Background and PurposeThe cannabinoid (CB1) receptor is among the most abundant G protein‐coupled receptors in brain. Allosteric ligands bind to a different site on receptors than the orthosteric ligand can have effects that are unique to the allosteric ligand and modulate orthosteric ligand activity. We propose a unified mathematical model describing the interaction effects of the allosteric ligand Org27569 and the orthosteric agonist CP55940 on CB1 receptor.Experimental ApproachA ternary complex model was constructed, which incorporated kinetic properties to describe the time course of effects of Org27569 and CP55940 reported in the literature: (i) enhanced receptor binding of CP55940, (ii) reduced internalisation and (iii), time‐dependent modulation of cAMP. Underlying mechanisms of time‐dependent modulation by Org27569 were evaluated by simulation.Key ResultsA hypothetical transitional state of CP55940–CB1–Org27569, which can internalise but cannot inhibit cAMP, was shown to be necessary and was sufficient to describe the allosteric modulation by Org27569, prior to receptors adopting an inactive conformation. The model indicated that the formation of this transitional CP55940–CB1–Org27569 state and final inactive CP55940–CB1–Org27569 state contributes to the enhanced CP55940 binding. The inactive CP55940–CB1–Org27569 cannot internalise or inhibit cAMP, leading to reduced internalisation and cessation of cAMP inhibition.Conclusions and ImplicationsIn conclusion, a kinetic mathematical model for CB1 receptor allosteric modulation was developed. However, a standard ternary complex model was not sufficient to capture the data and a hypothetical transitional state was required to describe the allosteric modulation properties of Org27569.
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
characterize 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 characterize ZCZ011 alone and in combination with orthosteric
agonists. Real-time BRET approaches were employed for G protein dissociation,
cAMP signaling, and β-arrestin translocation. Characterization
also included ERK1/2 phosphorylation (PerkinElmer AlphaLISA) and receptor
internalization. ZCZ011 is an allosteric agonist of CB1 in all pathways tested, with a similar signaling 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 internalization. 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
end points.
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