Tocrifluor T1117

Beyond radio-displacement techniques for identification of CB1 ligands: the first application of a fluorescence-quenching assay.[Pubmed:24441508]

Sci Rep. 2014 Jan 20;4:3757.

Cannabinoid type 1 Receptor (CB1) belongs to the GPCR family and it has been targeted, so far, for the discovery of drugs aimed at the treatment of neuropathic pain, nausea, vomit, and food intake disorders. Here, we present the development of the first fluorescent assay enabling the measurement of kinetic binding constants for CB1 orthosteric ligands. The assay is based on the use of T1117, a fluorescent analogue of AM251. We prove that T1117 binds endogenous and recombinant CB1 receptors with nanomolar affinity. Moreover, T1117 binding to CB1 is sensitive to the allosteric ligand ORG27569 and thus it is applicable to the discovery of new allosteric drugs. The herein presented assay constitutes a sustainable valid alternative to the expensive and environmental impacting radiodisplacement techniques and paves the way for an easy, fast and cheap high-throughput drug screening toward CB1 for identification of new orthosteric and allosteric modulators.

Cannabinoid- and lysophosphatidylinositol-sensitive receptor GPR55 boosts neurotransmitter release at central synapses.[Pubmed:23472002]

Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5193-8.

G protein-coupled receptor (GPR) 55 is sensitive to certain cannabinoids, it is expressed in the brain and, in cell cultures, it triggers mobilization of intracellular Ca(2+). However, the adaptive neurobiological significance of GPR55 remains unknown. Here, we use acute hippocampal slices and combine two-photon excitation Ca(2+) imaging in presynaptic axonal boutons with optical quantal analysis in postsynaptic dendritic spines to find that GPR55 activation transiently increases release probability at individual CA3-CA1 synapses. The underlying mechanism involves Ca(2+) release from presynaptic Ca(2+) stores, whereas postsynaptic stores (activated by spot-uncaging of inositol 1,4,5-trisphosphate) remain unaffected by GPR55 agonists. These effects are abolished by genetic deletion of GPR55 or by the GPR55 antagonist cannabidiol, a constituent of Cannabis sativa. GPR55 shows colocalization with synaptic vesicle protein vesicular glutamate transporter 1 in stratum radiatum. Short-term potentiation of CA3-CA1 transmission after a short train of stimuli reveals a presynaptic, Ca(2+) store-dependent component sensitive to cannabidiol. The underlying cascade involves synthesis of phospholipids, likely in the presynaptic cell, but not the endocannabinoids 2-arachidonoylglycerol or anandamide. Our results thus unveil a signaling role for GPR55 in synaptic circuits of the brain.

Fluorescent ligand binding reveals heterogeneous distribution of adrenoceptors and 'cannabinoid-like' receptors in small arteries.[Pubmed:20136833]

Br J Pharmacol. 2010 Feb;159(4):787-96.

BACKGROUND AND PURPOSE: Pharmacological analysis of synergism or functional antagonism between different receptors commonly assumes that interacting receptors are located in the same cells. We have now investigated the distribution of alpha-adrenoceptors, beta-adrenoceptors and cannabinoid-like (GPR55) receptors in the mouse arteries. EXPERIMENTAL APPROACH: Fluorescence intensity from vascular tissue incubated with fluorescent ligands (alpha(1)-adrenoceptor ligand, BODIPY-FL-prazosin, QAPB; beta-adrenoceptor ligand, TMR-CGP12177; fluorescent angiotensin II; a novel diarylpyrazole cannabinoid ligand (Tocrifluor 1117, T1117) was measured with confocal microscopy. Small mesenteric and tail arteries of wild-type and alpha(1B/D)-adrenoceptor-KO mice were used. KEY RESULTS: T1117, a fluorescent form of the cannabinoid CB(1) receptor antagonist AM251, was a ligand for GPR55, with low affinity for CB(1) receptors. In mesenteric arterial smooth muscle cells, alpha(1A)-adrenoceptors were predominantly located in different cells from those with beta-adrenoceptors, angiotensin receptors or cannabinoid-like (GPR55) receptors. Cells with beta-adrenoceptors predominated at arterial branches. Endothelial cells expressed beta-adrenoceptors, alpha-adrenoceptors and cannabinoid-like receptors. Only endothelial alpha-adrenoceptors appeared in clusters. Adventitia was a rich source of G protein-coupled receptors (GPCRs), particularly fibroblasts and nerve tracts, where Schwann cells bound alpha-adrenoceptor, beta-adrenoceptor and CB-receptor ligands, with a mix of separate receptor locations and co-localization. CONCLUSIONS AND IMPLICATIONS: Within each cell type, each GPCR had a distinctive heterogeneous distribution with limited co-localization, providing a guide to the possibilities for functional synergism, and suggesting a new paradigm for synergism in which interactions may be either between cells or involve converging intracellular signalling processes.