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

The cannabinoid type 2 receptor (CB2R), a G protein-coupled receptor (GPCR), is an important regulator of immune cell function and a promising target for to treat chronic inflammation and fibrosis. While CB2R is typically targeted by small organic molecules, including endo-, phyto- and synthetic cannabinoids, peptides may offer a different interaction space, owing to their larger size and greater contact area they facilitate differential interactions with a specific receptor target. 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 modulators of GPCR signaling 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 plant species. Employing pharmacology-guided fractionation and peptidomics we identified cyclotide vodo-C1 from sweet violet plant (Viola odorata) as a full agonist of CB2R with an affinity (Ki) of 1 µM and a potency (EC50) of 8 µM. Leveraging deep learning networks we verified the structural topology of vodo-C1 and modelled 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 new macrocyclic peptide phytocannabinoid, which served as template for the development of synthetic CB2R peptide modulators. These findings offer opportunities for future peptide-based probe and drug development at cannabinoid receptors.