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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
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology.
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 11, article id e0142711Article in journal (Refereed) Published
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Text
Abstract [en]

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 mu M, respectively), whereas the (S)-enantiomer of Ibu-AM5 (IC50 0.59 mu M) was more potent than the (R)-enantiomer (IC50 5.7 mu M). 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.

Place, publisher, year, edition, pages
2015. Vol. 10, no 11, article id e0142711
National Category
Medicinal Chemistry Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-114918DOI: 10.1371/journal.pone.0142711ISI: 000367628500049OAI: oai:DiVA.org:umu-114918DiVA, id: diva2:909137
Available from: 2016-03-04 Created: 2016-01-29 Last updated: 2018-06-07Bibliographically approved
In thesis
1. Endocannabinoid metabolism: the impact of inflammatory factors and pharmacological inhibitors
Open this publication in new window or tab >>Endocannabinoid metabolism: the impact of inflammatory factors and pharmacological inhibitors
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Endokannabinoid metabolism : påverkan av inflammatoriska faktorer och farmakologiska inhibitorer
Abstract [en]

The endocannabinoid (eCB) system is an endogenous signaling system consisting of ligands (referred to as endocannabinoids, eCBs), receptors and metabolic enzymes. The eCB system is involved in homeostatic control of a variety of biological functions such as neuronal signaling, mood, appetite and pathological conditions such as pain, inflammation and tumour progression. The main eCBs N- arachidonoylethanolamine (AEA, anandamide) and 2-arachidonoylglycerol (2-AG) are synthesised upon stimuli when and where their action is demanded. The signaling is brief and the eCBs are quickly degraded. The enzyme primarily responsible for eCB degradation is fatty acid amide hydrolase (FAAH) for AEA and monoacylglycerol lipase (MAGL) for 2-AG. In addition, both substances are substrates for cyclooxygenase-2 (COX-2). COX-2 is upregulated in inflammation, pain and in several tumours including prostate cancers, but it is not known whether COX-2 contribute significantly to eCB metabolism under these conditions.

Increasing endogenous levels of eCBs by inhibiting their degradation is exploited as a future therapy for pain conditions. One suggested therapeutic strategy is dual inhibition of enzymes FAAH and COX-2 to raise AEA levels. Paper I and II of this thesis investigates FAAH and COX inhibitory effects of: the major metabolites and enantiomers of derivatives (flu-AM1 and ibu-AM5) of the current clinically used NSAIDs ibuprofen and flurbiprofen. The metabolites 3 ́hydroxyibuprofen and 4 ́hydroxyflurbiprofen retained the FAAH and COX- inhibitory effects seen by the parent compounds although at lower potencies. Both enantiomers of flu-AM1 were equally potent as FAAH inhibitors and displayed a useful substrate selective COX-2 inhibition profile, favoring eCBs as substrates rather than arachidonic acid.

Paper III explores the impact of COX-2 and the effect of (R)-flu-AM1 upon AEA levels and degradation in mouse leukemic macrophage RAW264.7 cells. Despite the high inhibitory potency in enzyme assays, neither (R)-flu-AM1 nor the combination of a FAAH inhibitor with flurbiprofen increased AEA levels in the intact cells to any great extent. This suggests that the eCB turnover in these cells is rather slow. Further, in paper IV, induction of COX-2 did not unmask an ability of this enzyme to “gate” the uptake of AEA analogous to that seen with FAAH.

Paper IV and V focus upon the role of the eCB system in prostate cancer. The eCB system is altered in cancer and is linked to the progression and prognosis of prostate cancer. How and whereby this change occurs is unknown. This thesis explores the impact of the inflammatory factors TNFα, IL-6 and lactic acid induced low pH upon the mRNA levels of eCB related enzymes and the functional impact upon AEA degradation in human DU145 and rat AT-1 prostate cancer cells. TNFα treatment of DU145 and IL-6 and lactic acid induced low pH exposure of AT-1 changed the mRNA levels of 2-AG related enzymes leaving AEA rather unaffected other than for a substantial induction of COX-2 mRNA in DU145 cells. Thus, AEA homeostasis was not shifted in prostate cancer cell lines exposed to inflammatory factors. The results suggest that COX-2 does not gate the uptake of AEA and is a minor contributor to AEA degradation in intact cells. 

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2018. p. 82
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1958
Keywords
endocannabinoid, anandamide, 2-AG, fatty acid amide hydrolase, cyclooxygenase-2, prostate cancer, catabolism, inflammation, inflammatory factors
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-147503 (URN)978-91-7601-870-5 (ISBN)
Public defence
2018-06-01, Major Groove, NUS byggnad 6L, Umeå, 09:00 (English)
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Available from: 2018-05-09 Created: 2018-05-04 Last updated: 2018-06-09Bibliographically approved

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Karlsson, JessicaSvensson, MonaFowler, Christopher J.

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