The cannabinoid 1 (CB1) allosteric modulator, 5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide) (ORG27569), has the paradoxical effect of increasing the equilibrium binding of [3H](-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl] cyclohexan-1-ol (CP55,940, an orthosteric agonist), while at the same time decreasing its efficacy (in G protein-mediated signaling). ORG27569 also decreases basal signaling, acting as an inverse agonist for the G protein-mediated signaling pathway. In ligand displacement assays, ORG27569 can displace the CB1 antagonist/inverse agonist, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A). The goal of this work was to identify the ORG27569 binding site at CB1. To this end, we used computational, synthesis, mutation, and functional studies to identify the ORG27569 binding site in the CB1 TMH3-6-7 region. This site is consistent with K3.28(192)A, F3.36(200)A, W5.43(279)A, W6.48(356)A, and F3.25(189)A mutation studies which revealed that the ORG27569 binding site overlaps with our previously determined binding site of SR141716A, but extends extracellularly. Additionally, we identified a key electrostatic interaction between the ORG27569 piperidine ring nitrogen and K3.28(192) that is required for ORG27569 to act as an inverse agonist. At this putative allosteric binding site, ORG27569 promotes an active-like conformation of the CB1 receptor, explaining the ORG27569 ability to increase CP55,940 equilibrium binding. This site also explains the ORG27569 ability to antagonize CP55,940 efficacy in three complementary ways: (1) ORG27569 sterically blocks movements of the second extracellular loop that have been linked to receptor activation; (2) ORG27569 sterically blocks a key electrostatic interaction between the third extracellular loop residue K(373) and D2.63(176); and (3) ORG27569 packs against TMH6, sterically hindering movements of this helix that have been shown to be important for receptor activation.

PMID:

 

24366865

 

[PubMed – as supplied by publisher]