N-acylethanolamine-hydrolyzing Acid Amidase Research Articles

Combined in vivo effect of N-acylethanolamine-hydrolyzing acid amidase and glycogen synthase kinase-3β inhibition to treat multiple sclerosis

The current pharmacological approaches to multiple sclerosis (MS) target its inflammatory and autoimmune components, but effective treatments to foster remyelination and axonal repair are still lacking. We therefore selected two targets known to be involved in MS pathogenesis: N-acylethanolamine-hydrolyzing acid amidase (NAAA) and glycogen synthase kinase-3β (GSK-3β). We tested whether inhibiting these targets exerted a therapeutic effect against experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The combined inhibition of NAAA and GSK-3β by two selected small-molecule compounds, ARN16186 (an NAAA inhibitor) and AF3581 (a GSK-3β inhibitor), effectively mitigated disease progression, rescuing the animals from paralysis and preventing a worsening of the pathology. The complementary activity of the two inhibitors reduced the infiltration of immune cells into the spinal cord and led to the formation of thin myelin sheaths around the axons post-demyelination. Specifically, the inhibition of NAAA and GSK-3β modulated the over-activation of NF-kB and STAT3 transcription factors in the EAE-affected mice and induced the nuclear translocation of β-catenin, reducing the inflammatory insult and promoting the remyelination process. Overall, this work demonstrates that the dual-targeting of key aspects responsible for MS progression could be an innovative pharmacological approach to tackle the pathology.

A fluorogenic substrate for the detection of lipid amidases in intact cells

Lipid amidases of therapeutic relevance include acid ceramidase (AC), N-acylethanolamine-hydrolyzing acid amidase, and fatty acid amide hydrolase (FAAH). Although fluorogenic substrates have been developed for the three enzymes and high-throughput methods for screening have been reported, a platform for the specific detection of these enzyme activities in intact cells is lacking. In this article, we report on the coumarinic 1-deoxydihydroceramide RBM1-151, a 1-deoxy derivative and vinilog of RBM14-C12, as a novel substrate of amidases. This compound is hydrolyzed by AC (appKm = 7.0 μM; appVmax = 99.3 nM/min), N-acylethanolamine-hydrolyzing acid amidase (appKm = 0.73 μM; appVmax = 0.24 nM/min), and FAAH (appKm = 3.6 μM; appVmax = 7.6 nM/min) but not by other ceramidases. We provide proof of concept that the use of RBM1-151 in combination with reported irreversible inhibitors of AC and FAAH allows the determination in parallel of the three amidase activities in single experiments in intact cells.

Mulberry (Morus alba L.) leaf water extract attenuates type 2 diabetes mellitus by regulating gut microbiota dysbiosis, lipopolysaccharide elevation and endocannabinoid system disorder

Ethnopharmacological relevanceMulberry (Morus alba L.) leaf is a well-known herbal medicine and has been used to treat diabetes in China for thousands of years. Our previous studies have proven mulberry leaf water extract (MLWE) could improve type 2 diabetes mellitus (T2D). However, it is still unclear whether MLWE could mitigate T2D by regulating gut microbiota dysbiosis and thereof improve intestinal permeability and metabolic dysfunction through modulation of lipopolysaccharide (LPS) and endocannabinoid system (eCBs). Aim of studyThis study aims to explore the potential mechanism of MLWE on the regulation of metabolic function disorder of T2D mice from the aspects of gut microbiota, LPS and eCBs. Materials and methodsGut microbiota was analyzed by high-throughput 16S rRNA gene sequencing. LPS, N-arachidonoylethanolamine (AEA) and 2-ararchidonylglycerol (2-AG) contents in blood were determined by kits or liquid phase chromatography coupled with triple quadrupole tandem mass spectrometry, respectively. The receptors, enzymes or tight junction protein related to eCBs or gut barrier were detected by RT-PCR or Western blot, respectively. ResultsMLWE reduced the serum levels of AEA, 2-AG and LPS, decreased the expressions of N-acylphophatidylethanolamine phospholipase D, diacylglycerol lipase-α and cyclooxygenase 2, and increased the expressions of fatty acid amide hydrolase (FAAH), N-acylethanolamine-hydrolyzing acid amidase (NAAA), alpha/beta hydrolases domain 6/12 in the liver and ileum and occludin, monoacylglycerol lipase and cannabinoid receptor 1 in the ileum of T2D mice. Furthermore, MLWE could change the abundances of the genera including Acetatifactor, Anaerovorax, Bilophila, Colidextribacter, Dubosiella, Gastranaerophilales, Lachnospiraceae_NK4A136_group, Oscillibacter and Rikenella related to LPS, AEA and/or 2-AG. Moreover, obvious improvement of MLWE treatment on serum AEA level, ileum occludin expression, and liver FAAH and NAAA expression could be observed in germ-free-mimic T2D mice. ConclusionMLWE could ameliorate intestinal permeability, inflammation, and glucose and lipid metabolism imbalance of T2D by regulating gut microbiota, LPS and eCBs.

