Determinants of Cytochrome P450 2C8 Substrate Binding

Although a crystal structure and a pharmacophore model are available for cytochrome P450 2C8, the role of protein flexibility and specific ligand-protein interactions that govern substrate binding are poorly understood. X-ray crystal structures of P450 2C8 complexed with montelukast (2.8 Å), troglitazone (2.7 Å), felodipine (2.3 Å), and 9-cis-retinoic acid (2.6 Å) were determined to examine ligand-protein interactions for these chemically diverse compounds. Montelukast is a relatively large anionic inhibitor that exhibits a tripartite structure and complements the size and shape of the active-site cavity. The inhibitor troglitazone occupies the upper portion of the active-site cavity, leaving a substantial part of the cavity unoccupied. The smaller neutral felodipine molecule is sequestered with its dichlorophenyl group positioned close to the heme iron, and water molecules fill the distal portion of the cavity. The structure of the 9-cis-retinoic acid complex reveals that two substrate molecules bind simultaneously in the active site of P450 2C8. A second molecule of 9-cis-retinoic acid is located above the proximal molecule and can restrain the position of the latter for more efficient oxygenation. Solution binding studies do not discriminate between cooperative and noncooperative models for multiple substrate binding. The complexes with structurally distinct ligands further demonstrate the conformational adaptability of active site-constituting residues, especially Arg-241, that can reorient in the active-site cavity to stabilize a negatively charged functional group and define two spatially distinct binding sites for anionic moieties of substrates. Although a crystal structure and a pharmacophore model are available for cytochrome P450 2C8, the role of protein flexibility and specific ligand-protein interactions that govern substrate binding are poorly understood. X-ray crystal structures of P450 2C8 complexed with montelukast (2.8 Å), troglitazone (2.7 Å), felodipine (2.3 Å), and 9-cis-retinoic acid (2.6 Å) were determined to examine ligand-protein interactions for these chemically diverse compounds. Montelukast is a relatively large anionic inhibitor that exhibits a tripartite structure and complements the size and shape of the active-site cavity. The inhibitor troglitazone occupies the upper portion of the active-site cavity, leaving a substantial part of the cavity unoccupied. The smaller neutral felodipine molecule is sequestered with its dichlorophenyl group positioned close to the heme iron, and water molecules fill the distal portion of the cavity. The structure of the 9-cis-retinoic acid complex reveals that two substrate molecules bind simultaneously in the active site of P450 2C8. A second molecule of 9-cis-retinoic acid is located above the proximal molecule and can restrain the position of the latter for more efficient oxygenation. Solution binding studies do not discriminate between cooperative and noncooperative models for multiple substrate binding. The complexes with structurally distinct ligands further demonstrate the conformational adaptability of active site-constituting residues, especially Arg-241, that can reorient in the active-site cavity to stabilize a negatively charged functional group and define two spatially distinct binding sites for anionic moieties of substrates. Cytochrome P450 2C8 is one of the principal drug-metabolizing P450 4The abbreviations used are: P450, a generic term for a cytochrome P450 enzyme (individual P450s are identified using a number-letter-number format based on amino acid sequence relatedness); CHAPS, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid. 4The abbreviations used are: P450, a generic term for a cytochrome P450 enzyme (individual P450s are identified using a number-letter-number format based on amino acid sequence relatedness); CHAPS, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid. monooxygenases expressed in human liver. It is the predominant hepatic P450 catalyzing the 6α-hydroxylation of paclitaxel (1Rahman A. Korzekwa K.R. Grogan J. Gonzalez F.J. Harris J.W. Cancer Res. 1994; 54: 5543-5546PubMed Google Scholar) and the epoxidation of arachidonic acid (2Daikh B.E. Lasker J.M. Raucy J.L. Koop D.R. J. Pharmacol. Exp. Ther. 1994; 271: 1427-1433PubMed Google Scholar, 3Zeldin D.C. DuBois R.N. Falck J.R. Capdevila J.H. Arch. Biochem. Biophys. 1995; 322: 76-86Crossref PubMed Scopus (151) Google Scholar). Additionally, P450 2C8 contributes extensively to the metabolism of drugs such as pioglitazone, rosiglitazone, troglitazone, amodiaquine, amiodarone, and cerivastatin (4Totah R.A. Rettie A.E. Clin. Pharmacol. Ther. 2005; 77: 341-352Crossref PubMed Scopus (192) Google Scholar) as well as to the oxidation of retinoic acid (5Leo M.A. Lasker J.M. Raucy J.L. Kim C.-I. Black M. Lieber C.S. Arch. Biochem. Biophys. 1989; 269: 305-312Crossref PubMed Scopus (179) Google Scholar, 6McSorley L.C. Daly A.K. Biochem. Pharmacol. 2000; 60: 517-526Crossref PubMed Scopus (127) Google Scholar, J. Biochem. Pharmacol. PubMed Scopus Google Scholar, J. M. Pharmacol. 2000; PubMed Scopus Google Scholar). A of used drugs and identified of P450 2C8, and felodipine J. Clin. Pharmacol. 2005; PubMed Scopus (151) Google Scholar). the of is a inhibitor of P450 2C8 M. J. Pharmacol. Exp. Ther. 