

description: Raloxifene was approved in 2007 by the FDA for the chemoprevention of breast  cancer in postmenopausal women with osteoporosis and in postmenopausal women at  high risk for invasive breast cancer. The FDA's decision was based, in part, on a more  favorable safety profile for raloxifene, as compared to the standard treatment of  tamoxifen. However, recent studies have demonstrated the ability of raloxifene to form  reactive intermediates and to act as a mechanism-based inactivator of cytochrome P450  3A4 (CYP3A4). The reactive raloxifene intermediate was theorized to be produced  either through CYP3A4-mediated dehydrogenation to a di-quinone methide or through  the more common oxygenation route to an arene oxide. However, the operative  mechanism was never identified. The goal of this dissertation was to characterize the  reactive intermediate formation from raloxifene, specifically the ability of CYP3A4 to  catalyze the dehydrogenation of raloxifene to a di-quinone methide, using a combination  of convent•i onal b•i oc«h emi•c ali and-i computati• onal techn•i ques. In the current woir k,1 8O  incorporation studies were utilized to differentiate CYP3A4-mediated oxygenation versus  dehydrogenation of raloxifene. These studies established that 3'-hydroxyraloxifene is  produced exclusively via CYP3A4-mediated oxygenation with molecular O2. These  studies also provided convincing evidence for the mechanism of CYP3A4-mediated  dehydrogenation of raloxifene to a reactive di-quinone methide, and excluded the  alternative arene oxide pathway. Furthermore, it was demonstrated that 7-hydroxyraloxifene, which was previously believed to be a typical 02-derived metabolite  of CYP3A4, is in fact produced by a highly unusual hydrolysis pathway from a putative  ester, formed by conjugation of a carboxylic acid moiety of CYP3A4 or another protein,  with the di-quinone methide of raloxifene.  The use of molecular modeling in conjunction with site-directed mutagenesis has  extensively been used to study substrate orientation within the P450 active sites, and to  identify potential residues involved in positioning and/or catalysis of these substrates.  However, the effectiveness of these studies is highly dependent on the selection of the  most accurate enzyme crystal structure. In the current work, we compared the ability of  two commonly used CYP3A4 crystal structures, 1TQN and 1 WOE, to predict the sites of  metabolism of two known CYP3A4 substrates, indapamide and raloxifene. Indapamide  was used to evaluate the accuracy of the molecular model, while raloxifene was used to  investigate the effects of adding partial charges to the P450 heme moiety to improve  predictions of metabolic pathways by computation methods. The results demonstrated  that while docking studies with both 1TQN and 1 WOE crystal structures could accurately  predict the sites of indapamide metabolism, the interactions between the substrate and  active site residues were different for each crystal structure. The addition of partial  charges to the heme moiety of 1 WOE dramatically increased the predictive power of the  model, and dramatically increased the accuracy of the model, regarding substrate/active  site residue interactions.  To determine the validity of our models, active site residues involved in the  positioning and/or catalysis of the substrates were identified, and site-directed  mutagenesis studies were conducted. Mutations based on 1TQN and 1 WOE (without heme partial charges) models did not alter CYP3A4-mediated dehydrogenation of  raloxifene, demonstrating that these models were not accurate enough to correctly predict  enzyme/substrate interactions. However, the 1 WOE (with heme partial charges) model  identified the Phe215 residue as an important component of the enzyme tertiary structure  that would interact with raloxifene. Site-directed mutagenesis experiments with Phe215  confirmed that this residue positions raloxifene to favor the dehydrogenation pathway,  most likely through a combination of n bond T-stacking and steric interactions. These  results validate the use of the 1 WOE (with heme partial charges) model for docking  studies of raloxifene, and demonstrate the usefulness of molecular modeling in  conjunction with site-directed mutagenesis to study the mechanisms of P450-mediated  metabolism.