A simple stopped assay for fatty acid amide hydrolase avoiding the use of a chloroform extraction phase

doi:10.1016/j.jbbm.2004.04.020

Journal of Biochemical and Biophysical Methods

Volume 60, Issue 2, 31 August 2004, Pages 171-177

https://doi.org/10.1016/j.jbbm.2004.04.020 Get rights and content

Abstract

A stopped assay for fatty acid amide hydrolase (FAAH) has been developed, whereby the enzyme reaction product ([³H]ethanolamine) was separated from substrate (anandamide [ethanolamine-1-³H]), by differential adsorption to charcoal. The assay gave a better extraction efficiency when acidic rather than alkaline charcoal solutions were used to stop the reaction, and a very good ratio of sample/blank was also seen. The acidic charcoal assay gave the expected sensitivities to compounds known to inhibit FAAH (palmitoyltrifluoromethyl ketone, arvanil, AM404 and indomethacin). It is concluded that the acidic charcoal extraction method provides a robust and simple stopped assay for FAAH without the need to use potentially hazardous solvents like chloroform.

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Acknowledgements

C.F. would like to thank the Swedish Research Council (Grant no. 12158, medicine), Konung Gustav V's and Drottning Victorias Foundation, the Swedish Psoriasis Association, the Swedish Asthma and Allergy Association's Research Foundation, Gun and Bertil Stohne's Foundation and the Research Funds of the Medical Faculty, Umeå University, for financial support. This study was undertaken as a rotation project in the Biomedical Graduate School Programme, Umeå University.

