Discovery and development of macrocyclic peptide modulators of the cannabinoid 2 receptor

The cannabinoid type 2 receptor (CB₂R), a G protein-coupled receptor, is an important regulator of immune cell function and a promising target to treat chronic inflammation and fibrosis. While CB₂R is typically targeted by small molecules, including endo-, phyto-, and synthetic cannabinoids, peptides-owing to their size-may offer a different interaction space to facilitate differential interactions with the receptor. Here, we explore plant-derived cyclic cystine-knot peptides as ligands of the CB₂R. Cyclotides are known for their exceptional biochemical stability. Recently, they gained attention as G protein-coupled receptor modulators and as templates for designing peptide ligands with improved pharmacokinetic properties over linear peptides. Cyclotide-based ligands for CB₂R were profiled based on a peptide-enriched extract library comprising nine plants. Employing pharmacology-guided fractionation and peptidomics, we identified the cyclotide vodo-C1 from sweet violet (Viola odorata) as a full agonist of CB₂R with an affinity (K_i) of 1 microM and a potency (EC₅₀) of 8 microM. Leveraging deep learning networks, we verified the structural topology of vodo-C1 and modeled its molecular volume in comparison to the CB₂R ligand binding pocket. In a fragment-based approach, we designed and characterized vodo-C1-based bicyclic peptides (vBCL1-4), aiming to reduce size and improve potency. Opposite to vodo-C1, the vBCL peptides lacked the ability to activate the receptor but acted as negative allosteric modulators or neutral antagonists of CB₂R. This study introduces a macrocyclic peptide phytocannabinoid, which served as a template for the development of synthetic CB₂R peptide modulators. These findings offer opportunities for future peptide-based probe and drug development at cannabinoid receptors.