In vitro characterisation of 6-methyl-3-(2-nitro-1-(thiophen- 2-yl)ethyl)-2-phenyl-1Hindole (ZCZ011) at the type 1 cannabinoid receptor: allosteric agonist or allosteric modulator?

Orthosteric activation of CB1 is known to cause a plethora of adverse side effects in vivo. Allosteric modulation is an exciting therapeutic approach and is hoped to offer improved therapeutic potential and a reduced on-target side effect profile compared to orthosteric agonists. This study aimed to systematically characterise the in vitro activity of the positive allosteric modulator ZCZ011, explicitly considering its effects on receptor regulation. HEK293 cells expressing hCB1 receptors were used to characterise ZCZ011 alone and in combination with orthosteric agonists. Real-time BRET approaches were employed for G protein dissociation, cAMP signalling, and β-arrestin translocation. Characterisation also
included ERK1/2 phosphorylation (PerkinElmer AlphaLISA), and receptor internalisation. ZCZ011 is an allosteric agonist of CB1 in all pathways tested, with a similar signalling profile to that of the partial orthosteric agonist Δ9
-tetrahydrocannabinol. ZCZ011 also showed limited positive allosteric modulation in increasing the potency and efficacy of THC-induced ERK1/2 phosphorylation, β-arrestin translocation, and receptor internalisation. However, no positive allosteric modulation was observed for ZCZ011 in combination with either CP55940 or AMB-FUBINACA, in G protein dissociation nor cAMP inhibition. Our study suggests that ZCZ011 is an allosteric agonist, with effects that are often difficult to differentiate from those of orthosteric agonists. Together with its pronounced agonist activity, the limited extent of ZCZ011 positive allosteric modulation suggests that further investigation into the differences between allosteric and orthosteric agonism is required, especially in receptor
regulation endpoints.