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Cannabis sativa has been used therapeutically since early civilizations, with key cannabinoids Δ 9 -tetrahydrocannabinol (THC) 3.1 and cannabidiol characterized in the 1960s, leading to the discovery of cannabinoid receptors type 1 (CB 1 R) and type 2 (CB 2 R) and the endocannabinoid system (ECS) in the 1990s. The ECS, involving endogenous ligands like 2-arachidonoylglycerol (2-AG) 1.1, anandamide (N-arachidonoylethanolamine (AEA)) 1.2, and various proteins, regulates vital processes such as sleep, appetite, and memory, and holds significant therapeutic potential, especially for neurological disorders. Small molecule-derived pharmacological tools, or chemical probes, target key components of the ECS and are crucial for target validation, mechanistic studies, pathway elucidation, phenotypic screening, and drug discovery. These probes selectively interact with specific proteins or pathways, enabling researchers to modulate target activity and observe biological effects. When they carry an additional reporter group, they are referred to as labeled chemical probes. Developed through medicinal chemistry, structural biology, and high-throughput screening, effective chemical probes must be selective, potent, and depending on their purpose meet additional criteria such as cell permeability and metabolic stability.This chapter describes high-quality labeled and unlabeled chemical probes targeting ECS constituents that have been successfully applied for various research purposes. CB 1 R and CB 2 R, class A G protein-coupled receptors, are activated by 2-AG 1.1, AEA 1.2, and THC 3.1, with numerous ligands developed for these receptors. Imaging techniques like single-photon emission computed tomography, positron emission tomography, and fluorescently labeled CB 1 R and CB 2 R probes have enhanced CB receptor studies. CB 2 R activation generally results in immunosuppressive effects, limiting tissue injury. AEA 1.2 is mainly degraded by fatty acid amide hydrolase (FAAH) or N-acylethanolamine acid amidase (NAAA) into ethanolamine and arachidonic acid (AA) 1.3. FAAH inhibitors increase endogenous fatty acid amides, providing analgesic effects without adverse effects. NAAA inhibitors reduce inflammation and pain in animal models. Diacylglycerol lipase (DAGL) is essential for 2-AG 1.1 biosynthesis, while monoacylglycerol lipase (MAGL) degrades 2-AG 1.1 into AA 1.3, thus regulating cannabinoid signaling. Multiple inhibitors targeting FAAH and MAGL have been generated, though NAAA and DAGL probe development lags behind. Similarly, advancements in inhibitors targeting endocannabinoid (eCB) cellular uptake or trafficking proteins like fatty acid-binding proteins have been slower. The endocannabinoidome (eCBome) includes the ECS and related molecules and receptors, offering therapeutic opportunities from non-THC cannabinoids and eCBome mediators. Ongoing research aims to refine chemical tools for ECS and eCBome study, addressing unmet medical needs in central nervous system disorders and beyond., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Endocannabinoids [2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (AEA)], endogenously produced arachidonate-based lipids, are anti-inflammatory physiological ligands for two known cannabinoid receptors, CB1 and CB2, yet the molecular and cellular mechanisms underlying their effects after brain injury are poorly defined. In the present study, we hypothesize that traumatic brain injury (TBI)-induced loss of endocannabinoids exaggerates neurovascular injury, compromises brain-cerebrospinal fluid (CSF) barriers (BCB) and causes behavioral dysfunction. Preliminary analysis in human CSF and plasma indicates changes in endocannabinoid levels. This encouraged us to investigate the levels of endocannabinoid-metabolizing enzymes in a mouse model of controlled cortical impact (CCI). Reductions in endocannabinoid (2-AG and AEA) levels in plasma were supported by higher expression of their respective metabolizing enzymes, monoacylglycerol lipase (MAGL), fatty acid amide hydrolase (FAAH), and cyclooxygenase 2 (Cox-2) in the post-TBI mouse brain. Following increased metabolism of endocannabinoids post-TBI, we observed increased expression of CB2, non-cannabinoid receptor Transient receptor potential vanilloid-1 (TRPV1), aquaporin 4 (AQP4), ionized calcium binding adaptor molecule 1 (IBA1), glial fibrillary acidic protein (GFAP), and acute reduction in cerebral blood flow (CBF). The BCB and pericontusional cortex showed altered endocannabinoid expressions and reduction in ventricular volume. Finally, loss of motor functions and induced anxiety behaviors were observed in these TBI mice. Taken together, our findings suggest endocannabinoids and their metabolizing enzymes play an important role in the brain and BCB integrity and highlight the need for more extensive studies on these mechanisms. • TBI leads to chronic vascular, ventricular, and neurological deficits. • Brain barriers are compromised after traumatic brain injury. • Compromised brain-CSF barrier (BCB) shows altered cannabinoid regulation. • Altered endocannabinoid system (ECS) reduces endocannabinoids (2-AG and AEA). • Restoring cannabinoid homeostasis may promote neurological recovery after TBI. [ABSTRACT FROM AUTHOR]

