2‐Arachidonoylglycerol (2‐AG) is an important endogenous signaling lipid that activates the cannabinoid receptors (CB1R and CB2R), thereby regulating a diverse range of physiological processes ...including anxiety, appetite, inflammation, memory, pain sensation, and nociception. Diacylglycerol lipases (DAGLs) are the principle enzymes responsible for 2‐AG biosynthesis. Recently, the (patho)physiological functions of DAGLs have been explored by both genetic methods and chemical tools. This review will focus on the recent efforts to develop highly selective and in vivo active DAGLs inhibitors using activity‐based protein profiling.
The endocannabinoid system (ECS) is a widespread modulatory system composed of cannabinoid receptors, endogenous signaling lipids termed endocannabinoids and the enzymes for the biosynthesis and ...degradation of these endocannabinoids. It plays a crucial role in diverse (patho)physiological functions, such as neurotransmission, neural development, synaptic plasticity, mood, food intake, and inflammation. Enzymes involved in the biosynthesis (e. g. NAPE‐PLD, DAGLs) and degradation (e. g. FAAH, MAGL) of endocannabinoids have attracted attention from both academia and industry due to their therapeutic potential. The discovery of selective inhibitors for these enzymes is important for functional assignment and biomedical applications. Activity‐based protein profiling (ABPP) has emerged as a powerful technique, which to a large extend accelerated the development of selective inhibitors for ECS enzymes. This review summarizes the representative activity‐based probes (ABPs) and small molecular inhibitors developed in the past two decades for ECS enzymes and will discuss the biological discoveries attributed by the application of these small molecules.
Background and Purpose
Neurotransmission and neuroinflammation are controlled by local increases in both extracellular ATP and the endocannabinoid 2‐arachidonoyl glycerol (2‐AG). While it is known ...that extracellular ATP stimulates 2‐AG production in cells in culture, the dynamics and molecular mechanisms that underlie this response remain poorly understood. Detection of real‐time changes in eCB levels with the genetically encoded sensor, GRABeCB2.0, can address this shortfall.
Experimental Approach
2‐AG and arachidonoylethanolamide (AEA) levels in Neuro2a (N2a) cells were measured by LC‐MS, and GRABeCB2.0 fluorescence changes were detected using live‐cell confocal microscopy and a 96‐well fluorescence plate reader.
Key Results
2‐AG and AEA increased GRABeCB2.0 fluorescence in N2a cells with EC50 values of 81 and 58 nM, respectively; both responses were reduced by the cannabinoid receptor type 1 (CB1R) antagonist SR141617 and absent in cells expressing the mutant‐GRABeCB2.0. ATP increased only 2‐AG levels in N2a cells, as measured by LC‐MS, and induced a transient increase in the GRABeCB2.0 signal within minutes primarily via activation of P2X7 receptors (P2X7R). This response was dependent on diacylglycerol lipase β activity, partially dependent on extracellular calcium and phospholipase C activity, but not controlled by the 2‐AG hydrolysing enzyme, α/β‐hydrolase domain containing 6 (ABHD6).
Conclusions and Implications
Considering that P2X7R activation increases 2‐AG levels within minutes, our results show how these molecular components are mechanistically linked. The specific molecular components in these signalling systems represent potential therapeutic targets for the treatment of neurological diseases, such as chronic pain, that involve dysregulated neurotransmission and neuroinflammation.
Endocannabinoids, a class of lipid messengers, have emerged as crucial regulators of synaptic communication in the CNS. Dysregulation of these compounds has been implicated in many brain disorders. ...Although some studies have identified and quantified a limited number of target compounds, a method that provides comprehensive quantitative information on endocannabinoids and related N-acylethanolamines (NAEs) in cerebrospinal fluid (CSF) is currently lacking, as measurements are challenging due to low concentrations under normal physiological conditions. Here we developed and validated a high-throughput nano LC-ESI-MS/MS platform for the simultaneous quantification of endocannabinoids (anandamide and 2-arachidonoylglycerol), ten related NAEs, and eight additional putatively annotated NAEs in human CSF. Requiring only 200 μl of CSF, our method has limits of detection from 0.28 to 61.2 pM with precisions of relative SD <15% for most compounds. We applied our method to CSF from 45 healthy humans and demonstrated potential age and gender effects on concentrations of endocannabinoids and NAEs. Notably, our results show that docosahexaenoylethanolamide concentrations increase with age in males. Our method may offer new opportunities to gain insight into regulatory functions of endocannabinoids in the context of (ab)normal brain function.
Background and Purpose
Endocannabinoid (eCB) signalling gates many aspects of the stress response, including the hypothalamic–pituitary–adrenal (HPA) axis. The HPA axis is controlled by corticotropin ...releasing hormone (CRH) producing neurons in the paraventricular nucleus of the hypothalamus (PVN). Disruption of eCB signalling increases drive to the HPA axis, but the mechanisms subserving this process are poorly understood.
Experimental Approach
Using an array of cellular, endocrine and behavioural readouts associated with activation of CRH neurons in the PVN, we evaluated the contributions of tonic eCB signalling to the generation of a stress response.
