PSNCBAM-1, a novel allosteric antagonist at cannabinoid CB1 receptors with hypophagic effects in rats

Abstract

Background and purpose: Rimonabant (Acomplia, SR141716A), a cannabinoid CB1 receptor inverse agonist, has recently been approved for the treatment of obesity. There are, however, concerns regarding its side effect profile. Developing a CB1 antagonist with a different pharmacological mechanism may lead to a safer alternative. To this end we have screened a proprietary small molecule library and have discovered a novel class of allosteric antagonist at CB1 receptors. Herein, we have characterized an optimized prototypical molecule, PSNCBAM-1, and its hypophagic effects in vivo.

Experimental approach: A CB1 yeast reporter assay was used as a primary screen. PSNCBAM-1 was additionally characterized in [35S]-GTPgammaS, cAMP and radioligand binding assays. An acute rat feeding model was used to evaluate its effects on food intake and body weight in vivo.

Key results: In CB1 receptor yeast reporter assays, PSNCBAM-1 blocked the effects induced by agonists such as CP55,940, WIN55212-2, anandamide (AEA) or 2-arachidonoyl glycerol (2-AG). The antagonist characteristics of PSNCBAM-1 were confirmed in [35S]-GTPgammaS binding and cAMP assays and was shown to be non-competitive by Schild analyses. PSNCBAM-1 did not affect CB2 receptors. In radioligand binding assays, PSNCBAM-1 increased the binding of [3H]CP55,940 despite its antagonist effects. In an acute rat feeding model, PSNCBAM-1 decreased food intake and body weight.

Conclusions and implications: PSNCBAM-1 exerted its effects through selective allosteric modulation of the CB1 receptor. The acute effects on food intake and body weight induced in rats provide a first report of in vivo activity for an allosteric CB1 receptor antagonist.

Figure 1

Structure of PSNCBAM-1; 1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea.

Figure 2

Effect of compounds on receptor signalling in Saccharomyces cerevisiae stably expressing recombinant human CB₁ receptors. Antagonism of 100 nM CP55,940-induced signalling by SR141716A and PSNCBAM-1 (a). Effect of SR141716A and PSNCBAM-1 on constitutive hCB₁ signalling (b). Yeast cells were incubated with compounds at 30°C and reporter gene expression was measured by a fluorescent readout. Agonist concentrations used were determined by their EC₉₀ values in the assay. Data points are mean±s.e.m. from three experiments and curves are fitted to a four parameter, one-site, dose–response equation. PSNCBAM-1, 1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea; SR141716A, N-(piperidin-1-yl)-5-(4-cholrophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide.

Figure 3

Effect of PSNCBAM-1 and SR141716A on [³⁵S]GTPγS binding in HEK293-hCB₁ cell membranes stimulated by either 50 nM CP55,940 (a) or 2.5 μM AEA (b); and rat cerebellar membranes stimulated by either 200 nM CP55,940 (c) or 10 μM AEA (d). Agonist concentrations used were determined by their EC₉₀ values for each membrane type. [³⁵S]GTPγS and compounds were mixed with membranes and incubated at 30°C for 60 min. Plates were then filtered and the radioactivity counted. Data points are mean±s.e.m. from three experiments and curves are fitted to a four parameter, one-site, dose–response equation. AEA, arachidonoyl ethanolamide; HEK, human embryonic kidney; PSNCBAM-1, 1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea; SR141716A, N-(piperidin-1-yl)-5-(4-cholrophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide.

Figure 4

Effect of PSNCBAM-1 and SR141716A on [³⁵S]GTPγS binding in unstimulated HEK293-hCB₁ cell membranes. [³⁵S]GTPγS and compounds were mixed with membranes and incubated at 30°C for 60 min. Plates were then filtered and the radioactivity counted. Data points are mean±s.e.m. from four experiments and curves are fitted to a four parameter, one-site, dose–response equation. HEK, human embryonic kidney; PSNCBAM-1, 1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea; SR141716A, N-(piperidin-1-yl)-5-(4-cholrophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide.

Figure 5

CP55,940-stimulated [³⁵S]GTPγS binding in HEK293-hCB₁ membranes in the presence of a range of concentrations of PSNCBAM-1 (a) and SR141716A (b). Data for SR141716A was used to generate a Schild plot (c). [³⁵S]GTPγS and compounds were mixed with membranes and incubated at 30°C for 60 min. Plates were then filtered and the radioactivity counted. Curves are fitted to a four parameter, one-site, dose–response equation. HEK, human embryonic kidney; PSNCBAM-1, 1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea; SR141716A, N-(piperidin-1-yl)-5-(4-cholrophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide.

Figure 6

Effect of PSNCBAM-1 and SR141716A on either (a) 10 nM CP55,940 or (b) 1 μM AEA-stimulated inhibition of cAMP accumulation induced by 5 μM forskolin, in HEK293-hCB₁ cells. Agonist concentrations used were determined by their EC₉₀ values. Cells were incubated with compounds for 30 min at room temperature. Intracellular cAMP levels were subsequently measured using the AlphaScreen kits from Perkin Elmer Inc. Data are means±s.e.m. from three experiments. The direct effects of PSNCBAM-1 and SR141716A on forskolin-induced cAMP responses were investigated as part of the validation of this assay and although some effects were noted, in particular with SR141716A, they did not alter the data interpretation when the agonist CP55,940 was present. AEA, arachidonoyl ethanolamide; HEK, human embryonic kidney; PSNCBAM-1, 1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea; SR141716A, N-(piperidin-1-yl)-5-(4-cholrophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide.

Figure 7

Effect of compounds on the binding of 0.8 nM [³H]CP55,940 (a) and 2 nM [³H]SR141716A (b) to HEK293-hCB₁ cell membranes. Compounds were mixed with membranes and incubated at 30°C for 90 min. Plates were then filtered and the radioactivity counted. Data points are mean±s.e.m. from three experiments and curves are fitted to a four parameter, one-site, dose–response equation. HEK, human embryonic kidney; SR141716A, N-(piperidin-1-yl)-5-(4-cholrophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide.

Figure 8

Effect of PSNCBAM-1 and SR141716A on acute feeding (a) and body weight (b) in male SD rats. Compounds were administered by i.p. injection before the start of the dark phase. Food intake was monitored by weighing the feeding jars at 1, 2, 4, 6 and 24 h and animals were weighed at 24 h. Results are means±s.e.m. for groups of six rats. ^*P<0.05 and ^**P<0.01, analysis of variance followed by Dunnett's multiple comparison test. i.p., intraperitoneal; PSNCBAM-1, 1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea; SD, Sprague–Dawley; SR141716A, N-(piperidin-1-yl)-5-(4-cholrophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide.