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Ebola vaccine development has been accelerated in response to large outbreaks in West and Central Africa; those outbreaks have caused >32,000 cases and >13,500 deaths (1). One vaccine emerging from [...]

A decade ago, the drug-target residence time model has been (re-)introduced, which describes the importance of binding kinetics of ligands on their protein targets. Since then, it has been applied successfully for multiple protein targets, including GPCRs, for the development of lead compounds with slow dissociation kinetics (i.e. long target residence time) to increase in vivo efficacy or with short residence time to prevent on-target associated side effects. To date, this model has not been applied in the design and pharmacological evaluation of novel selective ligands for the cannabinoid CB 2 receptor (CB 2 R), a GPCR with therapeutic potential in the treatment of tissue injury and inflammatory diseases. Here, we have investigated the relationships between physicochemical properties, binding kinetics and functional activity in two different signal transduction pathways, G protein activation and β-arrestin recruitment. We synthesized 24 analogues of 3-cyclopropyl-1-(4-(6-((1,1-dioxidothiomorpholino)methyl)-5-fluoropyridin-2-yl)benzyl)imidazoleidine-2,4-dione (LEI101), our previously reported in vivo active and CB 2 R-selective agonist, with varying basicity and lipophilicity. We identified a positive correlation between target residence time and functional potency due to an increase in lipophilicity on the alkyl substituents, which was not the case for the amine substituents. Basicity of the agonists did not show a relationship with affinity, residence time or functional activity. Our findings provide important insights about the effects of physicochemical properties of the specific substituents of this scaffold on the binding kinetics of agonists and their CB 2 R pharmacology. This work therefore shows how CB 2 R agonists can be designed to have optimal kinetic profiles, which could aid the lead optimization process in drug discovery for the study or treatment of inflammatory diseases. [ABSTRACT FROM AUTHOR]

Diacylglycerol lipases (DAGLα and DAGLβ) convert diacylglycerol to the endocannabinoid 2-arachidonoylglycerol. Our understanding of DAGL function has been hindered by a lack of chemical probes that can perturb these enzymes in vivo. Here, we report a set of centrally active DAGL inhibitors and a structurally related control probe and their use, in combination with chemical proteomics and lipidomics, to determine the impact of acute DAGL blockade on brain lipid networks in mice. Within 2 h, DAGL inhibition produced a striking reorganization of bioactive lipids, including elevations in DAGs and reductions in endocannabinoids and eicosanoids. We also found that DAGLα is a short half-life protein, and the inactivation of DAGLs disrupts cannabinoid receptor-dependent synaptic plasticity and impairs neuroinflammatory responses, including lipopolysaccharide-induced anapyrexia. These findings illuminate the highly interconnected and dynamic nature of lipid signaling pathways in the brain and the central role that DAGL enzymes play in regulating this network. [ABSTRACT FROM AUTHOR]

Cannabinoid receptor 1 (CB 1 R) and cannabinoid receptor 2 (CB 2 R) are G protein-coupled receptors (GPCRs) that activate a variety of pathways upon activation by (partial) agonists including the G protein pathway and the recruitment of β-arrestins. Differences in the activation level of these pathways lead to biased signaling. Here, we describe a detailed protocol to characterize the potency and efficacy of ligands to induce or inhibit β-arrestin recruitment to the human CB 1 R and CB 2 R using the PathHunter ® assay. This is a cellular assay that uses a β-galactosidase complementation system which has a chemiluminescent read-out and can be performed in 384-well plates. We have successfully used this assay to characterize a set of reference ligands (both agonists, antagonists, and an inverse agonist) on human CB 1 R and CB 2 R, of which some examples will be presented here., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Using activity-based protein profiling (ABPP), functional proteins can be interrogated in their native environment. Despite their pharmaceutical relevance, G protein-coupled receptors (GPCRs) have been difficult to address through ABPP. In the current study, we took the prototypical human adenosine A 2A receptor (hA 2A R) as the starting point for the construction of a chemical toolbox allowing two-step affinity-based labeling of GPCRs. First, we equipped an irreversibly binding hA 2A R ligand with a terminal alkyne to serve as probe. We showed that our probe irreversibly and concentration-dependently labeled purified hA 2A R. Click-ligation with a sulfonated cyanine-3 fluorophore allowed us to visualize the receptor on SDS-PAGE. We further demonstrated that labeling of the purified hA 2A R by our probe could be inhibited by selective antagonists. Lastly, we showed successful labeling of the receptor in cell membranes overexpressing hA 2A R, making our probe a promising affinity-based tool compound that sets the stage for the further development of probes for GPCRs.

Introduction: Δ 9 -Tetrahydrocannabinol (THC), the principle psychoactive ingredient in Cannabis , is widely used for its therapeutic effects in a large variety of diseases, but it also has numerous neurological side effects. The cannabinoid receptors (CBRs) are responsible to a large extent for these, but not all biological responses are mediated via the CBRs. Objectives: The identification of additional target proteins of THC to enable a better understanding of the (adverse) physiological effects of THC. Methods: In this study, a chemical proteomics approach using a two-step photoaffinity probe is applied to identify potential proteins that may interact with THC. Results: Photoaffinity probe 1 , containing a diazirine as a photocrosslinker, and a terminal alkyne as a ligation handle, was synthesized in 14 steps. It demonstrated high affinity for both CBRs. Subsequently, two-step photoaffinity labeling in neuroblastoma cells led to identification of four potential novel protein targets of THC. The identification of these putative protein hits is a first step towards a better understanding of the protein interaction profile of THC, which could ultimately lead to the development of novel therapeutics based on THC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Chemical tools and methods that report on G protein-coupled receptor (GPCR) expression levels and receptor occupancy by small molecules are highly desirable. We report the development of LEI121 as a photoreactive probe to study the type 2 cannabinoid receptor (CB 2 R), a promising GPCR to treat tissue injury and inflammatory diseases. LEI121 is the first CB 2 R-selective bifunctional probe that covalently captures CB 2 R upon photoactivation. An incorporated alkyne serves as ligation handle for the introduction of reporter groups. LEI121 enables target engagement studies and visualization of endogenously expressed CB 2 R in HL-60 as well as primary human immune cells using flow cytometry. Our findings show that strategically functionalized probes allow monitoring of endogenous GPCR expression and engagement in human cells using tandem photoclick chemistry and hold promise as biomarkers in translational drug discovery.

Cannabinoid CB1 and CB2 receptors are G-protein-coupled receptors (GPCRs) that recruit β-arrestins upon activation by (partial) agonists. β-Arrestin recruitment is induced by phosphorylation of their C-terminal tails, and is associated with the termination of GPCR signaling; yet, it may also activate cellular signaling pathways independent of G-proteins. Here, we describe a detailed protocol to characterize the potency and efficacy of ligands to induce or inhibit β-arrestin recruitment to the human CB1 and CB2 receptors, by using the PathHunter(®) assay. The latter is a cellular assay that can be performed in plates with 384-wells. The PathHunter(®) assay makes use of β-galactosidase complementation, and has a chemiluminescent readout. We used this assay to characterize a set of reference ligands (both agonists and antagonists) on human CB1 and CB2 receptors.