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


METHODS
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.


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
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.
Centre for Brain Research and Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand.
Erin E
EE
Cawston
William J
WJ
Redmond
Courtney M
CM
Breen
Natasha L
NL
Grimsey
Mark
M
Connor
Michelle
M
Glass
D000818:Animals; D063386:Cannabinoid Receptor Agonists / Q000494:pharmacology*; D063387:Cannabinoid Receptor Antagonists / Q000494:pharmacology*; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000378:metabolism; D003511:Cyclohexanols / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D007700:Kinetics; D008024:Ligands; D008564:Membrane Potentials; D051379:Mice; D010671:Phenylurea Compounds / Q000494:pharmacology*; D010880:Piperidines / Q000494:pharmacology*; D024661:Potassium Channels, Inwardly Rectifying / Q000187:drug effects / Q000378:metabolism; D021381:Protein Transport; D011725:Pyridines / Q000494:pharmacology*; D051381:Rats; D043884:Receptor, Cannabinoid, CB1 / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
C526691:1-(4-chlorophenyl)-3-(3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl)urea; C572504:5-chloro-3-ethyl-1H-indole-2-carboxylic acid (2-(4-piperidin-1-yl-phenyl)ethyl)amide; C571130:CNR1 protein, human; D063386:Cannabinoid Receptor Agonists; D063387:Cannabinoid Receptor Antagonists; C543264:Cnr1 protein, rat; D003511:Cyclohexanols; D007211:Indoles; D008024:Ligands; D010671:Phenylurea Compounds; D010880:Piperidines; D024661:Potassium Channels, Inwardly Rectifying; D011725:Pyridines; D043884:Receptor, Cannabinoid, CB1; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol; D000242:Cyclic AMP
allosteric modulator; cannabinoid receptor; desensitization; internalization; receptor signalling