Aims Pro-inflammatory cytokines, growth and angiogenic factors released by leukocytes are involved in carcinogenesis and cancer progression, but they are also crucial for fighting tumour growth and spreading. We have previously demonstrated that endocannabinoids modulate cell-to-cell crosstalk during inflammation. Here, we investigated the inflammatory and tumourigenic properties of endocannabinoids in a human urinary bladder carcinoma cell line. Main methods Endocannabinoid-treated ECV304 cells were checked for tumour necrosis factor (TNF)-α secretion (by ELISA assay) and surface exposure of selectins (by in situ ELISA and FACS analysis). ECV304/Jurkat T cell interaction was assessed by adhesion and live imaging experiments. Proliferation rate, cell death and cell cycle were determined by FACS analysis. Key findings By binding to type-1 (CB 1 ) and type-2 (CB 2 ) cannabinoid receptors, the endocannabinoid 2-arachidonoylglycerol (2-AG) exacerbates the pro-inflammatory status surrounding bladder carcinoma ECV304 cells, by: (i) enhancing TNF-α release, (ii) increasing surface exposure of P- and E-selectins, and (iii) allowing Jurkat T lymphocytes to adhere to treated cancer cells. We also found that the CB 1 inverse agonist AM281, unlike 2-AG, decreases cancer proliferation by delaying cell cycle progression. Significance Our data suggest that 2-AG modulates the inflammatory milieu of cancer cells in vitro, while AM281 plays a more specific role in proliferation. Collectively, these findings suggest that CB receptors may play distinct roles in cancer biology, depending on the specific ligand employed. Conclusions The in vivo assessment of the role of CB receptors in inflammation and cancer might be instrumental in broadening the understanding about bladder cancer biology. [ABSTRACT FROM AUTHOR]

Abstract: Cannabinoids, the active components of cannabis (Cannabis sativa) extracts, have attracted the attention of human civilizations for centuries, much earlier than the discovery and characterization of their substrate of action, the endocannabinoid system (ECS). The latter is an ensemble of endogenous lipids, their receptors [in particular type-1 (CB1) and type-2 (CB2) cannabinoid receptors] and metabolic enzymes. Cannabinoid signaling regulates cell proliferation, differentiation and survival, with different outcomes depending on the molecular targets and cellular context involved. Cannabinoid receptors are expressed and functional from the very early developmental stages, when they regulate embryonic and trophoblast stem cell survival and differentiation, and thus may affect the formation of manifold adult specialized tissues derived from the three different germ layers (ectoderm, mesoderm and endoderm). In the ectoderm-derived nervous system, both CB1 and CB2 receptors are present in neural progenitor/stem cells and control their self-renewal, proliferation and differentiation. CB1 and CB2 show opposite patterns of expression, the former increasing and the latter decreasing along neuronal differentiation. Recently, endocannabinoid (eCB) signaling has also been shown to regulate proliferation and differentiation of mesoderm-derived hematopoietic and mesenchymal stem cells, with a key role in determining the formation of several cell types in peripheral tissues, including blood cells, adipocytes, osteoblasts/osteoclasts and epithelial cells. Here, we will review these new findings, which unveil the involvement of eCB signaling in the regulation of progenitor/stem cell fate in the nervous system and in the periphery. The developmental regulation of cannabinoid receptor expression and cellular/subcellular localization, together with their role in progenitor/stem cell biology, may have important implications in human health and disease. [Copyright &y& Elsevier]

Abstract: The endocannabinoid, N-arachidonoylethanolamine (AEA), is degraded by the enzyme fatty acid amide hydrolase (FAAH). This study examined whether the FAAH inhibitor, URB597, increases retinal ganglion cell (RGC) survival following optic nerve axotomy in young and aged animals. URB597 alone, or together with either a CB1 or CB2 receptor antagonist, was administered daily for 1 or 2 weeks post-axotomy. Histological assessment of retinas indicated that URB597 increased RGC survival in young retina at 1 and 2 weeks post-axotomy. The increase in RGC survival at 2 weeks was accompanied by a reduction in phagocytic microglia. The CB1 antagonist, AM281, but not the CB2 antagonist, AM630, ablated URB597-mediated RGC neuroprotection. CB1 or CB2 antagonism increased phagocytic microglia in URB597 and vehicle-treated animals. In aged animals, URB597 increased RGC survival at 1 week, but not at 2 weeks post-axotomy and had no effect on microglia. Retinal Iba-1 positive microglia were also decreased in URB597-treated axotomized young animals and this decrease was mitigated by CB1 but not CB2 antagonism. As seen with phagocytotic microglia, the CB2 antagonist, AM630, increased Iba-1 positive microglia in the absence of URB597 treatment. Measurement of retinal endocannabinoid levels in URB597-treated animals at 2 weeks post-axotomy revealed a significant increase in AEA levels, accompanied by a decrease in the AEA metabolite, N-arachidonoyl glycine, in young animals but not aged animals. 2-arachidonoylglycerol levels were similar across all experimental groups. These data demonstrate that URB597-mediated retinal neuroprotective effects are mediated primarily through CB1 receptors and that URB597 neuroprotective efficacy declines with age. [Copyright &y& Elsevier]

