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N-Acylethanolamine-hydrolyzing acid amidase inhibition increases colon N-palmitoylethanolamine levels and counteracts murine colitis.

ABSTRACT: N-Palmitoylethanolamine or palmitoylethanolamide (PEA) is an anti-inflammatory compound that was recently shown to exert peroxisome proliferator-activated receptor-α-dependent beneficial effects on colon inflammation. The actions of PEA are terminated following hydrolysis by 2 enzymes: fatty acid amide hydrolase (FAAH), and the less-studied N-acylethanolamine-hydrolyzing acid amidase (NAAA). This study aims to investigate the effects of inhibiting the enzymes responsible for PEA hydrolysis in colon inflammation in order to propose a potential therapeutic target for inflammatory bowel diseases (IBDs). Two murine models of IBD were used to assess the effects of NAAA inhibition, FAAH inhibition, and PEA on macroscopic signs of colon inflammation, macrophage/neutrophil infiltration, and the expression of proinflammatory mediators in the colon, as well as on the colitis-related systemic inflammation. NAAA inhibition increases PEA levels in the colon and reduces colon inflammation and systemic inflammation, similarly to PEA. FAAH inhibition, however, does not increase PEA levels in the colon and does not affect the macroscopic signs of colon inflammation or immune cell infiltration. This is the first report of an anti-inflammatory effect of a systemically administered NAAA inhibitor. Because NAAA is the enzyme responsible for the control of PEA levels in the colon, we put forth this enzyme as a potential therapeutic target in chronic inflammation in general and IBD in particular.

SUBMITTER: Alhouayek M

PROVIDER: S-EPMC4763939 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Endogenous molecules stimulating N-acylethanolamine-hydrolyzing acid amidase (NAAA).

Project description:Fatty acid amide hydrolase (FAAH) plays the central role in the degradation of bioactive N-acylethanolamines such as the endocannabinoid arachidonoylethanolamide (anandamide) in brain and peripheral tissues. A lysosomal enzyme referred to as N-acylethanolamine-hydrolyzing acid amidase (NAAA) catalyzes the same reaction with preference to palmitoylethanolamide, an endogenous analgesic and neuroprotective substance, and is therefore expected as a potential target of therapeutic drugs. In the in vitro assays thus far performed, the maximal activity of NAAA was achieved in the presence of both nonionic detergent (Triton X-100 or Nonidet P-40) and the SH reagent dithiothreitol. However, endogenous molecules that might substitute for these synthetic compounds remain poorly understood. Here, we examined stimulatory effects of endogenous phospholipids and thiol compounds on recombinant NAAA. Among different phospholipids tested, choline- or ethanolamine-containing phospholipids showed potent effects, and 1 mM phosphatidylcholine increased NAAA activity by 6.6-fold. Concerning endogenous thiol compounds, dihydrolipoic acid at 0.1-1 mM was the most active, causing 8.5-9.0-fold stimulation. These results suggest that endogenous phospholipids and dihydrolipoic acid may contribute in keeping NAAA active in lysosomes. Even in the presence of phosphatidylcholine and dihydrolipoic acid, however, the preferential hydrolysis of palmitoylethanolamide was unaltered. We also investigated a possible compensatory induction of NAAA mRNA in brain and other tissues of FAAH-deficient mice. However, NAAA expression levels in all the tissues examined were not significantly altered from those in wild-type mice.

| S-EPMC3382453 | biostudies-other

Biochemical and mass spectrometric characterization of human N-acylethanolamine-hydrolyzing acid amidase inhibition.

Project description:The mechanism of inactivation of human enzyme N-acylethanolamine-hydrolyzing acid amidase (hNAAA), with selected inhibitors identified in a novel fluorescent based assay developed for characterization of both reversible and irreversible inhibitors, was investigated kinetically and using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 1-Isothiocyanatopentadecane (AM9023) was found to be a potent, selective and reversible hNAAA inhibitor, while two others, 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701) and N-Benzyloxycarbonyl-L-serine ?-lactone (N-Cbz-serine ?-lactone), inhibited hNAAA in a covalent and irreversible manner. MS analysis of the hNAAA/covalent inhibitor complexes identified modification only of the N-terminal cysteine (Cys126) of the ?-subunit, confirming a suggested mechanism of hNAAA inactivation by the ?-lactone containing inhibitors. These experiments provide direct evidence of the key role of Cys126 in hNAAA inactivation by different classes of covalent inhibitors, confirming the essential role of cysteine for catalysis and inhibition in this cysteine N-terminal nucleophile hydrolase enzyme. They also provide a methodology for the rapid screening and characterization of large libraries of compounds as potential inhibitors of NAAA, and subsequent characterization or their mechanism through MALDI-TOF MS based bottom up-proteomics.

| S-EPMC3432061 | biostudies-literature

Selective N-acylethanolamine-hydrolyzing acid amidase inhibition reveals a key role for endogenous palmitoylethanolamide in inflammation.

