title: Conservative Secondary Shell Substitution In Cyclooxygenase-2 Reduces Inhibition by Indomethacin Amides and Esters via Altered Enzyme Dynamics.
abstract: The cyclooxygenase enzymes (COX-1 and COX-2) are the therapeutic targets of nonsteroidal anti-inflammatory drugs (NSAIDs). Neutralization of the carboxylic acid moiety of the NSAID indomethacin to an ester or amide functionality confers COX-2 selectivity, but the molecular basis for this selectivity has not been completely revealed through mutagenesis studies and/or X-ray crystallographic attempts. We expressed and assayed a number of divergent secondary shell COX-2 active site mutants and found that a COX-2 to COX-1 change at position 472 (Leu in COX-2, Met in COX-1) reduced the potency of enzyme inhibition by a series of COX-2-selective indomethacin amides and esters. In contrast, the potencies of indomethacin, arylacetic acid, propionic acid, and COX-2-selective diarylheterocycle inhibitors were either unaffected or only mildly affected by this mutation. Molecular dynamics simulations revealed identical equilibrium enzyme structures around residue 472; however, calculations indicated that the L472M mutation impacted local low-frequency dynamical COX constriction site motions by stabilizing the active site entrance and slowing constriction site dynamics. Kinetic analysis of inhibitor binding is consistent with the computational findings.
authors.affiliation: Departments of Biochemistry, ‡Chemistry, and §Pharmacology, Vanderbilt Institute of Chemical Biology, Center for Structural Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville Tennessee 37232-0146, United States.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Konkle
authors.affiliation: Departments of Biochemistry, ‡Chemistry, and §Pharmacology, Vanderbilt Institute of Chemical Biology, Center for Structural Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville Tennessee 37232-0146, United States.
authors.forename: Anna L
authors.initials: AL
authors.lastname: Blobaum
authors.affiliation: Departments of Biochemistry, ‡Chemistry, and §Pharmacology, Vanderbilt Institute of Chemical Biology, Center for Structural Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville Tennessee 37232-0146, United States.
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Moth
authors.affiliation: Departments of Biochemistry, ‡Chemistry, and §Pharmacology, Vanderbilt Institute of Chemical Biology, Center for Structural Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville Tennessee 37232-0146, United States.
authors.forename: Jeffery J
authors.initials: JJ
authors.lastname: Prusakiewicz
authors.affiliation: Departments of Biochemistry, ‡Chemistry, and §Pharmacology, Vanderbilt Institute of Chemical Biology, Center for Structural Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville Tennessee 37232-0146, United States.
authors.forename: Shu
authors.initials: S
authors.lastname: Xu
authors.affiliation: Departments of Biochemistry, ‡Chemistry, and §Pharmacology, Vanderbilt Institute of Chemical Biology, Center for Structural Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville Tennessee 37232-0146, United States.
authors.forename: Kebreab
authors.initials: K
authors.lastname: Ghebreselasie
authors.affiliation: Departments of Biochemistry, ‡Chemistry, and §Pharmacology, Vanderbilt Institute of Chemical Biology, Center for Structural Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville Tennessee 37232-0146, United States.
authors.forename: Dapo
authors.initials: D
authors.lastname: Akingbade
authors.affiliation: Departments of Biochemistry, ‡Chemistry, and §Pharmacology, Vanderbilt Institute of Chemical Biology, Center for Structural Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville Tennessee 37232-0146, United States.
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Jacobs
authors.affiliation: Departments of Biochemistry, ‡Chemistry, and §Pharmacology, Vanderbilt Institute of Chemical Biology, Center for Structural Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville Tennessee 37232-0146, United States.
authors.forename: Carol A
authors.initials: CA
authors.lastname: Rouzer
authors.affiliation: Departments of Biochemistry, ‡Chemistry, and §Pharmacology, Vanderbilt Institute of Chemical Biology, Center for Structural Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville Tennessee 37232-0146, United States.
authors.forename: Terry P
authors.initials: TP
authors.lastname: Lybrand
authors.affiliation: Departments of Biochemistry, ‡Chemistry, and §Pharmacology, Vanderbilt Institute of Chemical Biology, Center for Structural Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine , Nashville Tennessee 37232-0146, United States.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Marnett
mesh_terms: D000577:Amides / Q000737:chemistry*; D019295:Computational Biology; D051546:Cyclooxygenase 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004952:Esters / Q000737:chemistry*; D007213:Indomethacin / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
chemical_list: D000577:Amides; D004952:Esters; D051546:Cyclooxygenase 2; D007213:Indomethacin