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Following a further centrifugation, pellets were resuspended in 50 mM Tris-HCl buffer, pH 7.4, containing 1 mM EDTA and 3 mM MgCl2, and frozen at −80 °C in aliquots until used for the assay. The FAAH assay was conducted as described by Boldrup et al. [33]. Test compounds, homogenates and [3H]AEA (diluted with non-radioactive AEA to give a substrate concentration of 0.5 μM) in 10 mM Tris- HCl, 1 mM EDTA, pH 7.4, containing 1% w/v fatty acid-free bovine serum albumin were incubated for 10 min at 37 °C.
In experimental animals, inhibition of fatty acid amide hydrolase (FAAH) reduces the gastrointestinal damage produced by non-steroidal anti-inflammatory agents that act by inhibition of cyclooxygenase (COX). This suggests that compounds able to inhibit both enzymes may be potentially useful therapeutic agents. In the present study, we have investigated eight novel amide analogues of carprofen, ketoprofen and fenoprofen as potential FAAH/COX dual action inhibitors. Carpro-AM1 (2-(6-Chloro-9H-carbazol-2-yl)-N-(3-methylpyridin-2-yl)propenamide) and Carpro-AM6 (2-(6-Chloro-9H-carbazol-2-yl)-N-(3-chloropyridin-2-yl)propenamide) were found to be fully reversible inhibitors of the hydrolysis of 0.5 µM [³H]anandamide in rat brain homogenates with IC₅₀ values of 94 and 23 nM, respectively, i.e. 2–3 orders of magnitude more potent than carprofen in this respect. Both compounds inhibited the cyclooxygenation of arachidonic acid by ovine COX-1, and were more potent inhibitors of human recombinant COX-2 when 2-arachidonoylglycerol was used as substrate than when arachidonic acid was used. It is concluded that Carpro-AM1 and Carpro-AM6 are dual-acting FAAH/substrate-selective COX inhibitors.
Low mRNA expression and activity of monoacylglycerol lipase in human SH-SY5Y neuroblastoma cells
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Relatively little is known about the endocannabinoid system in human neuroblastoma cell lines. In the present study, we have investigated the expression of the genes coding for the enzymes involved in the synthesis and catabolism of endocannabinoids in the SH-SY5Y cell line. The expression of MGLL, the gene coding for the 2-arachidonoylglycerol hydrolytic enzyme monoacylglycerol lipase (MAGL), was found to be 85 and 340 fold lower than the expression levels for the genes coding for alpha/beta-hydrolase domain containing 6 and 12 (ABHD6, ABHD12), which are alternative hydrolytic enzymes for this endocannabinoid. In comparison, mRNA levels of MGLL were 1.5 fold higher than ABHD6 and 2 fold lower than the levels of ABHD12 in DU-145 human prostate cells. In functional assays, the hydrolysis of the 2-arachidonoylglycerol homologue 2-oleoylglycerol by intact SH-SY5Y cells was partially inhibited by the ABHD6 inhibitor WWL70, but not by the MAGL inhibitor JZL184, whereas the reverse was true in DU-145 cells. The combination of JZL184 + WWL70 did, however produce a significantly greater inhibition of 2-OG hydrolysis than seen with WWL70 alone in the SH-SY5Y cells. The low MGLL expression in the SH-SY5Y cells was not due to epigenetic silencing, since levels were not affected by treatment with the methylation inhibitor 5-aza-2′-deoxycytidine and/or the histone acetylase inhibitor trichostatin A. The low MGLL expression in SH-SY5Y cells should be taken into account when using these cells in experiments investigating the involvement of the endocannabinoid system in models of physiological and pathological processes.
n−3 polyunsaturated N-acylethanolamines are CB<inf>2</inf> cannabinoid receptor-preferring endocannabinoids
2018, Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
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While the synthetic pathway/s are more difficult to assay, the predominant hydrolytic enzyme, FAAH, has been investigated through a variety of different assays (for examples, see [21,40,41]). Some of these assays rely on synthetic substrates which generate spectrophotometrically-measurable products [41], while others use radiolabelled versions of a natural substrate [40]. While this latter assay has become a ‘Gold Standard’ for measurement of ex vivo enzyme activities, it lacks versatility in identifying kinetic characteristics of alternative endogenous substrates; that is, an alternative substrate cannot be distinguished from an inhibitor.
Anandamide, the first identified endogenous cannabinoid and TRPV1 agonist, is one of a series of endogenous N-acylethanolamines, NAEs. We have generated novel assays to quantify the levels of multiple NAEs in biological tissues and their rates of hydrolysis through fatty acid amide hydrolase. This range of NAEs was also tested in rapid response assays of CB₁, CB₂ cannabinoid and TRPV1 receptors. The data indicate that PEA, SEA and OEA are not endocannabinoids or endovanilloids, and that the higher endogenous levels of these metabolites compared to polyunsaturated analogues are a correlate of their slow rates of hydrolysis. The n−6 NAEs (AEA, docosatetraenoyl and docosapentaenoyl derivatives) activated both CB₁ and CB₂ receptors, as well as TRPV1 channels, suggesting them to be ‘genuine’ endocannabinoids and ‘endovanilloids’. The n−3 NAEs (eicosapentaenoyl, docosapentaenoyl and docosahexaenoyl derivatives) activated CB₂ receptors and some n−3 NAEs (docosapentaenoyl and docosahexaenoyl derivatives) also activated TRPV1 channels, but failed to activate the CB₁ receptor. We hypothesise that the preferential activation of CB₂ receptors by n−3 PUFA NAEs contributes, at least in some part, to their broad anti-inflammatory profile.
Novel propanamides as fatty acid amide hydrolase inhibitors
2017, European Journal of Medicinal Chemistry
