Identification of WIN55212‐3 as a competitive neutral antagonist of the human cannabinoid CB₂ receptor

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 p10 À4 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 5.370.2), 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 5.570.1) 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.

WIN55,212-2-Mediated Inhibition of HIV-1 Expression in Microglial Cells: Involvement of Cannabinoid Receptors

“…Microglia were treated with CB 1 /CB 2 receptor agonists (CP55,940, WIN55,212-2), a CB 2 -selective agonist (JWH 0-15), and CB 1 -selective (SR141716A) and CB 2 -selective (SR144528) receptor antagonists at concentrations ranging between 10 −9 and 10 −6 M (Howlett et al 2002). The inactive, neutral enantiomer of WIN55,212 (WIN55,212-3) was used as a control (Savinainen et al 2005). The results are shown for the most potent concentration (10 −6 M) for each of the agonists that had anti-HIV-1 activity (Fig.…”

“…Microglia were treated with WIN55,212-2 or its inactive, neutral enantiomer WIN55,212-3 (Savinainen et al 2005), and the percentage of microglia expressing CCR5 was compared to control cells as determined by flow cytometry (Fig. 3).…”

“…Drug treatment and viral infection Microglial cultures (1×10 5 cells/well in a 48-well plate) were pretreated without (control) or with CB 1 /CB 2 receptor agonists (WIN55,212-2 and CP55,940), CB 2 -selective agonist JWH 0-15, CB 1 -selective antagonists SR141716A and AM-251, CB 2 -selective antagonist SR144528, or the inactive enantiomer WIN55,212-3 (Savinainen et al 2005) for 3 h prior to HIV-1 infection. In one experiment SR144528 (a CB 2 -selective antagonist) (Howlett et al 2002) was added 30 min prior to treatment with CP55,940 or WIN55,212-2.…”

Cannabinoids and Multiple Sclerosis

“…More conclusive preclinical evidence that CB 1 and/or CB 2 receptor activation can indeed reduce signs and symptoms of multiple sclerosis or oppose its progression comes from reports that -R-(+)-WIN55212-induced amelioration of tremor and spasticity in CREAE/EAE mice (Table 2) can be attenuated by the CB 1 -selective antagonist SR141716A administered together with the CB 2 -selective antagonist, SR144528, albeit at doses that exacerbate tremor and spasticity in these lesioned animals in the absence of R-(+)-WIN55212 [49,51]; -amelioration by R-(+)-WIN55212 in EAE mice of tail limpness, hind limb weakness, hind limb paralysis and moribundity ( Table 2) and its attenuation in EAE mice of leukocyte rolling and adhesion (Table 3) is opposed by SR144528, although interestingly, not by SR141716A [45]; -neither (1) the ability of R-(+)-WIN55212 to ameliorate spasticity in CREAE mice or to decrease motor dysfunction and other clinical disease signs in TMEVinfected mice (Tables 2 and 3) nor (2) its ability to activate cannabinoid CB 1 and CB 2 receptors [61] is shared by the S-(-)-isomer of this aminoalkylindole [46,49]; -CB 1 −/− CREAE mice exhibit earlier onset of spasticity, more immobility and residual paresis, and greater neuronal/axonal loss, demyelination, and mortality than wild-type CREAE mice [62,63]; -signs of apoptosis and of axonal damage associated with IFN-γ-induced demyelination are more marked in cultured brain cells from CB 1 −/− mouse fetuses than from wild-type animals [64];…”

The Pharmacology of the Cannabinoid System—A Question of Efficacy and Selectivity

“…Similarly, an action produced by WIN55212-2 but not its purportedly inactive enantiomer WIN55212-3 can be interpreted as a CB receptor-mediated event (see e.g. [42]), although there is evidence in transfected cells that WIN 55,212-3 is, in fact, a weak neutral antagonist (pA 2 value, 6.1) at human CB 2 receptors, a partial inverse agonist (pIC 50 value, 5.5) at human CB 1 receptors, and can interact with the human MT 1 receptor melatonin at low micromolar concentrations [43]. Interpretation of the effects of CB receptor agonists assumes absolute selectivity of the compounds for the receptors in question.…”