Real-time characterization of cannabinoid receptor 1 (CB1 ) allosteric modulators reveals novel mechanism of action

Br J Pharmacol. 2013 Oct;170(4):893-907. doi: 10.1111/bph.12329.


Background and purpose: The cannabinoid receptor type 1 (CB1 ) has an allosteric binding site. The drugs ORG27569 {5-chloro-3-ethyl-N-[2-[4-(1-piperidinyl)phenyl]ethyl]-1H-indole-2-carboxamide} and PSNCBAM-1 {1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea} have been extensively characterized with regard to their effects on signalling of the orthosteric ligand CP55,940 {(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol}, and studies have suggested that these allosteric modulators increase binding affinity but act as non-competitive antagonists in functional assays. To gain a deeper understanding of allosteric modulation of CB1 , we examined real-time signalling and trafficking responses of the receptor in the presence of allosteric modulators.

Experimental approach: Studies of CB1 signalling were carried out in HEK 293 and AtT20 cells expressing haemagglutinin-tagged human and rat CB1 . We measured real-time accumulation of cAMP, activation and desensitization of potassium channel-mediated cellular hyperpolarization and CB1 internalization.

Key results: ORG27569 and PSNCBAM-1 produce a complex, concentration and time-dependent modulation of agonist-mediated regulation of cAMP levels, as well as an increased rate of desensitization of CB1 -mediated cellular hyperpolarization and a decrease in agonist-induced receptor internalization.

Conclusions and implications: Contrary to previous studies characterizing allosteric modulators at CB1, this study suggests that the mechanism of action is not non-competitive antagonism of signalling, but rather that enhanced binding results in an increased rate of receptor desensitization and reduced internalization, which results in time-dependent modulation of cAMP signalling. The observed effect of the allosteric modulators is therefore dependent on the time frame over which the signalling response occurs. This finding may have important consequences for the potential therapeutic application of these compounds.

Keywords: allosteric modulator; cannabinoid receptor; desensitization; internalization; receptor signalling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cannabinoid Receptor Agonists / pharmacology*
  • Cannabinoid Receptor Antagonists / pharmacology*
  • Cell Line, Tumor
  • Cyclic AMP / metabolism
  • Cyclohexanols / metabolism
  • Cyclohexanols / pharmacology*
  • Dose-Response Relationship, Drug
  • HEK293 Cells
  • Humans
  • Indoles / pharmacology*
  • Kinetics
  • Ligands
  • Membrane Potentials
  • Mice
  • Phenylurea Compounds / pharmacology*
  • Piperidines / pharmacology*
  • Potassium Channels, Inwardly Rectifying / drug effects
  • Potassium Channels, Inwardly Rectifying / metabolism
  • Protein Transport
  • Pyridines / pharmacology*
  • Rats
  • Receptor, Cannabinoid, CB1 / drug effects*
  • Receptor, Cannabinoid, CB1 / genetics
  • Receptor, Cannabinoid, CB1 / metabolism
  • Signal Transduction / drug effects
  • Transfection


  • 1-(4-chlorophenyl)-3-(3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl)urea
  • 5-chloro-3-ethyl-1H-indole-2-carboxylic acid (2-(4-piperidin-1-yl-phenyl)ethyl)amide
  • CNR1 protein, human
  • Cannabinoid Receptor Agonists
  • Cannabinoid Receptor Antagonists
  • Cnr1 protein, rat
  • Cyclohexanols
  • Indoles
  • Ligands
  • Phenylurea Compounds
  • Piperidines
  • Potassium Channels, Inwardly Rectifying
  • Pyridines
  • Receptor, Cannabinoid, CB1
  • 3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol
  • Cyclic AMP