Abstract: N-arachidonoylethanolamine (AEA) plays a crucial neuroprotective role in certain neurodegenerative diseases. Our recent studies suggested that AEA analog N-stearoyl-l-tyrosine (NsTyr) could protect neurons from apoptosis and improve hippocampus-dependent learning and memory deficits. The present study was designed to determine the neuroprotective effect of NsTyr on developmental sevoflurane neurotoxicity using primary hippocampal neuronal cultures and rat pups. We found that NsTyr could decrease cell viability and reduce apoptosis in sevoflurane treated neuronal cultures. In addition, NsTyr attenuated sevoflurane-induced apoptosis by modulating Caspase-3 and Bcl-2 in vivo. Moreover, sevoflurane exposure led to an inhibition of phospho-ERK1/2, which was rescued by NsTyr. Administration of U0126 (an inhibitor of MEK) abolished the neuroprotective effect of NsTyr on sevoflurane neurotoxicity both in vitro and in vivo. Finally, administration of NsTyr improved the learning and memory disorders induced by postnatal sevoflurane exposure without alteration in locomotor activity. These results indicated that AEA analog NsTyr protects developing brain against developmental sevoflurane neurotoxicity possibly through MEK/ERK1/2 MAPK signaling pathway. [Copyright &y& Elsevier]

Abstract: Class A G-protein coupled receptors (GPCRs) are thought to have a common topology that includes seven transmembrane alpha helices (TMHs) that are arranged to form a closed bundle. This bundle forms the ligand binding pocket into which ligands are commonly thought to enter via the extracellular milieu. This ligand approach direction makes sense for GPCRs that have small positively charged ligands, such as the beta-2-adrenergic or the dopamine D2 receptor. However, there is a growing sub-group of Class A GPCRs that bind lipid-derived endogenous ligands, such as the cannabinoid CB1 and CB2 receptors (with endogenous ligands, N-arachidonoylethanolamine (anandamide) and sn-2-arachidonylglycerol (2-AG)) and the S1P1–5 receptors (with endogenous ligand, sphingosine-1-phosphate). Even the widely studied Class A GPCR, rhodopsin, binds a highly lipophillic chromophore (11-cis-retinal). For these receptors, ligand approach from the extracellular milieu has seemed unlikely given that the ligands of these receptors readily partition into lipid or are actually synthesized in the lipid bilayer. The recent X-ray-crystal structure of the sub-type 1 sphingosine-1-phosphate receptor (S1P1) provides important information on the key structural variations that may be the hallmarks for a Class A GPCR that binds lipid-derived ligands. These include an extracellular domain that is closed off to the extracellular milieu and the existence of an opening between transmembrane helices that may serve as a portal for ligand entry via the lipid bilayer. This review examines structural aspects that the cannabinoid receptors may share with the S1P1 receptor based upon sequence homology. This review also examines experimental and simulation results that suggest ligand entry via a lipid portal is quite likely for this emerging sub-group. [Copyright &y& Elsevier]

Novel aromatic analogues of N-oleoylethanolamine and N-arachidonoylethanolamine (anandamide, AEA) were synthesized and, based on the capability of similar compounds to interact with proteins of the endocannabinoid and endovanilloid signaling systems, were tested on: (i) cannabinoid CB1 and CB2 receptors; (ii) vanilloid VR1 receptors; (iii) anandamide cellular uptake (ACU); and (iv) the fatty acid amide hydrolase (FAAH). The (R)- and, particularly, the (S)-1′-(4-hydroxybenzyl) derivatives of N-oleoylethanolamine and AEA (OMDM-1, OMDM-2, OMDM-3, and OMDM-4) inhibited to a varied extent ACU in RBL-2H3 cells (Ki ranging between 2.4 and 17.7 μM), the oleoyl analogues (OMDM-1 and OMDM-2, Ki 2.4 and 3.0 μM, respectively) being 6- to 7-fold more potent than the arachidonoyl analogues (OMDM-3 and OMDM-4). These four compounds exhibited: (i) poor affinity for either CB1 (Ki≥5 μM) or CB2 (Ki>10 μM) receptors in rat brain and spleen membranes, respectively; (ii) almost no activity at vanilloid receptors in the intracellular calcium assay carried out with intact cells over-expressing the human VR1 (ec50≥10 μM); and (iii) no activity as inhibitors of FAAH in N18TG2 cell membranes (Ki>50 μM). The oleoyl- and arachidonoyl-N′-(4-hydroxy-3-methoxybenzyl)hydrazines (OMDM-5 and OMDM-6), inhibited ACU (Ki 4.8 and 7.0 μM, respectively), and were more potent as VR1 agonists (ec50 75 and 50 nM, respectively), weakly active as CB1 receptor ligands (Ki 4.9 and 3.2 μM, respectively), and inactive as CB2 ligands (Ki>5 μM) as well as on FAAH (Ki≥40 μM). In conclusion, we report two novel potent and selective inhibitors of ACU (OMDM-1 and OMDM-2) and one “hybrid” agonist of CB1 and VR1 receptors (OMDM-6). Unlike other compounds of the same type, OMDM-1, OMDM-2, and OMDM-6 were very stable to enzymatic hydrolysis by rat brain homogenates. [Copyright &y& Elsevier]