Fatty acid amide hydrolase (FAAH) has a key role in the control of the cannabinoid signaling, through the hydrolysis of the endocannabinoids anandamide and in some tissues 2-arachidonoylglycerol. FAAH inhibition represents a promising strategy to activate the cannabinoid system, since it does not result in the psychotropic and peripheral side effects characterizing the agonists of the cannabinoid receptors. Here we present the discovery of a novel class of profen derivatives, the N-(heteroaryl)-2-(4-((2-(trifluoromethyl)pyridin-4-yl)amino)phenyl)propanamides, as FAAH inhibitors. Enzymatic assays showed potencies toward FAAH ranging from nanomolar to micromolar range, and the most compounds lack activity toward the two isoforms of cyclooxygenase. Extensive structure-activity studies and the definition of the binding mode for the lead compound of the series are also presented. Kinetic assays in rat and mouse FAAH on selected compounds of the series demonstrated that slight modifications of the chemical structure could influence the binding mode and give rise to competitive (TPA1) or non-competitive (TPA14) inhibition modes.
Inhibition of fatty acid amide hydrolase and cyclooxygenase by the N-(3-methylpyridin-2-yl)amide derivatives of flurbiprofen and naproxen
2013, European Journal of Pharmacology
Citation Excerpt :
Homogenates were again centrifuged, and the pellets were resuspended in 50 mM Tris–HCl buffer, pH 7.4, containing 1 mM EDTA and 3 mM MgCl2 and thereafter frozen at −80 °C in aliquots until used for assay. FAAH was assayed using the method of Boldrup et al. (2004) with [3H]AEA labelled in the ethanolamine part of the molecule as substrate (final concentration 0.5 µM, unless otherwise stated). Homogenates (usually 0.5 μg protein per assay, diluted with 10 mM Tris–HCl, 1 mM EDTA pH 7.4), test compounds (in ethanol) and 25 µL [3H]AEA (in 10 mM Tris–HCl, 1 mM EDTA, pH 7.4, containing 1% w/v fatty acid-free bovine serum albumin) were incubated for 10 min at 37 °C (final assay volume 200 μL).
Inhibitors of the metabolism of the endogenous cannabinoid ligand anandamide by fatty acid amide hydrolase (FAAH) reduce the gastric damage produced by non-steroidal anti-inflammatory agents and synergise with them in experimental pain models. This motivates the design of compounds with joint FAAH/cyclooxygenase (COX) inhibitory activity. Here we present data on the N-(3-methylpyridin-2-yl)amide derivatives of flurbiprofen and naproxen (Flu-AM1 and Nap-AM1, respectively) with respect to their properties towards these two enzymes. Flu-AM1 and Nap-AM1 inhibited FAAH-catalysed hydrolysis of [³H]anandamide by rat brain homogenates with IC₅₀ values of 0.44 and 0.74 µM. The corresponding values for flurbiprofen and naproxen were 29 and >100 µM, respectively. The inhibition by Flu-AM1 was reversible, mixed-type, with Kⁱ_slope and Kⁱ_intercept values of 0.21 and 1.4 µM, respectively. Flurbiprofen and Flu-AM1 both inhibited COX in the same manner with the order of potencies COX-2 vs. 2-arachidonoylglycerol>COX-1 vs. arachidonic acid>COX-2 vs. arachidonic acid with flurbiprofen being approximately 2–3 fold more potent than Flu-AM1 in the assays. Nap-AM1 was a less potent inhibitor of COX. Flu-AM1 at low micromolar concentrations inhibited the FAAH-driven uptake of [³H]anandamide into RBL2H3 basophilic leukaemia cells in vitro, but did not penetrate the brain in vivo sufficiently to block the binding of [¹⁸F]DOPP to brain FAAH. It is concluded that Flu-AM1 is a dual-action inhibitor of FAAH and COX that may be useful in exploring the optimal balance of effects on these two enzyme systems in producing peripheral alleviation of pain and inflammation in experimental models.
Development and characterization of a promising fluorine-18 labelled radiopharmaceutical for in vivo imaging of fatty acid amide hydrolase
2013, Bioorganic and Medicinal Chemistry
Citation Excerpt :
The synthesis took 52–55 min from end-of-bombardment while the radiochemical purity was >97% with specific activities of 74–144 GBq/μmol at end-of-synthesis. The ability of 5 to inhibit FAAH activity was measured using rat brain homogenates and [3H]anandamide as substrate50 (Fig. 1). Following a 60 min preincubation phase, the compound inhibited FAAH with a pI50 value of 9.09 ± 0.04, corresponding to an IC50 value of 0.82 nM (Fig. 1A).
Fatty acid amide hydrolase (FAAH), the enzyme responsible for terminating signaling by the endocannabinoid anandamide, plays an important role in the endocannabinoid system, and FAAH inhibitors are attractive drugs for pain, addiction, and neurological disorders. The synthesis, radiosynthesis, and evaluation, in vitro and ex vivo in rat, of an ¹⁸F-radiotracer designed to image FAAH using positron emission tomography (PET) is described.
Fluorine-18 labelled 3-(4,5-dihydrooxazol-2-yl)phenyl (5-fluoropentyl)carbamate, [¹⁸F]5, was synthesized at high specific activity in a one-pot three step reaction using a commercial module with a radiochemical yield of 17–22% (from [¹⁸F]fluoride). In vitro assay using rat brain homogenates showed that 5 inhibited FAAH in a time-dependent manner, with an IC₅₀ value of 0.82 nM after a preincubation of 60 min. Ex vivo biodistribution studies and ex vivo autoradiography in rat brain demonstrated that [¹⁸F]5 had high brain penetration with standard uptake values of up to 4.6 and had a regional distribution which correlated with reported regional FAAH enzyme activity. Specificity of binding to FAAH with [¹⁸F]5 was high (>90%) as demonstrated by pharmacological challenges with potent and selective FAAH inhibitors and was irreversible as demonstrated by radioactivity measurements on homogenized brain tissue extracts.
We infer from these results that [¹⁸F]5 is a highly promising candidate radiotracer with which to image FAAH in human subjects using PET and clinical studies are proceeding.

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Short communicationA simple stopped assay for fatty acid amide hydrolase avoiding the use of a chloroform extraction phase

Abstract

Section snippets

Acknowledgements

Prog. Neurobiol.

Brain Res. Rev.

Biochem. Pharmacol.

Biochem. Pharmacol.

J. Biol. Chem.

Anal. Biochem.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

Life Sci.

Anal. Biochem.

Short communication
A simple stopped assay for fatty acid amide hydrolase avoiding the use of a chloroform extraction phase