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Updated: Aug 18, 2026

BRET-based G Protein Biosensors for Measuring G Protein-Coupled Receptor Activity in Live Cells
Published on: November 7, 2025
Identification of WIN55212-3 as a competitive neutral antagonist of the human cannabinoid CB2 receptor
Juha R Savinainen1, Tarja Kokkola, Outi M H Salo
1Department of Physiology, University of Kuopio, POB 1627, FIN-70211 Kuopio, Finland. juha.savinainen@uku.fi
Abstract:
1. Several G protein-coupled receptors (GPCRs), including cannabinoid CB(1) and CB(2) receptors, show constitutive activity under heterologous expression. Such a tonic response is generated in the absence of an activating ligand, and can be inhibited by inverse agonists. Neutral antagonists, however, are silent at such receptors, but can reverse both agonist and inverse agonist responses. To date, no neutral antagonist for the CB(2) receptor has been reported. 2. Here, by monitoring receptor-dependent G protein activation, we demonstrate that WIN55212-3 acts as a neutral antagonist at the human CB(2) (hCB(2)) receptor. WIN55212-3 alone, at concentrations =10(-4) M, behaved as a silent ligand exhibiting no agonist or inverse agonist activity. However, WIN55212-3 competitively antagonized cannabinoid agonist CP-55,940-stimulated responses (pA(2) 6.1). Importantly, the inverse agonism evoked by SR144528 in hCB(2) was dose-dependently reversed by WIN55212-3 (pEC(50) 5.3+/-0.2), indicating true neutral antagonist behavior. 3. Furthermore, WIN55212-3 also antagonized CB(1) receptor signaling in a competitive manner (pA(2) 5.6), but behaved as a partial inverse agonist (pIC(50) 5.5+/-0.1) at the constitutively active human CB(1). 4. Additionally, WIN55212-3 antagonized signaling of the human melatonin MT(1) receptor, with modest activity at the human muscarinic M4 receptor, but it was inactive towards several other GPCRs. 5. These data identify WIN55212-3 as a true neutral hCB(2) receptor antagonist. WIN55212-3 offers a valuable tool for further characterization of ligand activities at the CB(2) receptor and may serve as a lead compound in further efforts to develop more potent and selective neutral CB(2) receptor antagonists.
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