Characterization and Preliminary In Vitro Antioxidant Activity of a New Multidrug Formulation Based on the Co-Encapsulation of Rutin and the α-Acylamino-β-Lactone NAAA Inhibitor URB894 within PLGA Nanoparticles.

A biodegradable and biocompatible polymeric matrix made up of poly(d,l-lactide-co-glycolide) (PLGA) was used for the simultaneous delivery of rutin and the (S)-N-(2-oxo-3-oxetanyl)biphenyl-4-carboxamide derivative (URB894). The goal was to exploit the well-known radical scavenging properties of rutin and the antioxidant features recently reported for the molecules belonging to the class of N-acylethanolamine-hydrolyzing acid amidase (NAAA) inhibitors, such as URB894. The use of the compounds, both as single agents or in association promoted the development of negatively-charged nanosystems characterized by a narrow size distribution and an average diameter of ~200 nm when 0.2-0.6 mg/mL of rutin or URB894 were used. The obtained multidrug carriers evidenced an entrapment efficiency of ~50% and 40% when 0.4 and 0.6 mg/mL of rutin and URB894 were associated during the sample preparation, respectively. The multidrug formulation evidenced an improved in vitro dose-dependent protective effect against H2O2-related oxidative stress with respect to that of the nanosystems containing the active compounds as a single agent, confirming the rationale of using the co-encapsulation approach to obtain a novel antioxidant nanomedicine.

Assay of NAAA Activity.

N-acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal hydrolase degrading various N-acylethanolamines at acidic pH. NAAA prefers anti-inflammatory and analgesic palmitoylethanolamide to other N-acylethanolamines as a substrate, and its specific inhibitors are shown to exert anti-inflammatory and analgesic actions in animal models. Therefore, these inhibitors are expected as a new class of therapeutic agents. Here, we introduce an NAAA assay system, using [14C]palmitoylethanolamide and thin-layer chromatography. The preparation of NAAA enzyme from native and recombinant sources as well as the chemical synthesis of N-[1'-14C]palmitoyl-ethanolamine is also described.

Inhibition of N-acylethanolamine-hydrolyzing acid amidase reduces T cell infiltration in a mouse model of multiple sclerosis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), in which myeloid cells sustain inflammation, take part in priming, differentiation, and reactivation of myelin-specific T cells, and cause direct myelin damage. N-Acylethanolamine-hydrolyzing acid amidase (NAAA) is a proinflammatory enzyme induced by phlogosis and overexpressed in macrophages and microglia of EAE mice. Targeting these cell populations by inhibiting NAAA may be a promising pharmacological strategy to modulate the inflammatory aspect of MS and manage disease progression. To address this goal, we used ARN16186, a small molecule specifically designed and synthesized as a pharmacological tool to inhibit NAAA. We assessed whether enzyme inhibition affected the severity of neurological symptoms and modulated immune cell infiltration into the central nervous system of EAE mice. We found that preventive chronic treatment with ARN16186 was efficacious in slowing disease progression and preserving locomotor activity in EAE mice. Furthermore, NAAA inhibition reduced the number of immune cells infiltrating the spinal cord and modulated the overactivation of NF-kB and STAT3 transcription factors, leading to less expansion of Th17 cells over the course of the disease.

