N-acylethanolamine Acid Amidase Inhibition Research Articles

Pharmacological inhibition of N-Acylethanolamine acid amidase (NAAA) mitigates intestinal fibrosis through modulation of macrophage activity.

Intestinal fibrosis, a frequent complication of inflammatory bowel disease, is characterized by stricture formation with no pharmacological treatment to date. N-acylethanolamine acid amidase (NAAA) is responsible of acylethanolamides (AEs, e.g., palmitoylethanolamide and oleoylethanolamide) hydrolysis. Here, we investigated NAAA and AEs signalling in gut fibrosis. NAAA and AEs signalling were evaluated in human intestinal specimens from stenotic Crohn's diseases (CD) patients. Gut fibrosis was induced by TNBS, monitored by colonoscopy and unascertained by qRT-PCR, histological analyses, and confocal microscopy. Immune cells were analysed in mesenteric lymph nodes by FACS. Colonic fibroblasts were cultured in conditioned media derived from polarized or not bone marrow-derived macrophages (BMDM). IL-23 signalling was evaluated by qRT-PCR, ELISA, FACS, and western blot in BMDM and in lamina propria CX3CR1+ cells. In ileocolonic human CD strictures, increased transcript expression of NAAA was observed with a decrease of its substrates OEA and PEA. NAAA inhibition reduced intestinal fibrosis in vivo, as revealed by decrease in inflammatory parameters, collagen deposition and fibrosis genes, including epithelial to mesenchymal transition. More in-depth studies revealed modulation of the immune response related to IL-23 following NAAA inhibition. The antifibrotic actions of NAAA inhibition are mediated by Mφ and M2 macrophages that indirectly affect fibroblast collagenogenesis. NAAA inhibitor AM9053 normalized IL-23 signalling in BMDM and in lamina propria CX3CR1+ cells. Our findings provide new insights into the pathophysiological mechanism of intestinal fibrosis and identify NAAA as a promising target for the development of therapeutic treatments to alleviate CD fibrosis.

Inhibiting immunoregulatory amidase NAAA blocks ZIKV maturation in Human Neural Stem Cells

Recent evidence suggests that lipids play a crucial role in viral infections beyond their traditional functions of supplying envelope and energy, and creating protected niches for viral replication. In the case of Zika virus (ZIKV), it alters host lipids by enhancing lipogenesis and suppressing β-oxidation to generate viral factories at the endoplasmic reticulum (ER) interface. This discovery prompted us to hypothesize that interference with lipogenesis could serve as a dual antiviral and anti-inflammatory strategy to combat the replication of positive sense single-stranded RNA (ssRNA+) viruses.To test this hypothesis, we examined the impact of inhibiting N-Acylethanolamine acid amidase (NAAA) on ZIKV-infected human Neural Stem Cells. NAAA is responsible for the hydrolysis of palmitoylethanolamide (PEA) in lysosomes and endolysosomes. Inhibition of NAAA results in PEA accumulation, which activates peroxisome proliferator-activated receptor-α (PPAR-α), directing β-oxidation and preventing inflammation.Our findings indicate that inhibiting NAAA through gene-editing or drugs moderately reduces ZIKV replication by approximately one log10 in Human Neural Stem Cells, while also releasing immature virions that have lost their infectivity. This inhibition impairs furin-mediated prM cleavage, ultimately blocking ZIKV maturation. In summary, our study highlights NAAA as a host target for ZIKV infection.

Ramulus Cinnamomi essential oil exerts an anti-inflammatory effect on RAW264.7 cells through N-acylethanolamine acid amidase inhibition

