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Scheme 1 Reagents and conditions: (a) p-toluenesulfonic acid, TFA, room temperature, 15 min; (b) S7, DIPEA, dry DCM, room temperature, 15 h; (c) 5-azidopentyl chloroformate, DIPEA, dry DCM, room temperature, 15 h; (d) BODIPY alkyne 10, CuSO 4 Á5H 2 O, sodium ascorbate, DMF, room temperature, 15 h.
Source publication
The cysteine hydrolase, N-acylethanolamine acid amidase (NAAA) is a promising target for analgesic and anti-inflammatory drugs. Here, we describe the development of two unprecedented NAAA-reactive activity-based probes as research tools...
Citations
... 60 The N-O-aryl β-lactam 21 provided the starting point for the design of two fluorescent molecules that contained either a boron-dipyrromethene (BODIPY; 23) or norbornene moiety (24). 108 The latter's ability to cross cell membranes may allow its application to monitor, for example, the acid-catalyzed generation of active NAAA in its natural setting. ...
N-Acylethanolamine acid amidase (NAAA) is an N-terminal cysteine hydrolase primarily found in the endosomal-lysosomal compartment of innate and adaptive immune cells. NAAA catalyzes the hydrolytic deactivation of palmitoylethanolamide (PEA), a lipid-derived peroxisome proliferator-activated receptor-α (PPAR-α) agonist that exerts profound anti-inflammatory effects in animal models. Emerging evidence points to NAAA-regulated PEA signaling at PPAR-α as a critical control point for the induction and the resolution of inflammation, and to NAAA itself as a target for anti-inflammatory medicines. The present perspective discusses three key aspects of this hypothesis: the role of NAAA in controlling the signaling activity of PEA; the structural bases for NAAA function and inhibition by covalent and non-covalent agents; and, finally, the potential value of NAAA-targeting drugs in the treatment of human inflammatory disorders.
... All probes have a distinct PUFA or endocannabinoid structure that serves as the basis of the probe. We recognise that next to these PUFA and endocannabinoid derived probes, many groups have focussed on the development of chemical probes for CB receptors [110][111][112] and endocannabinoid-related enzymes (like FAAH, NAAA, COX, and LOX, for example) [113][114][115][116]. These probes are specifically designed in order to have specific and high binding interactions with their protein target, and do therefore not contain a clear PUFA or endocannabinoid related structure. ...
Notwithstanding the ongoing debate on their full potential in health and disease, there is general consensus that n-3 PUFAs play important physiological roles. Increasing dietary n-3 PUFA intake results in increased DHA and EPA content in cell membranes as well as an increase in n-3 derived oxylipin and -endocannabinoid concentrations, like fatty acid amides and glycerol-esters. These shifts are believed to (partly) explain the pharmacological and anti-inflammatory effects of n-3 PUFAs. Recent studies discovered that n-3 PUFA-derived endocannabinoids can be further metabolized by the oxidative enzymes CYP-450, LOX and COX, similar to the n-6 derived endocannabinoids. Interestingly, these oxidized n-3 PUFA derived endocannabinoids of eicosapentaenoyl ethanolamide (EPEA) and docosahexaenoyl ethanolamide (DHEA) have higher anti-inflammatory and anti-proliferative potential than their precursors. In this review, an overview of recently discovered n-3 PUFA derived endocannabinoids and their metabolites is provided. In addition, the use of chemical probes will be presented as a promising technique to study the n-3 PUFA and n-3 PUFA metabolism within the field of lipid biochemistry.
... This cannot be excluded, as this region of the enzyme demonstrates significant flexibility, and additional rearrangements may occur upon contact with a lipid bilayer. The modeled ethanolamine portion is solvent exposed, explaining the enzyme's ability to hydrolyze a substrate bearing a bulky fluorescent head group (49) or react with probes containing a large head group substituent (61). These moieties would take the place of ethanolamine, projecting outside of the cavity into the solvent (Fig. 3E). ...
Significance
There is a strong need for new analgesic and antiinflammatory medicines that are both effective and safe. Animal studies have shown that inhibition of N -acylethanolamine acid amidase (NAAA)—an intracellular enzyme that degrades the lipid mediator palmitoylethanolamide—causes profound analgesic and antiinflammatory effects. To facilitate the discovery of drugs targeting this protein and to better understand its mechanism of action, we determined its 3D structure. Our results illustrate the sequential steps leading to the activation of NAAA at lipid membranes, and reveal how current inhibitors block this enzyme.
