title: Improved cytochrome P450 3A4 molecular models accurately predict the Phe215 requirement for raloxifene dehydrogenation selectivity.
abstract: 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.
authors.affiliation: Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Chad D
authors.initials: CD
authors.lastname: Moore
authors.forename: Kiumars
authors.initials: K
authors.lastname: Shahrokh
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Sontum
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Cheatham
authors.forename: Garold S
authors.initials: GS
authors.lastname: Yost
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D006418:Heme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D010649:Phenylalanine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020849:Raloxifene Hydrochloride / Q000737:chemistry* / Q000378:metabolism
chemical_list: D006418:Heme; D010649:Phenylalanine; D020849:Raloxifene Hydrochloride; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human
references.citation: Toxicol In Vitro. 2003 Apr;17(2):179-90
references.pmid: 12650672
references.citation: Chem Res Toxicol. 2002 Jul;15(7):907-14
references.pmid: 12119000
references.citation: Chem Res Toxicol. 2001 Jun;14(6):611-50
references.pmid: 11409933
references.citation: Eur J Biochem. 2000 May;267(10):2916-20
references.pmid: 10806389
references.citation: Arch Biochem Biophys. 2008 Mar 15;471(2):134-45
references.pmid: 18206979
references.citation: J Pharmacol Exp Ther. 1999 Aug;290(2):594-602
references.pmid: 10411567
references.citation: Chem Res Toxicol. 2007 Jun;20(6):954-64
references.pmid: 17497897
references.citation: Adv Exp Med Biol. 2001;500:53-62
references.pmid: 11764993
references.citation: Chem Res Toxicol. 2007 Oct;20(10):1373-8
references.pmid: 17867646
references.citation: J Inorg Biochem. 2001 Dec 15;87(4):209-13
references.pmid: 11744058
references.citation: Biochemistry. 2010 Jun 1;49(21):4466-75
references.pmid: 20405834
references.citation: Chem Res Toxicol. 2001 Aug;14(8):1014-24
references.pmid: 11511175
references.citation: Chem Res Toxicol. 1991 Jul-Aug;4(4):413-21
references.pmid: 1912327
references.citation: J Comput Chem. 2005 Jun;26(8):818-26
references.pmid: 15812779
references.citation: J Am Chem Soc. 2004 Apr 28;126(16):5072-3
references.pmid: 15099082
references.citation: Life Sci. 1997;61(26):2507-20
references.pmid: 9416773
references.citation: Science. 2004 Jul 30;305(5684):683-6
references.pmid: 15256616
references.citation: Biochemistry. 2008 Sep 16;47(37):9756-70
references.pmid: 18717595
references.citation: J Am Chem Soc. 2005 Oct 5;127(39):13634-42
references.pmid: 16190729
references.citation: J Biol Chem. 1964 Jul;239:2370-8
references.pmid: 14209971
references.citation: Eur J Biochem. 2003 Sep;270(18):3684-95
references.pmid: 12950252
references.citation: Chem Res Toxicol. 2004 Jan;17(1):3-16
references.pmid: 14727914
references.citation: Drug Metab Rev. 2002 Feb-May;34(1-2):83-448
references.pmid: 11996015
references.citation: Xenobiotica. 2004 Jun;34(6):549-69
references.pmid: 15277015
references.citation: Arch Biochem Biophys. 1998 Feb 15;350(2):223-32
references.pmid: 9473295
references.citation: Curr Drug Metab. 2001 Jun;2(2):93-115
references.pmid: 11469727
references.citation: Chem Rev. 2005 Jun;105(6):2279-328
references.pmid: 15941215
references.citation: J Biol Chem. 2004 Sep 10;279(37):38091-4
references.pmid: 15258162
references.citation: Mol Pharmacol. 2002 Mar;61(3):495-506
references.pmid: 11854429
references.citation: Drug Metab Rev. 2002 Feb-May;34(1-2):17-35
references.pmid: 11996009
references.citation: Nat Rev Drug Discov. 2004 Nov;3(11):935-49
references.pmid: 15520816
references.citation: J Med Chem. 2003 Jul 3;46(14):2895-907
references.pmid: 12825931
references.citation: Chem Res Toxicol. 2004 Jul;17(7):879-88
references.pmid: 15257612
references.citation: Drug Metab Dispos. 2009 Mar;37(3):672-84
references.pmid: 19074530
references.citation: J Biol Chem. 1989 May 5;264(13):7584-9
references.pmid: 2708380