title: Interaction of the N-(3-Methylpyridin-2-yl)amide Derivatives of Flurbiprofen and Ibuprofen with FAAH: Enantiomeric Selectivity and Binding Mode.
abstract: BACKGROUND
  Combined fatty acid amide hydrolase (FAAH) and cyclooxygenase (COX) inhibition is a promising approach for pain-relief. The Flu-AM1 and Ibu-AM5 derivatives of flurbiprofen and ibuprofen retain similar COX-inhibitory properties and are more potent inhibitors of FAAH than the parent compounds. However, little is known as to the nature of their interaction with FAAH, or to the importance of their chirality. This has been explored here.
  
  
  RESULTS
  FAAH inhibitory activity was measured in rat brain homogenates and in lysates expressing either wild-type or FAAH(T488A)-mutated enzyme. Molecular modelling was undertaken using both docking and molecular dynamics. The (R)- and (S)-enantiomers of Flu-AM1 inhibited rat FAAH with similar potencies (IC50 values of 0.74 and 0.99 μM, respectively), whereas the (S)-enantiomer of Ibu-AM5 (IC50 0.59 μM) was more potent than the (R)-enantiomer (IC50 5.7 μM). Multiple inhibition experiments indicated that both (R)-Flu-AM1 and (S)-Ibu-AM5 inhibited FAAH in a manner mutually exclusive to carprofen. Computational studies indicated that the binding site for the Flu-AM1 and Ibu-AM5 enantiomers was located between the acyl chain binding channel and the membrane access channel, in a site overlapping the carprofen binding site, and showed a binding mode in line with that proposed for carprofen and other non-covalent ligands. The potency of (R)-Flu-AM1 was lower towards lysates expressing FAAH mutated at the proposed carprofen binding area than in lysates expressing wild-type FAAH.
  
  
  CONCLUSIONS
  The study provides kinetic and structural evidence that the enantiomers of Flu-AM1 and Ibu-AM5 bind in the substrate channel of FAAH. This information will be useful in aiding the design of novel dual-action FAAH: COX inhibitors.
authors.affiliation: Department of Pharmacology and Clinical Neuroscience, Pharmacology Unit, Umeå University, Umeå, Sweden.
authors.forename: Jessica
authors.initials: J
authors.lastname: Karlsson
authors.affiliation: Department of Pharmacy, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Carmine M
authors.initials: CM
authors.lastname: Morgillo
authors.affiliation: Department of Life and Environmental Sciences, Unit of Pharmaceutical, Pharmacological and Nutraceutical Sciences, University of Cagliari, via Ospedale 72, Cagliari, I-09124, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Deplano
authors.affiliation: Institute of Biostructures and Bioimaging, CNR, Naples, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Smaldone
authors.affiliation: Institute of Biostructures and Bioimaging, CNR, Naples, Italy.
authors.forename: Emilia
authors.initials: E
authors.lastname: Pedone
authors.affiliation: Departament de Fisicoquímica and Institut de Biomedicina (IBUB), Facultat de Farmàcia, Universitat de Barcelona, Santa Coloma de Gramenet, Spain.
authors.forename: F Javier
authors.initials: FJ
authors.lastname: Luque
authors.affiliation: Department of Pharmacology and Clinical Neuroscience, Pharmacology Unit, Umeå University, Umeå, Sweden.
authors.forename: Mona
authors.initials: M
authors.lastname: Svensson
authors.affiliation: Department of Pharmacy, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Ettore
authors.initials: E
authors.lastname: Novellino
authors.affiliation: Department of Life and Environmental Sciences, Unit of Pharmaceutical, Pharmacological and Nutraceutical Sciences, University of Cagliari, via Ospedale 72, Cagliari, I-09124, Italy.
authors.forename: Cenzo
authors.initials: C
authors.lastname: Congiu
authors.affiliation: Department of Life and Environmental Sciences, Unit of Pharmaceutical, Pharmacological and Nutraceutical Sciences, University of Cagliari, via Ospedale 72, Cagliari, I-09124, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Onnis
authors.affiliation: Department of Pharmacy, Università degli Studi di Napoli Federico II, Napoli, Italy.
authors.forename: Bruno
authors.initials: B
authors.lastname: Catalanotti
authors.affiliation: Department of Pharmacology and Clinical Neuroscience, Pharmacology Unit, Umeå University, Umeå, Sweden.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Fowler
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000631:Aminopyridines / Q000737:chemistry*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D016861:Cyclooxygenase Inhibitors / Q000737:chemistry; D015195:Drug Design; D005480:Flurbiprofen / Q000031:analogs & derivatives* / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D007052:Ibuprofen / Q000031:analogs & derivatives* / Q000737:chemistry; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008297:Male; D051379:Mice; D056004:Molecular Dynamics Simulation; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000378:metabolism; D013237:Stereoisomerism; D000099252:Fatty Acid Amide Hydrolases
chemical_list: D000581:Amidohydrolases; D000631:Aminopyridines; D016861:Cyclooxygenase Inhibitors; D005480:Flurbiprofen; D007052:Ibuprofen; D011451:Prostaglandin-Endoperoxide Synthases; D011994:Recombinant Proteins; D000099252:Fatty Acid Amide Hydrolases; C000608610:flurbiprofen 2-amino-3-methylpyridine amide; C000608609:ibuprofen 2-amino-3-methylpyridine amide