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Interaction of the N-(3-Methylpyridin-2-yl)amide Derivatives of Flurbiprofen and Ibuprofen with FAAH: Enantiomeric Selectivity and Binding Mode.

Karlsson J, Morgillo CM, Deplano A, Smaldone G, Pedone E, Luque FJ, Svensson M, Novellino E, Congiu C, Onnis V, Catalanotti B, Fowler CJ - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Clinical Neuroscience, Pharmacology Unit, Umeå University, Umeå, Sweden.

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.

Methodology/principal findings: 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/significance: 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.

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Inhibition of FAAH by the enantiomers of Flu-AM1 and Ibu-AM5 and by Ibufenac-AM1.Structures of the compounds are shown in Panels A-C: A, Flu-AM1; B, Ibu-AM5; C, ibufenac-AM1. The asterisks show the chiral centres. In Panels D-F, the inhibition of 0.5 μM [3H]AEA hydrolysis in rat brain homogenates by the compounds is shown. Data are means ± SEM (when not enclosed by the symbols), N = 3 for the enantiomers of D, Flu-AM1; E, Ibu-AM5 and F, racemic Ibu-AM5 and Ibufenac-AM1.
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pone.0142711.g001: Inhibition of FAAH by the enantiomers of Flu-AM1 and Ibu-AM5 and by Ibufenac-AM1.Structures of the compounds are shown in Panels A-C: A, Flu-AM1; B, Ibu-AM5; C, ibufenac-AM1. The asterisks show the chiral centres. In Panels D-F, the inhibition of 0.5 μM [3H]AEA hydrolysis in rat brain homogenates by the compounds is shown. Data are means ± SEM (when not enclosed by the symbols), N = 3 for the enantiomers of D, Flu-AM1; E, Ibu-AM5 and F, racemic Ibu-AM5 and Ibufenac-AM1.

Mentions: Radioactive arachidonoyl ethanolamide[1-3H] ([3H]-AEA) was obtained from American Radiolabeled Chemicals, Inc (St Louis, MO, USA). (R)(-)-Ibuprofen and (S)(+)-ibuprofen, were purchased from Santa Cruz Biotechnology Inc. (Dallas, Texas, USA). The enantiomers of Flu-AM1 (structures, see Fig 1) were synthesised as described elsewhere [26]). AEA and URB597 (cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester) were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Carprofen was obtained from Sigma-Aldrich Inc, St. Louis, (MO, U.S.A.). Substrates were dissolved in ethanol or DMSO as appropriate.


Interaction of the N-(3-Methylpyridin-2-yl)amide Derivatives of Flurbiprofen and Ibuprofen with FAAH: Enantiomeric Selectivity and Binding Mode.

Karlsson J, Morgillo CM, Deplano A, Smaldone G, Pedone E, Luque FJ, Svensson M, Novellino E, Congiu C, Onnis V, Catalanotti B, Fowler CJ - PLoS ONE (2015)

Inhibition of FAAH by the enantiomers of Flu-AM1 and Ibu-AM5 and by Ibufenac-AM1.Structures of the compounds are shown in Panels A-C: A, Flu-AM1; B, Ibu-AM5; C, ibufenac-AM1. The asterisks show the chiral centres. In Panels D-F, the inhibition of 0.5 μM [3H]AEA hydrolysis in rat brain homogenates by the compounds is shown. Data are means ± SEM (when not enclosed by the symbols), N = 3 for the enantiomers of D, Flu-AM1; E, Ibu-AM5 and F, racemic Ibu-AM5 and Ibufenac-AM1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643906&req=5

pone.0142711.g001: Inhibition of FAAH by the enantiomers of Flu-AM1 and Ibu-AM5 and by Ibufenac-AM1.Structures of the compounds are shown in Panels A-C: A, Flu-AM1; B, Ibu-AM5; C, ibufenac-AM1. The asterisks show the chiral centres. In Panels D-F, the inhibition of 0.5 μM [3H]AEA hydrolysis in rat brain homogenates by the compounds is shown. Data are means ± SEM (when not enclosed by the symbols), N = 3 for the enantiomers of D, Flu-AM1; E, Ibu-AM5 and F, racemic Ibu-AM5 and Ibufenac-AM1.
Mentions: Radioactive arachidonoyl ethanolamide[1-3H] ([3H]-AEA) was obtained from American Radiolabeled Chemicals, Inc (St Louis, MO, USA). (R)(-)-Ibuprofen and (S)(+)-ibuprofen, were purchased from Santa Cruz Biotechnology Inc. (Dallas, Texas, USA). The enantiomers of Flu-AM1 (structures, see Fig 1) were synthesised as described elsewhere [26]). AEA and URB597 (cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester) were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Carprofen was obtained from Sigma-Aldrich Inc, St. Louis, (MO, U.S.A.). Substrates were dissolved in ethanol or DMSO as appropriate.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Clinical Neuroscience, Pharmacology Unit, Umeå University, Umeå, Sweden.

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.

Methodology/principal findings: 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/significance: 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.

Show MeSH
Related in: MedlinePlus