Ethnopharmacological relevanceRamulus Cinnamomi, the dried twig of Cinnamomum cassia (L.) J.Presl., is a traditional Chinese medicine (TCM) with anti-inflammatory effects. The medicinal functions of Ramulus Cinnamomi essential oil (RCEO) have been confirmed, although the potential mechanisms by which RCEO exerts its anti-inflammatory effects have not been fully elucidated. Aim of the studyTo investigate whether N-acylethanolamine acid amidase (NAAA) mediates the anti-inflammatory effects of RCEO. Materials and methodsRCEO was extracted by steam distillation of Ramulus Cinnamomi, and NAAA activity was detected using HEK293 cells overexpressing NAAA. N-Palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), both of which are NAAA endogenous substrates, were detected by liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The anti-inflammatory effects of RCEO were analyzed in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and the cell viability was measured with a Cell Counting Kit-8 (CCK-8) kit. The nitric oxide (NO) in the cell supernatant was measured using the Griess method. The level of tumor necrosis factor-α (TNF-α) in the RAW264.7 cell supernatant was determined using an enzyme-linked immunosorbent assay (ELISA) kit. The chemical composition of RCEO was assessed by gas chromatography-mass spectroscopy (GC-MS). The molecular docking study for (E)-cinnamaldehyde and NAAA was performed by using Discovery Studio 2019 software (DS2019). ResultsWe established a cell model for evaluating NAAA activity, and we found that RCEO inhibited the NAAA activity with an IC50 of 5.64 ± 0.62 μg/mL. RCEO significantly elevated PEA and OEA levels in NAAA-overexpressing HEK293 cells, suggesting that RCEO might prevent the degradation of cellular PEA and OEA by inhibiting the NAAA activity in NAAA-overexpressing HEK293 cells. In addition, RCEO also decreased NO and TNF-α cytokines in lipopolysaccharide (LPS)-stimulated macrophages. Interestingly, the GC-MS assay revealed that more than 93 components were identified in RCEO, of which (E)-cinnamaldehyde accounted for 64.88%. Further experiments showed that (E)-cinnamaldehyde and O-methoxycinnamaldehyde inhibited NAAA activity with an IC50 of 3.21 ± 0.03 and 9.62 ± 0.30 μg/mL, respectively, which may represent key components of RCEO that inhibit NAAA activity. Meanwhile, docking assays revealed that (E)-cinnamaldehyde occupies the catalytic cavity of NAAA and engages in a hydrogen bond interaction with the TRP181 and hydrophobic-related interactions with LEU152 of human NAAA. ConclusionsRCEO showed anti-inflammatory effects by inhibiting NAAA activity and elevating cellular PEA and OEA levels in NAAA-overexpressing HEK293 cells. (E)-cinnamaldehyde and O-methoxycinnamaldehyde, two components in RCEO, were identified as the main contributors of the anti-inflammatory effects of RCEO by modulating cellular PEA levels through NAAA inhibition.

Genetic Blockade of NAAA Cell-specifically Regulates Fatty Acid Ethanolamides (FAEs) Metabolism and Inflammatory Responses.

N-Acylethanolamine acid amidase (NAAA) is a lysosomal enzyme responsible for the hydrolysis of fatty acid ethanolamides (FAEs). However, the role of NAAA in FAEs metabolism and regulation of pain and inflammation remains mostly unknown. Here, we generated NAAA-deficient (NAAA-/-) mice using CRISPR-Cas9 technique, and found that deletion of NAAA increased PEA and AEA levels in bone marrow (BM) and macrophages, and elevated AEA levels in lungs. Unexpectedly, genetic blockade of NAAA caused moderately effective anti-inflammatory effects in lipopolysaccharides (LPS)-induced acute lung injury (ALI), and poor analgesic effects in carrageenan-induced hyperalgesia and sciatic nerve injury (SNI)-induced mechanical allodynia. These data contrasted with acute (single dose) or chronic NAAA inhibition by F96, which produced marked anti-inflammation and analgesia in these models. BM chimera experiments indicated that these phenotypes were associated with the absence of NAAA in non-BM cells, whereas deletion of NAAA in BM or BM-derived cells in rodent models resulted in potent analgesic and anti-inflammatory phenotypes. When combined, current study suggested that genetic blockade of NAAA regulated FAEs metabolism and inflammatory responses in a cell-specifical manner.

N-Acylethanolamine acid amidase (NAAA) is dysregulated in colorectal cancer patients and its inhibition reduces experimental cancer growth.

Background and Purpose N‐Acylethanolamine acid amidase (NAAA) is a lysosomal enzyme accountable for the breakdown of N‐acylethanolamines (NAEs) and its pharmacological inhibition has beneficial effects in inflammatory conditions. The knowledge of NAAA in cancer is fragmentary with an unclarified mechanism, whereas its contribution to colorectal cancer (CRC) is unknown to date.Experimental ApproachCRC xenograft and azoxymethane models were used to assess the in vivo effect of NAAA inhibition. Further, the tumour secretome was evaluated by an oncogenic array, CRC cell lines were used for in vitro studies, cell cycle was analysed by cytofluorimetry, NAAA was knocked down with siRNA, human biopsies were obtained from surgically resected CRC patients, gene expression was measured by RT‐PCR and NAEs were measured by LC–MS.Key ResultsThe NAAA inhibitor AM9053 reduced CRC xenograft tumour growth and counteracted tumour development in the azoxymethane model. NAAA inhibition affected the composition of the tumour secretome inhibiting the expression of EGF family members. In CRC cells, AM9053 reduced proliferation with a mechanism mediated by PPAR‐α and TRPV1. AM9053 induced cell cycle arrest in the S phase associated with cyclin A2/CDK2 down‐regulation. NAAA knock‐down mirrored the effects of NAAA inhibition with AM9053. NAAA expression was down‐regulated in human CRC tissues, with a consequential augmentation of NAE levels and dysregulation of some of their targets.Conclusion and ImplicationsOur results show novel data on the functional importance of NAAA in CRC progression and the mechanism involved. We propose that this enzyme is a valid drug target for the treatment of CRC growth and development.