Project description:Identifying points of control in inflammation is essential to discovering safe and effective antiinflammatory medicines. Palmitoylethanolamide (PEA) is a naturally occurring lipid amide that, when administered as a drug, inhibits inflammatory responses by engaging peroxisome proliferator-activated receptor-alpha (PPAR-alpha). PEA is preferentially hydrolyzed by the cysteine amidase N-acylethanolamine-hydrolyzing acid amidase (NAAA), which is highly expressed in macrophages. Here we report the discovery of a potent and selective NAAA inhibitor, N-[(3S)-2-oxo-3-oxetanyl]-3-phenylpropanamide [(S)-OOPP], and show that this inhibitor increases PEA levels in activated leukocytes and blunts responses induced by inflammatory stimuli both in vitro and in vivo. These effects are stereoselective, mimicked by exogenous PEA, and abolished by PPAR-alpha deletion. (S)-OOPP also attenuates inflammation and tissue damage and improves recovery of motor function in mice subjected to spinal cord trauma. The results suggest that PEA activation of PPAR-alpha in leukocytes serves as an early stop signal that contrasts the progress of inflammation. The PEA-hydrolyzing amidase NAAA may provide a previously undescribed target for antiinflammatory medicines.

| S-EPMC2791595 | biostudies-literature

Design and synthesis of potent N-acylethanolamine-hydrolyzing acid amidase (NAAA) inhibitor as anti-inflammatory compounds.

Project description:N-acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme involved in biological deactivation of N-palmitoylethanolamide (PEA), which exerts anti-inflammatory and analgesic effects through the activation of nuclear receptor peroxisome proliferator-activated receptor-alpha (PPAR-?). To develop selective and potent NAAA inhibitors, we designed and synthesized a series of derivatives of 1-pentadecanyl-carbonyl pyrrolidine (compound 1), a general amidase inhibitor. Structure activity relationship (SAR) studies have identified a compound 16, 1-(2-Biphenyl-4-yl)ethyl-carbonyl pyrrolidine, which has shown the highest inhibition on NAAA activity (IC(50)?=2.12 ± 0.41 µM) and is characterized as a reversible and competitive NAAA inhibitor. Computational docking analysis and mutagenesis study revealed that compound 16 interacted with Asparagine 209 (Asn(209)) residue flanking the catalytic pocket of NAAA so as to block the substrate entrance. In vitro pharmacological studies demonstrated that compound 16 dose-dependently reduced mRNA expression levels of iNOS and IL-6, along with an increase of intracellular PEA levels, in mouse macrophages with lipopolysaccharides (LPS) induced inflammation. Our study discovered a novel NAAA inhibitor, compound 16, that could serve as a potential anti-inflammatory agent.

| S-EPMC3423427 | biostudies-literature

Interferon γ treatment increases endocannabinoid and related N-acylethanolamine levels in T84 human colon carcinoma cells.

Project description:Background and purposeEndocannabinoids and related N-acylethanolamines (NAEs) are involved in regulation of gut function, but relatively little is known as to whether inflammatory cytokines such as IFNγ affect their levels. We have investigated this in vitro using cultures of T84 colon cancer cells.Experimental approachT84 cells, when cultured in monolayers, differentiate to form adult colonic crypt-like cells with excellent permeability barrier properties. The integrity of the permeability barrier in these monolayers was measured using transepithelial electrical resistance (TEER). NAE levels were determined by ultra-performance liquid chromatography-tandem mass spectrometric analysis. Expression of the enzymes involved in NAE and 2-arachidonoylglycerol (2-AG) turnover were assessed with qPCR.Key resultsIFNγ treatment for 8 or 24 h increased levels of both endocannabinoids (anandamide and 2-AG) and the related NAEs. The treatment did not affect the rate of hydrolysis of either anandamide or palmitoylethanolamide by intact cells, and in both cases, fatty acid amide hydrolase (FAAH) rather than NAE-hydrolysing acid amidase (NAAA) was mainly responsible for the hydrolysis of these NAEs. IFNγ treatment reduced the TEER of the cells in a manner that was not prevented by inhibition of either FAAH or NAAA but was partially reversed by apical administration of the NAE palmitoylethanolamide.Conclusion and implicationsIFNγ treatment mobilized endocannabinoid and related NAE levels in T84 cells. However, blockade of anandamide or NAE hydrolysis was insufficient to negate the deleterious effects of this cytokine upon the permeability barrier of the cell monolayers.Linked articlesThis article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