... The first described probe, ARN14686 ( Figure Ie), was based on the NAAA inhibitor ARN726, and requires addition of the reporting group to the probe via click chemistry after the probe reacted with the proteome [76]. Additional probes (for example Figure If) that do not require this two-step labeling procedure were developed [77]. Among the advantages of ABPP is the possibility to test potency and selectivity of an enzyme within a single experiment and to detect the enzyme in its active form ex vivo after administration of the probe to animals. ...
N-acylethanolamines (NAEs) (e.g., N-palmitoylethanolamine, N-arachidonoylethanolamine, N-oleoylethanolamine) are bioactive lipids involved in many physiological processes including pain, inflammation, anxiety, cognition and food intake. Two enzymes are responsible for the hydrolysis of NAEs and therefore regulate their endogenous levels and effects: fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase (NAAA). As discussed here, extensive biochemical characterization of NAAA was carried out over the years that contributed to a better understanding of NAAA enzymology. An increasing number of studies describe the synthesis and pharmacological characterization of NAAA inhibitors. Recent medicinal chemistry efforts have led to the development of potent and stable inhibitors that enable studying the effects of NAAA inhibition in preclinical disease models, notably in the context of pain and inflammation.
ConspectusSignaling lipids, such as the endocannabinoids, play an important role in the brain. They regulate synaptic transmission and control various neurophysiological processes, including pain sensation, appetite, memory formation, stress, and anxiety. Unlike classical neurotransmitters, lipid messengers are produced on demand and degraded by metabolic enzymes to control their lifespan and signaling actions. Chemical biology approaches have become one of the main driving forces to study and unravel the physiological role of lipid messengers in the brain. Here, we review how the development and use of chemical probes has allowed one to study endocannabinoid signaling by (i) inhibiting the biosynthetic and metabolic enzymes; (ii) visualizing the activity of these enzymes; and (iii) controlling the release and transport of the endocannabinoids. Activity-based probes were instrumental to guide the discovery of highly selective and in vivo active inhibitors of the biosynthetic (DAGL, NAPE-PLD) and metabolic (MAGL, FAAH) enzymes of endocannabinoids. These inhibitors allowed one to study the role of these enzymes in animal models of disease. For instance, the DAGL-MAGL axis was shown to control neuroinflammation and the NAPE-PLD-FAAH axis to regulate emotional behavior. Activity-based protein profiling and chemical proteomics were essential to guide the drug discovery and development of compounds targeting MAGL and FAAH, such as ABX-1431 (Lu AG06466) and PF-04457845, respectively. These experimental drugs are now in clinical trials for multiple indications, including multiple sclerosis and post-traumatic stress disorders. Activity-based probes have also been used to visualize the activity of these lipid metabolizing enzymes with high spatial resolution in brain slices, thereby showing the cell type-specific activity of these lipid metabolizing enzymes. The transport, release, and uptake of signaling lipids themselves cannot, however, be captured by activity-based probes in a spatiotemporal controlled manner. Therefore, bio-orthogonal lipids equipped with photoreactive, photoswitchable groups or photocages have been developed. These chemical probes were employed to investigate the protein interaction partners of the endocannabinoids, such as putative membrane transporters, as well as to study the functional cellular responses within milliseconds upon irradiation. Finally, genetically encoded sensors have recently been developed to monitor the real-time release of endocannabinoids with high spatiotemporal resolution in cultured neurons, acute brain slices, and in vivo mouse models. It is anticipated that the combination of chemical probes, highly selective inhibitors, and sensors with advanced (super resolution) imaging modalities, such as PharmacoSTORM and correlative light-electron microscopy, will uncover the fundamental basis of lipid signaling at nanoscale resolution in the brain. Furthermore, chemical biology approaches enable the translation of these fundamental discoveries into clinical solutions for brain diseases with aberrant lipid signaling.
Lipids perform a wide range of functions inside the cell, ranging from structural building block of membranes and energy storage to cell signaling. The mode of action of many signaling lipids has remained elusive due to their low abundance, high lipophilicity, and inherent instability. Various chemical biology approaches, such as photoaffinity or activity-based protein profiling methods, have been employed to shed light on the biological role of lipids and the lipid–protein interaction profile. In this review, we will summarize the recent developments in the field of chemical probes to study lipid biology, especially in immunology, and indicate potential avenues for future research.