N-Acylethanolamine Acid Amidase Inhibition Potentiates Morphine Analgesia and Delays the Development of Tolerance

Opioids are essential drugs for pain management, although long-term use is accompanied by tolerance, necessitating dose escalation, and dependence. Pharmacological treatments that enhance opioid analgesic effects and/or attenuate the development of tolerance (with a desirable opioid-sparing effect in treating pain) are actively sought. Among them, N-palmitoylethanolamide (PEA), an endogenous lipid neuromodulator with anti-inflammatory and neuroprotective properties, was shown to exert anti-hyperalgesic effects and to delay the emergence of morphine tolerance. A selective augmentation in endogenous PEA levels can be achieved by inhibiting N-acylethanolamine acid amidase (NAAA), one of its primary hydrolyzing enzymes. This study aimed to test the hypothesis that NAAA inhibition, with the novel brain permeable NAAA inhibitor AM11095, modulates morphine’s antinociceptive effects and attenuates the development of morphine tolerance in rats. We tested this hypothesis by measuring the pain threshold to noxious mechanical stimuli and, as a neural correlate, we conducted in vivo electrophysiological recordings from pain-sensitive locus coeruleus (LC) noradrenergic neurons in anesthetized rats. AM11095 dose-dependently (3–30 mg/kg) enhanced the antinociceptive effects of morphine and delayed the development of tolerance to chronic morphine in behaving rats. Consistently, AM11095 enhanced morphine-induced attenuation of the response of LC neurons to foot-shocks and prevented the attenuation of morphine effects following chronic treatment. Behavioral and electrophysiological effects of AM11095 on chronic morphine were paralleled by a decrease in glial activation in the spinal cord, an index of opioid-induced neuroinflammation. NAAA inhibition might represent a potential novel therapeutic approach to increase the analgesic effects of opioids and delay the development of tolerance.

N-acylethanolamine acid amidase (NAAA) inhibition decreases the motivation for alcohol in Marchigian Sardinian alcohol-preferring rats.

N-acylethanolamine acid amidase (NAAA) is an intracellular cysteine hydrolase that terminates the biological actions of oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), two endogenous lipid-derived agonists of the nuclear receptor, and peroxisome proliferator-activated receptor-α. OEA and PEA are important regulators of energy balance, pain, and inflammation, but recent evidence suggests that they might also contribute to the control of reward-related behaviors. In the present study, we investigated the effects of systemic and intracerebral NAAA inhibition in the two-bottle choice model of voluntary alcohol drinking and on operant alcohol self-administration. Intraperitoneal injections of the systemically active NAAA inhibitor ARN19702 (3 and 10mg/kg) lowered voluntary alcohol intake in a dose-dependent manner, achieving ≈47% reduction at the 10mg/kg dose (p< 0.001). Water, food, or saccharin consumption was not affected by the inhibitor. Similarly, ARN19702 dose-dependently attenuated alcohol self-administration under both fixed ratio 1 (FR-1) and progressive ratio schedules of reinforcement. Furthermore, microinjection of ARN19702 (1, 3 and 10μg/μl) or of two chemically different NAAA inhibitors, ARN077 and ARN726 (both at 3 and 10μg/μl), into the midbrain ventral tegmental area produced dose-dependent decreases in alcohol self-administration under FR-1 schedule. Microinjection of ARN19702 into the nucleus accumbens had no such effect. Collectively, the results point to NAAA as a possible molecular target for the treatment of alcohol use disorder.