Key Results
The CB1 receptor antagonist/inverse agonist AM251, neutral antagonist NESS243 and NAPE PLD inhibitor LEI401 all uniformly increased Fos in the PVN, unmasked stress‐linked behaviours, such as grooming, and increased circulating CORT, recapitulating the effects of stress. Similar effects were also seen after direct administration of AM251 into the PVN, while optogenetic inhibition of PVN CRH neurons ameliorated stress‐like behavioural changes produced by disruption of eCB signalling.
Conclusions and Implications
These data indicate that under resting conditions, constitutive eCB signalling restricts activation of the HPA axis through local regulation of CRH neurons in the PVN.
The cannabinoid receptor type 2 (CB2R) is a G protein‐coupled receptor with therapeutic potential for the treatment of inflammatory disorders. Fluorescent probes are desirable to study its receptor ...localization, expression and occupancy. Previously, we have reported a photoaffinity probe LEI‐121 that stabilized the inactive conformation of the CB2R. Here, we report the structure‐based design of a novel bifunctional probe that captures the active conformation of the CB2R upon irradiation with light. An alkyne handle was incorporated to visualize the receptor using click‐chemistry with fluorophore‐azides. These probes may hold promise to study different receptor conformations in relation to their cellular localization and function.
We present a series of bifunctional photoaffinity CB2R selective probes based on the 5‐fluoropyridin‐2‐yl‐benzyl‐imidazoleidine‐2,4‐dione derivative LEI‐102, with both inverse agonist and partial agonist behaviour in vitro. These photoaffinity probes have improved affinity and potency compared to previously published LEI‐121.
Competitive activity-based protein profiling is a highly efficient chemical biology technique to determine target engagement and selectivity profiles of enzyme inhibitors in complex proteomes. ...Fluorophosphonate-based fluorescent inhibitors are widely used as broad-spectrum probes for serine hydrolases. However, diacylglycerol lipase-α is not labeled by fluorophosphonate-based probes. To overcome this problem, we have developed a tailor-made activity-based probe that reacts with diacylglycerol lipase-α. Here we describe a case study in which we apply competitive activity-based protein profiling using a broad-spectrum and a tailor-made activity-based probe to establish selectivity and activity profiles of inhibitors targeting diacylglycerol lipase-α in the mouse brain proteome.
Ground-breaking research in disease biology and continuous efforts in method development have uncovered a range of potential new drug targets. Increasingly, the drug discovery process is informed by ...technologies involving chemical probes as tools. Applications for chemical probes comprise target identification and assessment, as well as the qualification of small molecules as chemical starting points and drug candidates. Progress in probe chemistry has opened the way to novel assay formats and pharmaceutical compound classes. The European Federation of Medicinal Chemistry and Chemical Biology (EFMC) has launched the Chemical Biology Initiative to advance science in the field of medicinal chemistry and chemical biology, while representing all members of this extended scientific community. This review provides an overview of the many important developments in the field of chemical biology that have happened at the lively interface of academic and industrial research.
Individuals with alcohol use disorder exhibit compulsive habitual behaviors that are thought to be, in part, a consequence of chronic and persistent use of alcohol. The endocannabinoid system plays a ...critical role in habit learning and in ethanol self‐administration, but the role of this neuromodulatory system in the expression of habitual alcohol seeking is unknown. Here, we investigated the role of the endocannabinoid system in established alcohol habits using contingency degradation in male C57BL/6 mice. We found that administration of the novel diacyl glycerol lipase inhibitor DO34, which decreases the biosynthesis of the endocannabinoid 2‐arachidonoyl glycerol (2‐AG), reduced habitual responding for ethanol and ethanol approach behaviors. Moreover, administration of the endocannabinoid transport inhibitor AM404 or the cannabinoid receptor type 1 antagonist AM251 produced similar reductions in habitual responding for ethanol and ethanol approach behaviors. Notably, AM404 was also able to reduce ethanol seeking and consumption in mice that were insensitive to lithium chloride‐induced devaluation of ethanol. Conversely, administration of JZL184, a monoacyl glycerol lipase inhibitor that increases levels of 2‐AG, increased motivation to respond for ethanol on a progressive ratio schedule of reinforcement. These results demonstrate an important role for endocannabinoid signaling in the motivation to seek ethanol, in ethanol‐motivated habits, and suggest that pharmacological manipulations of endocannabinoid signaling could be effective therapeutics for treating alcohol use disorder.
Here, we found that pharmacologically reducing levels of the endocannabinoid 2‐arachidonoyl glycerol (2‐AG) with DO34 reduced habitual responding for ethanol in mice. Conversely, we found that pharmacologically increasing levels of 2‐AG with JZL184 increased breakpoint for ethanol in a progressive ratio paradigm. We additionally found that pharmacologically increasing levels of the endocannabinoid anandamide had no effect on habitual responding for ethanol. Together, these results demonstrate an important and selective role for 2‐AG signaling in the motivation for ethanol.
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