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The distal portion of the cavity a of amino acid and and for of water molecules in the distal portion of the cavity. the binding of water in the portion of the cavity is to and binding of the substrate in the proximal portion of the cavity the heme to a molecule of troglitazone, two molecules of 9-cis-retinoic acid bind to P450 2C8 and the of the cavity and fill the available A between the two molecules of retinoic acid is the two molecules in the portion of the cavity the and The between the two molecules of retinoic acid the of molecule in the active The binding of the proximal molecule of 9-cis-retinoic acid in the portion of the cavity the retinoic acid for oxidation the site the The of montelukast is positioned close to the iron, that the Montelukast is a and relatively inhibitor of P450 2C8 in J. Clin. Pharmacol. 2005; PubMed Scopus (151) Google Scholar, 2005; PubMed Scopus Google montelukast not to the of in is P450 2C8 M. M. J. Clin. Pharmacol. PubMed Scopus Google Scholar, M. Clin. Pharmacol. Ther. PubMed Scopus Google Scholar). is to the of montelukast that the in of montelukast available for P450 2C8 binding. The of montelukast is to in M.A. M. A. J.H. Google Scholar) and in M. J.H. Google to the of the oxidation of montelukast P450 2C8 not is to a inhibitor of P450 2C8 J. Clin. Pharmacol. 2005; PubMed Scopus (151) Google Scholar). The dichlorophenyl is positioned in 2C8 for oxidation with the heme iron, are not of for the oxidation of the P450 2C8. is to the of the is P450 Pharmacol. PubMed Scopus Google Scholar, J. Google Scholar). troglitazone is not positioned for is to P450 2C8 A. M. M. Google Scholar). of the a position for oxidation to is not The moieties of montelukast and retinoic acid are to negatively charged The group of montelukast is located the and in exhibits with the of and the of The of the proximal retinoic acid is positioned to the of and A of the in the the available and the the substrate for The of portion of the cavity is to the binding of the moieties of to these neutral It is that a such as in the and not The binding interactions for these anionic are in to the binding of to P450 the occupies the site of a charged protein PubMed Scopus Google Scholar). binding with the interactions for the binding of the negatively charged group of troglitazone and for the binding of the of the distal retinoic acid The binding of the proximal retinoic acid in the portion of the cavity close to the heme for a second molecule of retinoic acid to bind in the portion of the cavity. 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It is that two molecules of acid bind to P450 2C8 and that the the binding of the distal molecule and the binding of the proximal substrate these the of P450 2C8 in the binding of anionic ligands that not with the pharmacophore model A. M. M.A. PubMed Scopus Google Scholar). The P450 2C8 complex structures demonstrate that two binding in the of of substrate inhibitor binding. The binding studies are with the binding of two molecules of 9-cis-retinoic acid to the for structure to the enzyme in The binding are to the of the to a well in the of the binding a of to protein and a to the binding models that two binding The can well a model with two binding sites binding models with for The relatively of to for of the protein that are to are to between these models and of the binding The for the of 9-cis-retinoic acid P450 2C8 is to J. Biochem. Pharmacol. PubMed Scopus Google is the for the the binding and for the of acid P450 2C8 L.C. Daly A.K. Biochem. Pharmacol. 2000; 60: 517-526Crossref PubMed Scopus (127) Google Scholar, J. M. Pharmacol. 2000; PubMed Scopus Google Scholar) and these the of the retinoic acid in as well as the for of retinoic acid in in these The binding of two molecules in the active site of P450 is to the the enzyme with a of substrates. The of can to and of oxidation and K.R. M. A. R.A. Rettie A.E. Gonzalez F.J. PubMed Google Scholar). studies a of binding to P450 in the molecule to a site that not a in the of the and binding of a second molecule the of the enzyme PubMed Scopus Google Scholar, 2005; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). that the molecule of retinoic acid that to P450 2C8 in a binding model a in the of the A structure of P450 that two molecules of the inhibitor in the active-site cavity M. A. PubMed Scopus Google Scholar). a structure of P450 B.E. Lasker J.M. Raucy J.L. Koop D.R. J. Pharmacol. Exp. Ther. 1994; 271: 1427-1433PubMed Google Scholar) exhibits two molecules of in the cavity A. D.C. M. M. M. Falck J.R. J. 2005; PubMed Scopus Google the of the two the enzyme P450 exhibits for the binding of and structures of the enzyme that two molecules of bind in the relatively large active site of the enzyme J. A. 2000; PubMed Scopus Google Scholar). The active-site of P450 and are relatively large and that the ligands can on the of P450 2C8, the two retinoic acid molecules in a portion of the cavity bind to more sites in the cavity of the shape of the cavity. the of the binding to P450 2C8 to P450 the of substrate for oxidation of P450 2C8 A. M. PubMed Scopus Google Scholar) the binding of a second substrate molecule to the distal site with a on the oxidation of the substrate to the proximal The structure of the troglitazone complex that binding of in a for a second molecule to P450 2C8 complexes with troglitazone, and 9-cis-retinoic acid two binding for anionic functional with of the active-site cavity, The P450 complex structure the inhibitor to sequestered close to the heme and water molecules in the distal portion of the active-site cavity. the active-site cavity is of binding structurally conformational protein conformational are to specific with the in the and the in A is in complexes with troglitazone and retinoic to stabilize the negatively charged functional The large size of the P450 2C8 active site and the of the of a pharmacophore The the structures determined in of P450 2C8 and the of more pharmacophore and for and the of the and for with