N-Acylethanolamine Acid Amidase contributes to disease progression in a mouse model of multiple sclerosis

N-Acylethanolamine acid amidase (NAAA) deactivates the endogenous peroxisome proliferator-activated receptor-α (PPAR-α) agonist palmitoylethanolamide (PEA). NAAA-regulated PEA signaling participates in the control of peripheral inflammation, but evidence suggests also a role in the modulation of neuroinflammatory pathologies such as multiple sclerosis (MS). Here we show that disease progression in the mouse experimental autoimmune encephalomyelitis (EAE) model of MS is accompanied by induction of NAAA expression in spinal cord, which in presymptomatic animals is confined to motor neurons and oligodendrocytes but, as EAE progresses, extends to microglia/macrophages and other cell types. As previously reported for NAAA inhibition, genetic NAAA deletion delayed disease onset and attenuated symptom intensity in female EAE mice, suggesting that accrued NAAA expression may contribute to pathology. To further delineate the role of NAAA in EAE, we generated a mouse line that selectively overexpresses the enzyme in macrophages, microglia and other monocyte-derived cells. Non-stimulated alveolar macrophages from these NaaaCD11b+ mice contain higher-than-normal levels of inducible nitric oxide synthase and display an activated morphology. Furthermore, intranasal lipopolysaccharide injections cause greater alveolar leukocyte accumulation in NaaaCD11b+ than in control mice. NaaaCD11b+ mice also display a more aggressive clinical response to EAE induction, compared to their wild-type littermates. The results identify NAAA as a critical control step in EAE pathogenesis, and point to this enzyme as a possible target for the treatment of MS.

P.204 Effect of N-acylethanolamine acid amidase inhibition on locus coeruleus noradrenergic neuronal responses to morphine

P.204 Effect of N-acylethanolamine acid amidase inhibition on locus coeruleus noradrenergic neuronal responses to morphine

328 – Preclinical Drug Evaluation Reveals Inhibition of Nacylethanolamine Acid Amidase As a Potential Treatment for Inflammatory Bowel Disease

328 – Preclinical Drug Evaluation Reveals Inhibition of Nacylethanolamine Acid Amidase As a Potential Treatment for Inflammatory Bowel Disease

Inhibition of N-acylethanolamine acid amidase reduces nicotine-induced dopamine activation and reward

Tobacco smoke is the leading preventable cause of death in the world and treatments aimed to increase success rate in smoking cessation by reducing nicotine dependence are sought. Activation of peroxisome proliferator-activated receptor-alpha (PPARα) by synthetic or endogenous agonists was shown to suppress nicotine-induced activation of mesolimbic dopamine system, one of the major neurobiological substrates of nicotine dependence, and nicotine-seeking behavior in rats and monkeys. An alternative indirect way to activate PPARα is inhibition of N-acylethanolamine acid amidase (NAAA), one of the major hydrolyzing enzyme for its endogenous agonists palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). We synthetized a novel specific brain permeable NAAA inhibitor, AM11095. We administered AM11095 to rats and carried out brain lipid analysis, a functional observational battery (FOB) to assess toxicity, in vivo electrophysiological recording from dopamine cells in the ventral tegmental area, brain microdialysis in the nucleus accumbens shell and behavioral experiments to assess its effect on nicotine -induced conditioned place preference (CPP).AM11095 (5 and 25 mg/kg, i.p.) was devoid of neurotoxic and behavioral effects and did not affect motor behavior and coordination. This NAAA inhibitor (5 mg/kg i.p.) increased OEA and PEA levels in the hippocampus and cortex, prevented nicotine-induced activation of mesolimbic dopamine neurons in the ventral tegmental area, nicotine-induced elevation of dopamine levels in the nucleus accumbens shell and decreased the expression of nicotine CPP.Our results indicate that NAAA inhibitors represent a new class of pharmacological tools to modulate brain PEA/PPARα signalling and show potential in the treatment of nicotine dependence.

Identification of highly potent N-acylethanolamine acid amidase (NAAA) inhibitors: Optimization of the terminal phenyl moiety of oxazolidone derivatives

N-acylethanolamine acid amidase (NAAA) is a cysteine hydrolase that participates in the deactivation of fatty acid ethanolamides, such as palmitoylethanolamide (PEA). NAAA inhibition may provide a potential therapeutic strategy for the treatment of diseases in which higher PEA level is desired. In the present study, we reported the structure-activity relationship (SAR) studies for oxazolidone derivatives as NAAA inhibitors. A series of substituents or alkyl replacements for the terminal phenyl ring of oxazolidone derivatives were examined. The results showed that the inhibition potency of these oxazolidone derivatives towards NAAA depends on the sizes, flexibility, and lipophilicity of the terminal groups. SAR results suggested that small lipophilic 3-phenyl substituents or hydroxy-containing 4-phenyl substituents were preferable for optimal potency. Furthermore, the distal aliphatic replacement is also preferred for high inhibitory potency. Rapid dilution and kinetic analysis suggested that oxazolidone derivatives with different terminal phenyl moieties inhibited NAAA via different mechanisms. This study identified several highly potent NAAA inhibitors, including 1a (F215, IC50 = 0.009 μM), 1o (IC50 = 0.061 μM) and 2e (IC50 = 0.092 μM), and also determined structural requirements of oxazolidone derivatives for potent inhibition against NAAA.

