title: 13-Methylarachidonic acid is a positive allosteric modulator of endocannabinoid oxygenation by cyclooxygenase.
abstract: Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide to prostaglandins, prostaglandin glyceryl esters, and prostaglandin ethanolamides, respectively. A structural homodimer, COX-2 acts as a conformational heterodimer with a catalytic and an allosteric monomer. Prior studies have demonstrated substrate-selective negative allosteric regulation of 2-AG oxygenation. Here we describe AM-8138 (13(S)-methylarachidonic acid), a substrate-selective allosteric potentiator that augments 2-AG oxygenation by up to 3.5-fold with no effect on AA oxygenation. In the crystal structure of an AM-8138·COX-2 complex, AM-8138 adopts a conformation similar to the unproductive conformation of AA in the substrate binding site. Kinetic analysis suggests that binding of AM-8138 to the allosteric monomer of COX-2 increases 2-AG oxygenation by increasing kcat and preventing inhibitory binding of 2-AG. AM-8138 restored the activity of COX-2 mutants that exhibited very poor 2-AG oxygenating activity and increased the activity of COX-1 toward 2-AG. Competition of AM-8138 for the allosteric site prevented the inhibition of COX-2-dependent 2-AG oxygenation by substrate-selective inhibitors and blocked the inhibition of AA or 2-AG oxygenation by nonselective time-dependent inhibitors. AM-8138 selectively enhanced 2-AG oxygenation in intact RAW264.7 macrophage-like cells. Thus, AM-8138 is an important new tool compound for the exploration of allosteric modulation of COX enzymes and their role in endocannabinoid metabolism.
authors: From the A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry.
authors: Shalley N
authors: SN
authors: Kudalkar
authors: the Department of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, and.
authors: Spyros P
authors: SP
authors: Nikas
authors: From the A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry.
authors: Philip J
authors: PJ
authors: Kingsley
authors: From the A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry.
authors: Shu
authors: S
authors: Xu
authors: From the A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry.
authors: James J
authors: JJ
authors: Galligan
authors: From the A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Vanderbilt Institute of Chemical Biology.
authors: Carol A
authors: CA
authors: Rouzer
authors: the Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, and the Northeastern Collaborative Access Team, Argonne National Laboratory, Argonne, Illinois 60439.
authors: Surajit
authors: S
authors: Banerjee
authors: the Department of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, and.
authors: Lipin
authors: L
authors: Ji
authors: the Department of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, and.
authors: Marsha R
authors: MR
authors: Eno
authors: the Department of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, and.
authors: Alexandros
authors: A
authors: Makriyannis
authors: From the A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry, Vanderbilt Institute of Chemical Biology, Chemistry, and Pharmacology, Vanderbilt Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, and Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, larry.marnett@vanderbilt.edu.
authors: Lawrence J
authors: LJ
authors: Marnett
mesh_terms: D000494:Allosteric Regulation; D001095:Arachidonic Acids / Q000494:pharmacology*; D063388:Endocannabinoids / Q000378:metabolism*; D007700:Kinetics; D010100:Oxygen / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*
chemical_list: C000598216:13-methylarachidonic acid; D001095:Arachidonic Acids; D063388:Endocannabinoids; D011451:Prostaglandin-Endoperoxide Synthases; D010100:Oxygen
keywords: Allosteric Regulation; Cyclooxygenase (COX); Endocannabinoid; Enzyme Kinetics; Site-directed Mutagenesis
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