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

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.

Natural Potent NAAA Inhibitor Atractylodin Counteracts LPS-Induced Microglial Activation.

N-acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme that inhibits the degradation of palmitoylethanolamide (PEA), an endogenous lipid that induces analgesic, anti-inflammation, and anti-multiple sclerosis through PPARα activation. Only a few potent NAAA inhibitors have been reported to date, which is mainly due to the restricted substrate-binding site of NAAA. Here, we established a high-throughput fluorescence-based assay for NAAA inhibitor screening. Several new classes of NAAA inhibitors were discovered from a small library of natural products. One of these is atractylodin, a polyethylene alkyne compound from the root of Atractylodes lancea (Thunb) DC., which significantly inhibits NAAA activity and has an IC50 of 2.81 µM. Kinetic analyses and dialysis assays suggested that atractylodin engages in competitive inhibition via reversible reaction to the enzyme. Docking assays revealed that atractylodin occupies the catalytic cavity of NAAA, where the atractylodin furan head group has a hydrophobic-related interaction with the backbone of the Trp181 and Leu152 residues of human NAAA. Further investigation indicated that atractylodin significantly increases PEA and OEA levels and dose-dependently inhibits LPS-induced nitrate, TNF-α, IL-1β, and IL-6 pro-inflammatory cytokine release in BV-2 microglia. Our results show that atractylodin elevates cellular PEA levels and inhibits microglial activation by inhibiting NAAA activity, which in turn could contribute to NAAA functional research.

Joint Synovial Fluid Metabolomics Method to Decipher the Metabolic Mechanisms of Adjuvant Arthritis and Geniposide Intervention.

Rheumatoid arthritis (RA), a chronic systemic autoimmune disease, is mainly characterized by joint lesions and permanent loss of joint function. To discover the metabolic characteristics of RA and the underlying mechanisms in treatment with geniposide (GE), untargeted metabolomic analysis based on hydrophilic interaction liquid chromatography coupled to high-resolution mass spectrometry (HILIC-HRMS) was performed using the joint synovial fluid samples from adjuvant arthritis (AA) rats. Microdialysis (MD) was utilized to collect the dialysate samples precisely from the articular cavity of AA rats. Multivariate statistical analysis was then conducted to discover the metabolite changes induced by AA and to differentiate GE-related biomarkers. The mass spectrometry data are available on the Chorus website (https://chorusproject.org/pages/index.html) with the data set identifier 1680. The results showed that 20 metabolites differed significantly between AA rats and normal rats. GE treatment recovered the altered levels of the 13 metabolites mentioned above, such as palmitoylethanolamide (PEA), Cer (d18:0/22:0), and PC (18:1(11Z)/16:1(9Z)), and normalized glycerophospholipid metabolism. As evidenced by western blotting, the changes in PEA levels adjusted by GE were associated with the down-regulated expression of N-acylethanolamine-hydrolyzing acid amidase (NAAA) in synovial tissues. Taken together, the elucidation of metabolic changes of joint synovial fluid and how this is influenced by GE will promote future therapeutic interventions of RA.

N-Palmitoylethanolamine-Oxazoline as a New Therapeutic Strategy to Control Neuroinflammation: Neuroprotective Effects in Experimental Models of Spinal Cord and Brain Injury.

Modulation of N-acylethanolamine-hydrolyzing acid amidase (NAAA) represents a potential alternative strategy in the treatment of neuroinflammation. Recent studies showed that pharmacological modulation of NAAA could be achieved with the oxazoline of palmitoylethanolamide (PEA; PEA-OXA). The aim of this study was to evaluate the neuroprotective effects of PEA-OXA in the secondary neuroinflammatory events induced by spinal and brain trauma in mice. Animals were subjected to spinal cord and brain injury models and PEA-OXA (10 mg/kg) was administered both intraperitoneally and orally 1 h and 6 h after trauma. PEA-OXA treatment markedly reduced the histological alterations induced by spinal cord injury (SCI) and traumatic brain injury (TBI) and ameliorated the motor function and behavioral deficits, as well. In addition, the expression of neurotrophic factors, such as glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, and neurotrophin-3 were increased by PEA-OXA treatment. Moreover, PEA-OXA also significantly decreased glial fibrillary acidic protein hyperexpression, the nuclear translocation of nuclear factor (NF)-κB, phosphorylation of Ser536 on the NF-κB subunit p65, and degradation of IκB-α, as well as diminished the expression of pro-inflammatory mediators such as cyclooxygenase-2 (COX-2), inducible nitric oxide synthase, tumor necrosis factor (TNF)-α and interleukin (IL)-1β. The modulation of intracellular NAAA by PEA-OXA treatment could thus represent a novel therapy to control neuroinflammatory conditions associated with SCI and TBI.

