ABSTRACT
We have previously identified allosteric modulators of the cannabinoid CB(1) receptor (Org 27569, PSNCBAM-1) that display a contradictory pharmacological profile: increasing the specific binding of the CB(1) receptor agonist [(3)H]CP55940 but producing a decrease in CB(1) receptor agonist efficacy. Here we investigated the effect one or both compounds in a broad range of signaling endpoints linked to CB(1) receptor activation. We assessed the effect of these compounds on CB(1) receptor agonist-induced [(35)S]GTPγS binding, inhibition, and stimulation of forskolin-stimulated cAMP production, phosphorylation of extracellular signal-regulated kinases (ERK), and ß-arrestin recruitment. We also investigated the effect of these allosteric modulators on CB(1) agonist binding kinetics. Both compounds display ligand dependence, being significantly more potent as modulators of CP55940 signaling as compared with WIN55212 and having little effect on [(3)H]WIN55212 binding. Org 27569 displays biased antagonism whereby it inhibits: agonist-induced guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding, simulation (Gα(s)-mediated), and inhibition (Gα(i)-mediated) of cAMP production and ß-arrestin recruitment. In contrast, it acts as an enhancer of agonist-induced ERK phosphorylation. Alone, the compound can act also as an allosteric agonist, increasing cAMP production and ERK phosphorylation. We find that in both saturation and kinetic-binding experiments, the Org 27569 and PSNCBAM-1 appeared to influence only orthosteric ligand maximum occupancy rather than affinity. The data indicate that the allosteric modulators share a common mechanism whereby they increase available high-affinity CB(1) agonist binding sites. The receptor conformation stabilized by the allosterics appears to induce signaling and also selectively traffics orthosteric agonist signaling via the ERK phosphorylation pathway.
Subject(s)
Allosteric Regulation/drug effects , Receptor, Cannabinoid, CB1/agonists , Receptor, Cannabinoid, CB1/metabolism , Animals , Arrestins/metabolism , Benzoxazines/pharmacology , Brain/drug effects , Brain/metabolism , CHO Cells , Cell Line , Cell Membrane/drug effects , Cell Membrane/metabolism , Colforsin/pharmacology , Cricetinae , Cyclic AMP/metabolism , Cyclohexanols/pharmacology , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , HEK293 Cells , Humans , Indoles/pharmacology , Kinetics , Ligands , MAP Kinase Signaling System/drug effects , Male , Mice , Morpholines/pharmacology , Naphthalenes/pharmacology , Phosphorylation/drug effects , Piperidines/pharmacology , Protein Binding/drug effects , Signal Transduction/drug effects , beta-ArrestinsABSTRACT
A series of potent carboxylic acid DGAT1 inhibitors with high permeability were developed from a virtual screening hit. Strategies were employed to reduce Pgp substrate recognition and increase passive permeability, resulting in the discovery of a series showing good inhibition of cellular triglyceride synthesis. The mutagenic potential of prospective metabolites was evaluated in the Ames assay, and one aniline was shown to be devoid of mutagenicity.
Subject(s)
Diacylglycerol O-Acyltransferase/antagonists & inhibitors , Drug Design , Enzyme Inhibitors/pharmacology , Caco-2 Cells , Diacylglycerol O-Acyltransferase/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Molecular Structure , Permeability/drug effects , Structure-Activity RelationshipABSTRACT
1-(4-Chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl] urea (PSNCBAM-1) has recently been described as a cannabinoid CB1 receptor allosteric antagonist associated with hypophagic effects in vivo; however, PSNCBAM-1 effects on CB(1) ligand-mediated modulation of neuronal excitability remain unknown. Here, we investigate PSNCBAM-1 actions on CB(1) receptor-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding in cerebellar membranes and on CB(1) ligand modulation of presynaptic CB(1) receptors at inhibitory interneuron-Purkinje cell synapses in the cerebellum using whole-cell electrophysiology. PSNCBAM-1 caused noncompetitive antagonism in [(35)S]GTPγS binding studies, with higher potency against the CB receptor agonist (-)-cis-3-[2-hydroxy-4-(1,1-dimethyl heptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55940) than for R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1,2,3,-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate] [WIN55,212-2 (WIN55)]. In electrophysiological studies, WIN55 and CP55940 reduced miniature inhibitory postsynaptic currents (mIPSCs) frequency but not amplitude. PSNCBAM-1 application alone had no effect on mIPSCs; however, PSNCBAM-1 pretreatment revealed agonist-dependent functional antagonism, abolishing CP55940-induced reductions in mIPSC frequency but having no clear effect on WIN55 actions. The CB(1) antagonist/inverse agonist N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1H-multipyrazole-3-carboxamide (AM251) increased mIPSC frequency beyond control; this effect was reversed by PSNCBAM-1. PSNCBAM-1 pretreatment also attenuated AM251 effects. Thus, PSNCBAM-1 reduced CB(1) receptor ligand functional efficacy in the cerebellum. The differential effect of PSNCBAM-1 on CP55940 versus WIN55 actions in [(35)S]GTPγS binding and electrophysiological studies and the attenuation of AM251 effects are consistent with the ligand-dependence associated with allosteric modulation. These data provide the first description of functional PSNCBAM-1 allosteric antagonist effects on neuronal excitability in the mammalian central nervous system (CNS). PSNCBAM-1 allosteric antagonism may provide viable therapeutic alternatives to orthosteric CB(1) antagonists/inverse agonists in the treatment of CNS disease.
