Chad D. Moore

The use of molecular modeling in conjunction with site-directed mutagenesis has been extensively used to study substrate orientation within cytochrome P450 active sites and to identify potential residues involved in the positioning and catalytic mechanisms of these substrates. However, because docking studies utilize static models to simulate dynamic P450 enzymes, the effectiveness of these studies is strongly dependent on accurate enzyme models. This study employed a cytochrome P450 3A4 (CYP3A4) crystal structure (Protein Data Bank entry 1W0E) to predict the sites of metabolism of the known CYP3A4 substrate raloxifene. In addition, partial charges were incorporated into the P450 heme moiety to investigate the effect of the modified CYP3A4 model on metabolite prediction with the ligand docking program Autodock. Dehydrogenation of raloxifene to an electrophilic diquinone methide intermediate has been linked to the potent inactivation of CYP3A4. Active site residues involved in the positioning and/or catalysis of raloxifene supporting dehydrogenation were identified with the two models, and site-directed mutagenesis studies were conducted to validate the models. The addition of partial charges to the heme moiety improved the accuracy of the docking studies, increasing the number of conformations predicting dehydrogenation and facilitating the identification of substrate−active site residue interactions. On the basis of the improved model, the Phe215 residue was hypothesized to play an important role in orienting raloxifene for dehydrogenation through a combination of electrostatic and steric interactions. Substitution of this residue with glycine or glutamine significantly decreased dehydrogenation rates without concurrent changes in the rates of raloxifene oxygenation. Thus, the improved structural model predicted novel enzyme−substrate interactions that control the selective dehydrogenation of raloxifene to its protein-binding intermediate.

Raloxifene was approved in 2007 by the FDA for the chemoprevention of breast cancer in postmenopausal women at high risk for invasive breast cancer. Approval was based in part on the improved safety profile for raloxifene relative to the standard treatment of tamoxifen. However, recent studies have demonstrated the ability of raloxifene to form reactive intermediates and act as a mechanism-based inhibitor of cytochrome P450 3A4 (CYP3A4) by forming adducts with the apoprotein. However, previous studies could not differentiate between dehydrogenation to a diquinone methide and the more common oxygenation pathway to an arene oxide as the most likely intermediate to inactivate CYP3A4. In the current work, ¹⁸O-incorporation studies were utilized to carefully elucidate CYP3A4-mediated oxygenation versus dehydrogenation of raloxifene. These studies established that 3′-hydroxyraloxifene is produced exclusively via CYP3A4-mediated oxygenation and provide convincing evidence for the mechanism of CYP3A4-mediated dehydrogenation of raloxifene to a reactive diquinone methide, while excluding the alternative arene oxide pathway. Furthermore, it was demonstrated that 7-hydroxyraloxifene, which was previously believed to be a typical O₂-derived metabolite of CYP3A4, is in fact produced by a highly unusual hydrolysis pathway from a putative ester, formed by the conjugation of raloxifene diquinone methide with a carboxylic acid moiety of CYP3A4, or other proteins in the reconstituted system. These findings not only confirm CYP3A4-mediated dehydrogenation of raloxifene to a reactive diquinone methide but also suggest a novel route of raloxifene toxicity.