Optimization of a Medium‐Throughput Cathepsin K Inhibition Assay for NAAA Inhibitors

Cathepsin K and N‐acylethanolamine‐hydrolyzing acid amidase (NAAA) are members of the cysteine protease family. While NAAA is a metabolic enzyme in the endocannabinoid system, Cathepsin K is involved in bone reabsorption and the release of calcium into the bloodstream. Our interest in the endocannabinoid system has resulted in significant NAAA inhibitor development for the treatment of inflammation and the resulting chronic pain and has prompted our interest in Cathepsin K for off‐target selectivity screening. A medium‐throughput fluorescent assay to determine Cathepsin K inhibition was optimized to screen NAAA‐lead compounds using Omnicathepsin (aka Z‐Phe‐Arg‐AMC) as the fluorogenic substrate. The concentrations of enzyme (2 nM Cathepsin K) and substrate (25 μM Omnicathepsin) were optimized and the Km of Cathepsin K and Omnicathepsin was found to be 9.8 μM. Two published compounds, Odanacatib and BML‐244 with IC50 values of 0.54 ± 0.4 nM and 317 ± 41 nM respectively, were developed to be reference compounds in the inhibition 3‐point screen. The optimized assay was then used to screen NAAA‐inhibitors for competitive inhibition of Cathepsin K in order to create more potent, specific drugs for NAAA.Support or Funding InformationThis project was funded by NIH: 2‐R44‐DK104636, NAAA Inhibitors as Anti‐Inflammatory Agents, Shakiru Alapafuja, PI; Northeastern PI, JodiAnne T. Wood.

N-acylethanolamine-hydrolyzing acid amidase and fatty acid amide hydrolase inhibition differentially affect N-acylethanolamine levels and macrophage activation

N-acylethanolamines (NAEs) such as N-palmitoylethanolamine and anandamide are endogenous bioactive lipids having numerous functions, including the control of inflammation. Their levels and therefore actions can be controlled by modulating the activity of two hydrolytic enzymes, N-acylethanolamine-hydrolyzing acid amidase (NAAA) and fatty acid amide hydrolase (FAAH). As macrophages are key to inflammatory processes, we used lipopolysaccharide-activated J774 macrophages, as well as primary mouse alveolar macrophages, to study the effect of FAAH and NAAA inhibition, using PF-3845 and AM9053 respectively, on macrophage activation and NAE levels measured by HPLC-MS. Markers of macrophage activation were measured by qRT-PCR and ELISA. Activation of macrophages decreased NAAA expression and NAE hydrolytic activity. FAAH and NAAA inhibition increased the levels of the different NAEs, although with different magnitudes, whether in control condition or following LPS-induced macrophage activation. Both inhibitors reduced several markers of macrophage activation, such as mRNA expression of inflammatory mediators, as well as cytokine and prostaglandin production, with however some differences between FAAH and NAAA inhibition. Most of the NAEs tested – including N-docosatetraenoylethanolamine and N-docosahexaenoylethanolamine – also reduced LPS-induced mRNA expression of inflammatory mediators, again with differences depending on the marker and the NAE, thus offering a potential explanation for the differential effect of the inhibitors on macrophage activation markers. In conclusion, we show different and complementary effects of NAE on lipopolysaccharide-induced macrophage activation. Our results support an important role for inhibition of NAE hydrolysis and NAAA inhibition in particular in controlling macrophage activation, and thus inflammation.

Development of new inhibitors for N-acylethanolamine-hydrolyzing acid amidase as promising tool against bladder cancer

The endocannabinoid system is a signaling system involved in a wide range of biological effects. Literature strongly suggests the endocannabinoid system role in the pathogenesis of cancer and that its pharmacological activation produces therapeutic benefits. Last research promotes the endocannabinoid system modulation by inhibition of endocannabinoids hydrolytic enzymes instead of direct activation of endocannabinoid receptors to avoid detrimental effects on cognition and motor control. Here we report the identification of N-acylethanolamine-hydrolyzing acid amidase (NAAA) inhibitors able to reduce cell proliferation and migration and cause cell death on different bladder cancer cell lines. These molecules were designed, synthesized and characterized and active compounds were selected by a fluorescence high-throughput screening method set-up on human recombinant NAAA that also allows to characterize the mechanism of inhibition. Together our results suggest an important role for NAAA in cell migration and in inducing tumor cell death promoting this enzyme as pharmacological target against bladder cancer.

2-pentadecyl-2-oxazoline: Identification in coffee, synthesis and activity in a rat model of carrageenan-induced hindpaw inflammation

N-acylethanolamines (NAEs) comprise a family of bioactive lipid molecules present in animal and plant tissues, with N-palmitoylethanolamine (PEA) having received much attention owing to its anti-inflammatory, analgesic and neuroprotective activities.2-Pentadecyl-2-oxazoline (PEA-OXA), the oxazoline of PEA, reportedly modulates activity of N-acylethanolamine-hydrolyzing acid amidase (NAAA), which catabolizes PEA. Because PEA is produced on demand and exerts pleiotropic effects on non-neuronal cells implicated in neuroinflammation, modulating the specific amidases for NAEs (NAAA in particular) could be a way to preserve PEA role in maintaining cellular homeostasis through its rapid on-demand synthesis and equally rapid degradation. This study provides the first description of PEA-OXA in both green and roasted coffee beans and Moka infusions, and its synthesis. In an established model of carrageenan (CAR)-induced rat paw inflammation, PEA-OXA was orally active in limiting histological damage and thermal hyperalgesia 6h after CAR intraplantar injection in the right hindpaw and the accumulation of infiltrating inflammatory cells. PEA-OXA appeared to be more potent compared to ultramicronized PEA given orally at the same dose (10mg/kg). PEA-OXA markedly reduced also the increase in hindpaw myeloperoxidase activity, an index of polymorphonuclear cell accumulation in inflammatory tissues. NAAA modulators like PEA-OXA may serve to maximize availability of NAEs (e.g. PEA) while providing for recycling of the NAE components for further resynthesis.

Assay of NAAA Activity.

N-acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal hydrolase degrading various N-acylethanolamines at acidic pH. Since NAAA prefers anti-inflammatory and analgesic palmitoylethanolamide to other N-acylethanolamines as a substrate, its specific inhibitors are expected as a new class of anti-inflammatory and analgesic agents. Here, we introduce an NAAA assay system, using [(14)C]palmitoylethanolamide and thin-layer chromatography. The preparation of NAAA enzyme from native and recombinant sources as well as the chemical synthesis of N-[1'-(14)C]palmitoyl-ethanolamine is also described.

Atom based 3D-QSAR studies on 2,4-dioxopyrimidine-1-carboxamide analogs: Validation of experimental inhibitory potencies towards acid ceramidase