Inhibition of N-acylethanolamine acid amidase reverses effects of chronic binge ethanol drinking on ventral tegmental area dopamine neuron firing

Inhibition of N-acylethanolamine acid amidase reverses effects of chronic binge ethanol drinking on ventral tegmental area dopamine neuron firing

Design, synthesis, and biological evaluation of oxazolidone derivatives as highly potent N-acylethanolamine acid amidase (NAAA) inhibitors

Preventing PEA degradation by inhibition of NAAA is a novel strategy for the treatment of inflammation and pain. We reported the discovery of oxazolidone derivative as highly potent NAAA inhibitors, including 2f, 3h, 3i and 3j.

Potential analgesic effects of a novel N-acylethanolamine acid amidase inhibitor F96 through PPAR-α.

Pharmacological blockade of N-acylethanolamine acid amidase (NAAA) activity is an available approach for inflammation and pain control through restoring the ability of endogenous PEA. But the recently reported NAAA inhibitors suffer from the chemical and biological unstable properties, which restrict functions of NAAA inhibition in vivo. It is still unrevealed whether systematic inhibition of NAAA could modulate PEA-mediated pain signalings. Here we reported an oxazolidinone imide compound 3-(6-phenylhexanoyl) oxazolidin-2-one (F96), which potently and selectively inhibited NAAA activity (IC50 = 270 nM). Intraperitoneal (i.p.) injection of F96 (3–30 mg/kg) dose-dependently reduced ear edema and restored PEA levels of ear tissues in 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced ear edema models. Furthermore, F96 inhibited acetic acid-induced writhing and increased spared nerve injury induced tactile allodynia thresholds in a dose-dependent manner. Pharmacological effects of F96 (10 mg/kg, i.p.) on various animal models were abolished in PPAR-α−/− mice, and were prevented by PPAR-α antagonist MK886 but not by canabinoid receptor type 1 (CB1) antagonist Rimonabant nor canabinoid receptor type 2 (CB2) antagonist SR144528. Zebrafish embryos experiments showed better security and lower toxicity for F96 than ibuprofen. These results revealed that F96 might be useful in treating inflammatory and neuropathic pain by NAAA inhibition depending on PPAR-α receptors.

3-Aminoazetidin-2-one derivatives as N-acylethanolamine acid amidase (NAAA) inhibitors suitable for systemic administration.

N-Acylethanolamine acid amidase (NAAA) is a cysteine hydrolase that catalyzes the hydrolysis of endogenous lipid mediators such as palmitoylethanolamide (PEA). PEA has been shown to exert anti-inflammatory and antinociceptive effects in animals by engaging peroxisome proliferator-activated receptor α (PPAR-α). Thus, preventing PEA degradation by inhibiting NAAA may provide a novel approach for the treatment of pain and inflammatory states. Recently, 3-aminooxetan-2-one compounds were identified as a class of highly potent NAAA inhibitors. The utility of these compounds is limited, however, by their low chemical and plasma stabilities. In the present study, we synthesized and tested a series of N-(2-oxoazetidin-3-yl)amides as a novel class of NAAA inhibitors with good potency and improved physicochemical properties, suitable for systemic administration. Moreover, we elucidated the main structural features of 3-aminoazetidin-2-one derivatives that are critical for NAAA inhibition.

N-(2-Oxo-3-oxetanyl)carbamic Acid Esters as N-Acylethanolamine Acid Amidase Inhibitors: Synthesis and Structure–Activity and Structure–Property Relationships

The β-lactone ring of N-(2-oxo-3-oxetanyl)amides, a class of N-acylethanolamine acid amidase (NAAA) inhibitors endowed with anti-inflammatory properties, is responsible for both NAAA inhibition and low compound stability. Here, we investigate the structure-activity and structure-property relationships for a set of known and new β-lactone derivatives, focusing on the new class of N-(2-oxo-3-oxetanyl)carbamates. Replacement of the amide group with a carbamate one led to different stereoselectivity for NAAA inhibition and higher intrinsic stability, because of the reduced level of intramolecular attack at the lactone ring. The introduction of a syn methyl at the β-position of the lactone further improved chemical stability. A tert-butyl substituent in the side chain reduced the reactivity with bovine serum albumin. (2S,3R)-2-Methyl-4-oxo-3-oxetanylcarbamic acid 5-phenylpentyl ester (27, URB913/ARN077) inhibited NAAA with good in vitro potency (IC(50) = 127 nM) and showed improved stability. It is rapidly cleaved in plasma, which supports its use for topical applications.