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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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Related in: MedlinePlus

Kinetics of inhibition of FAAH by A, (R)-Flu-AM1; B, (S)-Flu-AM1; C, (R)-Ibu-AM5 and D, (S)-Ibu-AM5.Left panels shown are means ± SEM, N = 3–4, when not enclosed within the symbol, of the FAAH activity at the added concentrations shown of AEA. Right panels show Dixon plots of the mean data. The dotted lines show the projections of the median intersection point on the axes.
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pone.0142711.g003: Kinetics of inhibition of FAAH by A, (R)-Flu-AM1; B, (S)-Flu-AM1; C, (R)-Ibu-AM5 and D, (S)-Ibu-AM5.Left panels shown are means ± SEM, N = 3–4, when not enclosed within the symbol, of the FAAH activity at the added concentrations shown of AEA. Right panels show Dixon plots of the mean data. The dotted lines show the projections of the median intersection point on the axes.

Mentions: The inhibition of the hydrolysis of 0.5–4 μM of added [3H]AEA by the (R)- and (S)-enantiomers of Flu-AM1 are shown in Fig 3. The data for the untransformed curves (left panels for the figures) were in both cases better fitted to a model assuming a linear mixed inhibition (see Statistics section of Materials and Methods for a description of the model) than a model assuming a pure competitive interaction. For (R)-Flu-AM1, the model returned Ki and α values of 0.63±0.17 μM and 3.2±1.3, respectively (Fig 3A, left panel). For (S)-Flu-AM1, the corresponding values were 0.79±0.28 μM and 5.6±3.6, respectively (Fig 3B, left panel). Dixon plots were also constructed for the data (Fig 3A and 3B right panels). For a mixed-type inhibitor, the straight lines obtained at each substrate concentration should intersect at a value which, when projected onto the x-axis, corresponds to -Ki [38,39]. For eight substrate concentrations as here, there are 28 different intersections. In an analogous situation, the direct linear plot of Eisenthal and Cornish-Bowden [40], those authors used the median values to solve the issue of undue influence of outliers on intersections. Using this approach, Ki values for (R)- and (S)-Flu-AM1 of 0.28 and 0.86 μM, respectively, were found.


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)

Kinetics of inhibition of FAAH by A, (R)-Flu-AM1; B, (S)-Flu-AM1; C, (R)-Ibu-AM5 and D, (S)-Ibu-AM5.Left panels shown are means ± SEM, N = 3–4, when not enclosed within the symbol, of the FAAH activity at the added concentrations shown of AEA. Right panels show Dixon plots of the mean data. The dotted lines show the projections of the median intersection point on the axes.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142711.g003: Kinetics of inhibition of FAAH by A, (R)-Flu-AM1; B, (S)-Flu-AM1; C, (R)-Ibu-AM5 and D, (S)-Ibu-AM5.Left panels shown are means ± SEM, N = 3–4, when not enclosed within the symbol, of the FAAH activity at the added concentrations shown of AEA. Right panels show Dixon plots of the mean data. The dotted lines show the projections of the median intersection point on the axes.
Mentions: The inhibition of the hydrolysis of 0.5–4 μM of added [3H]AEA by the (R)- and (S)-enantiomers of Flu-AM1 are shown in Fig 3. The data for the untransformed curves (left panels for the figures) were in both cases better fitted to a model assuming a linear mixed inhibition (see Statistics section of Materials and Methods for a description of the model) than a model assuming a pure competitive interaction. For (R)-Flu-AM1, the model returned Ki and α values of 0.63±0.17 μM and 3.2±1.3, respectively (Fig 3A, left panel). For (S)-Flu-AM1, the corresponding values were 0.79±0.28 μM and 5.6±3.6, respectively (Fig 3B, left panel). Dixon plots were also constructed for the data (Fig 3A and 3B right panels). For a mixed-type inhibitor, the straight lines obtained at each substrate concentration should intersect at a value which, when projected onto the x-axis, corresponds to -Ki [38,39]. For eight substrate concentrations as here, there are 28 different intersections. In an analogous situation, the direct linear plot of Eisenthal and Cornish-Bowden [40], those authors used the median values to solve the issue of undue influence of outliers on intersections. Using this approach, Ki values for (R)- and (S)-Flu-AM1 of 0.28 and 0.86 μM, respectively, were found.

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