Ceramide (Cer), the central lipid molecule in sphingolipid biosynthesis and degradation, which plays a key role in sphingolipid signaling, induces cell differentiation and apoptosis. Cellular degradation of ceramide to sphingosine is catalyzed by a family of ceramidases (CDases). Pharmacological inhibition of ceramidases and more particularly, acid ceramidase (aCDase) is suggestive of a chemotherapeutic approach as it increases the cellular concentration of ceramide inducing apoptosis. In the present report, we have utilized atom-based 3D-QSAR method to analyze the structural aspects on a series of 2,4-dioxopyrimidine-1-carboxamide (carmofur) derivatives as potent inhibitors of aCDase. In this approach the experimental dataset was divided into training (83%) and test (17%) sets and the best model was chosen based on randomized trial distributions consisting of five compounds in a test set with a wide range of activity profile and superior values of statistical parameters such as Q2 and R2 values. The reported experimental results by Piomelli and co-workers on the inhibition of aCDase by the carmofur derivatives were correlated using robust 3D-QSAR as well as docking methods. With careful structure–activity correlation studies the carmofur analogs were classified into four sub-categories (Set 1–4) to understand the effect of each structural features separately. This approach led us to short-list most active carmofur derivatives such as compounds 26, 30 and 32 with the incorporation of more than one structural features in a single molecule. However, the inhibition potency might further be enhanced by designing compound 33 upon the incorporation of all features in a single compound. Compound 33 that was missing in the experimental study by Piomelli and co-workers (J. Med. Chem. 2013, 56, 3518), could be identified using 3D-QSAR studies. Moreover, the importance of structural features in lead inhibitors such as 26, 30 and 32 along with 33 was further justified by their efficient molecular interactions at the active site of homology modeled protein human N-acyl ethanolamine hydrolyzing acid amidase (hNAAA) as evidenced by molecular docking study. Furthermore, efficient molecular interaction of some representative inhibitors with hNAAA led to the understanding that hNAAA could be a possible alternative of aCDase for developing potent inhibitors.

Activity of neutral and alkaline ceramidases on fluorogenic N-acylated coumarin-containing aminodiols

Ceramidases catalyze the cleavage of ceramides into sphingosine and fatty acids. Previously, we reported on the use of the RBM14 fluorogenic ceramide analogs to determine acidic ceramidase activity. In this work, we investigated the activity of other amidohydrolases on RBM14 compounds. Both bacterial and human purified neutral ceramidases (NCs), as well as ectopically expressed mouse neutral ceramidase hydrolyzed RBM14 with different selectivity, depending on the N-acyl chain length. On the other hand, microsomes from alkaline ceramidase (ACER)3 knockdown cells were less competent at hydrolyzing RBM14C12, RBM12C14, and RBM14C16 than controls, while microsomes from ACER2 and ACER3 overexpressing cells showed no activity toward the RBM14 substrates. Conversely, N-acylethanolamine-hydrolyzing acid amidase (NAAA) overexpressing cells hydrolyzed RBM14C14 and RBM14C16 at acidic pH. Overall, NC, ACER3, and, to a lesser extent, NAAA hydrolyze fluorogenic RBM14 compounds. Although the selectivity of the substrates toward ceramidases can be modulated by the length of the N-acyl chain, none of them was specific for a particular enzyme. Despite the lack of specificity, these substrates should prove useful in library screening programs aimed at identifying potent and selective inhibitors for NC and ACER3.

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

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.

Diacerein is a potent and selective inhibitor of palmitoylethanolamide inactivation with analgesic activity in a rat model of acute inflammatory pain

Palmitoylethanolamide (PEA) is produced by mammalian cells from its biosynthetic precursor, N-palmitoyl-phosphatidyl-ethanolamine, and inactivated by enzymatic hydrolysis to palmitic acid and ethanolamine. Apart from fatty acid amide hydrolase (FAAH), the N-acylethanolamine-hydrolyzing acid amidase (NAAA), a lysosomal enzyme, was also shown to catalyze the hydrolysis of PEA and to limit its analgesic and anti-inflammatory action. Here we report the finding of a new potential inhibitor of NAAA, EPT4900 (4,5-diacetyloxy-9,10-dioxo-anthracene-2-carboxylic acid, diacerein). EPT4900 exhibited a high inhibitory activity on human recombinant NAAA over-expressed in HEK293 cells (HEK-NAAA cells). EPT4900 selectively increased the levels of PEA in intact HEK-NAAA cells, and inhibited inflammation as well as hyperalgesia in rats treated with an intraplantar injection of carrageenan. This latter effect was accompanied by elevation of PEA endogenous levels in the paw skin.

Design and Synthesis of Potent N-Acylethanolamine-hydrolyzing Acid Amidase (NAAA) Inhibitor as Anti-Inflammatory Compounds

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-α). 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 (IC50 = 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 (Asn209) 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.