Therapeutic targeting of CB1 has been limited by adverse effects of orthosteric ligands. Allosteric modulation and biased signalling have been proposed to achieve pathway-selective control, but the receptor's complex coupling complicates interpretation of ligand actions.

In this study, we systematically compared orthosteric and allosteric CB1 ligands in transfected HEK293T cells by measuring G-protein coupling and downstream cAMP regulation in real time using NanoBiT and GloSensor assays.

Orthosteric agonists CP 55,940 and methanandamide activated both Gi and Gs with weak and transient β-arrestin2 recruitment, producing potent inhibition of forskolin-stimulated cAMP and modest increases in basal levels. The inverse agonist AM251 reduced basal Gi coupling and increased basal cAMP, consistent with inverse agonism at constitutively active CB1 receptors. Under forskolin stimulation, AM251 attenuated agonist-induced inhibition of cAMP and produced concentration-dependent modulation of forskolin-stimulated responses. AM4113 marginally reduced G-protein coupling and increased basal, while modestly reducing forskolin-stimulated cAMP, consistent with very low inverse efficacy. Among allosteric ligands, the positive allosteric modulator GAT229 increased Gi coupling and, with orthosteric agonists, further enhanced Gi and Gs signalling, elevating basal cAMP, without affecting β-arrestin2. The negative allosteric modulators PSNCBAM-1 and ORG27569 each reduced Gi- and Gs-coupling, elevated basal cAMP, and attenuated agonist-induced inhibition of forskolin-stimulated cAMP. Cannabidiol showed no intrinsic activity at CB1 and marginally reduced orthosteric G-protein signalling, consistent with indirect interaction with the receptor.

In summary, this study shows that orthosteric and allosteric CB1 ligands differ in G-protein coupling and cAMP regulation, clarifying key aspects of cannabinoid receptor signalling.