| S-EPMC6487551 | biostudies-literature

Activity-Based Probe for N-Acylethanolamine Acid Amidase.

Project description:N-Acylethanolamine acid amidase (NAAA) is a lysosomal cysteine hydrolase involved in the degradation of saturated and monounsaturated fatty acid ethanolamides (FAEs), a family of endogenous lipid signaling molecules that includes oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). Among the reported NAAA inhibitors, ?-amino-?-lactone (3-aminooxetan-2-one) derivatives have been shown to prevent FAE hydrolysis in innate-immune and neural cells and to reduce reactions to inflammatory stimuli. Recently, we disclosed two potent and selective NAAA inhibitors, the compounds ARN077 (5-phenylpentyl-N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl]carbamate) and ARN726 (4-cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate). The former is active in vivo by topical administration in rodent models of hyperalgesia and allodynia, while the latter exerts systemic anti-inflammatory effects in mouse models of lung inflammation. In the present study, we designed and validated a derivative of ARN726 as the first activity-based protein profiling (ABPP) probe for the in vivo detection of NAAA. The newly synthesized molecule 1 is an effective in vitro and in vivo click-chemistry activity based probe (ABP), which is able to capture the catalytically active form of NAAA in Human Embryonic Kidney 293 (HEK293) cells overexpressing human NAAA as well as in rat lung tissue. Competitive ABPP with 1 confirmed that ARN726 and ARN077 inhibit NAAA in vitro and in vivo. Compound 1 is a useful new tool to identify activated NAAA both in vitro and in vivo and to investigate the physiological and pathological roles of this enzyme.

| S-EPMC4575644 | biostudies-literature

N-Acylethanolamine-Hydrolyzing Acid Amidase Inhibition, but Not Fatty Acid Amide Hydrolase Inhibition, Prevents the Development of Experimental Autoimmune Encephalomyelitis in Mice.

Project description:N-acylethanolamines (NAEs) are endogenous bioactive lipids reported to exert anti-inflammatory and neuroprotective effects mediated by cannabinoid receptors and peroxisome proliferator-activated receptors (PPARs), among others. Therefore, interfering with NAE signaling could be a promising strategy to decrease inflammation in neurological disorders such as multiple sclerosis (MS). Fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase (NAAA) are key modulators of NAE levels. This study aims to investigate and compare the effect of NAAA inhibition, FAAH inhibition, and dual inhibition of both enzymes in a mouse model of MS, namely the experimental autoimmune encephalomyelitis (EAE). Our data show that NAAA inhibition strongly decreased the hallmarks of the pathology. Interestingly, FAAH inhibition was less efficient in decreasing inflammatory hallmarks despite the increased NAE levels. Moreover, the inhibition of both NAAA and FAAH, using a dual-inhibitor or the co-administration of NAAA and FAAH inhibitors, did not show an added value compared to NAAA inhibition. Furthermore, our data suggest an important role of decreased activation of astrocytes and microglia in the effects of NAAA inhibition on EAE, while NAAA inhibition did not affect T cell recall. This work highlights the beneficial effects of NAAA inhibition in the context of central nervous system inflammation and suggests that the simultaneous inhibition of NAAA and FAAH has no additional beneficial effect in EAE.

| S-EPMC8609003 | biostudies-literature

Secretion, isotopic labeling and deglycosylation of N-acylethanolamine acid amidase for biophysical studies.

