Positive allosteric modulation of the type 1 cannabinoid receptor potentiates endocannabinoid signalling and changes ERK1/2 phosphorylation kinetics.

Background and Purpose: Activation of CB1 by exogenous agonists causes adverse effects in
vivo. Positive allosteric modulation may offer improved therapeutic potential and a reduced
on-target adverse effect profile compared to orthosteric agonists, due to reduced
desensitisation/tolerance, but this has not been directly tested. This study investigated the
ability of PAMs/ago-PAMs to induce receptor regulation pathways, including desensitisation
and receptor internalisation.

Experimental Approach: Bioluminescence resonance energy transfer assays in HEK293 cells were performed to investigate G protein dissociation, ERK1/2 phosphorylation and β-arrestin 2 translocation, while immunocytochemistry was performed to measure internalisation of CB1 in response to the PAMs ZCZ011, GAT229, and ABD1236 alone and in combination with the orthosteric agonists AEA, 2-AG, and AMB-FUBINACA.

Key Results: ZCZ011, GAT229, and ABD1236 were found to be allosteric agonists in all
pathways tested. The ago-PAM ZCZ011 induced a biphasic ERK1/2 phosphorylation time
course compared to transient activation by orthosteric agonists. In combination with 2-AG but not AEA or AMB-FUBINACA, ZCZ011 and ABD1236 caused the transient peak of ERK1/2
phosphorylation to become sustained. All PAMs increased the potency and efficacy of AEAinduced signalling in all pathways tested, however no notable potentiation of 2-AG or AMBFUBINACA was observed.
Conclusion and Implications: ago PAMs can potentiate endocannabinoid CB1 agonism by
AEA to a larger extent compared to 2-AG. However, all compounds were found to be allosteric
agonists, and induce activation of CB1 in the absence of endocannabinoid, including arrestin
recruitment and internalisation. Thus, the spatiotemporal signalling of endogenous
cannabinoids will not be retained in vivo.