Project description:N-acylethanolamine acid amidase (NAAA) is an N-terminal nucleophile (Ntn) enzyme with a catalytic cysteine residue that has highest activity at acidic pH. The most prominent substrate hydrolyzed is palmitoylethanolamine (PEA), which regulates inflammation. Inhibitors of NAAA have been shown to increase endogenous levels of PEA, and are of interest as potential treatments for inflammatory disorders and other maladies. Currently, there are no X-ray or NMR structures of NAAA available to inform medicinal chemistry. Additionally, there are a limited number of enzyme structures available that are within the Ntn-hydrolase family, have a catalytic cysteine residue, and have a high sequence homology. For these reasons, we developed expression and purification methods for the production of enzyme samples amenable to structural characterization. Mammalian cells are necessary for post-translational processing, including signal sequence cleavage and glycosylation, that are required for a correctly folded zymogen before conversion to active, and mature enzyme. We have identified an expression construct, mammalian cell line, specific media and additives to express and secrete hNAAA zymogen and we further optimized propagation conditions and show this secretion method is suitable for isotopic labeling of the protein. We refined purification methods to achieve a high degree of protein purity potentially suited to crystallography. Glycosylated proteins can present challenges to biophysical methods. Therefore we deglycosylate the enzyme and show that the activity of the mature enzyme is not affected by deglycosylation.

| S-EPMC6022831 | biostudies-literature

Discovery and SAR Evolution of Pyrazole Azabicyclo[3.2.1]octane Sulfonamides as a Novel Class of Non-Covalent <i>N</i>-Acylethanolamine-Hydrolyzing Acid Amidase (NAAA) Inhibitors for Oral Administration.

Project description:Inhibition of intracellular N-acylethanolamine-hydrolyzing acid amidase (NAAA) activity is a promising approach to manage the inflammatory response under disabling conditions. In fact, NAAA inhibition preserves endogenous palmitoylethanolamide (PEA) from degradation, thus increasing and prolonging its anti-inflammatory and analgesic efficacy at the inflamed site. In the present work, we report the identification of a potent, systemically available, novel class of NAAA inhibitors, featuring a pyrazole azabicyclo[3.2.1]octane structural core. After an initial screening campaign, a careful structure-activity relationship study led to the discovery of endo-ethoxymethyl-pyrazinyloxy-8-azabicyclo[3.2.1]octane-pyrazole sulfonamide 50 (ARN19689), which was found to inhibit human NAAA in the low nanomolar range (IC50 = 0.042 μM) with a non-covalent mechanism of action. In light of its favorable biochemical, in vitro and in vivo drug-like profile, sulfonamide 50 could be regarded as a promising pharmacological tool to be further investigated in the field of inflammatory conditions.

| S-EPMC8474119 | biostudies-literature

Antinociceptive effects of the N-acylethanolamine acid amidase inhibitor ARN077 in rodent pain models.

Project description:Fatty acid ethanolamides (FAEs), which include palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), are endogenous agonists of peroxisome proliferator-activated receptor-? (PPAR-?) and important regulators of the inflammatory response. They are degraded in macrophages by the lysosomal cysteine amidase, N-acylethanolamine acid amidase (NAAA). Previous studies have shown that pharmacological inhibition of NAAA activity suppresses macrophage activation in vitro and causes marked anti-inflammatory effects in vivo, which is suggestive of a role for NAAA in the control of inflammation. It is still unknown, however, whether NAAA-mediated FAE deactivation might regulate pain signaling. The present study examined the effects of ARN077, a potent and selective NAAA inhibitor recently disclosed by our group, in rodent models of hyperalgesia and allodynia caused by inflammation or nerve damage. Topical administration of ARN077 attenuated, in a dose-dependent manner, heat hyperalgesia and mechanical allodynia elicited in mice by carrageenan injection or sciatic nerve ligation. The antinociceptive effects of ARN077 were prevented by the selective PPAR-? antagonist GW6471 and did not occur in PPAR-?-deficient mice. Furthermore, topical ARN077 reversed the allodynia caused by ultraviolet B radiation in rats, and this effect was blocked by pretreatment with GW6471. Sciatic nerve ligation or application of the proinflammatory phorbol ester 12-O-tetradecanoylphorbol 13-acetate decreased FAE levels in sciatic nerve and skin tissue, respectively. ARN077 reversed these biochemical effects. The results identify ARN077 as a potent inhibitor of intracellular NAAA activity, which is active in vivo by topical administration. The findings further suggest that NAAA regulates peripheral pain initiation by interrupting endogenous FAE signaling at PPAR-?.

| S-EPMC3723234 | biostudies-literature

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