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

The cannabinoid-type 2 receptor (CB2 R), a G protein-coupled receptor (GPCR), is an important regulator of immune cell function and a promising target to treat chronic inflammation and fibrosis. While CB2 R 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 CB2 R. Cyclotides are known for their exceptional biochemical stability. Recently they gained attention as GPCR modulators and as templates for designing peptide ligands with improved pharmacokinetic properties over linear peptides. Cyclotide-based ligands for CB2 R were profiled based on a peptide-enriched extract library comprising nine plants. Employing pharmacology-guided fractionation and peptidomics we identified cyclotide vodo-C1 from sweet violet (Viola odorata) as a full agonist of CB2 R with an affinity (Ki ) of 1μM and a potency (EC50 ) of 8μM. Leveraging deep learning networks we verified the structural topology of vodo-C1 and modelled its molecular volume in comparison to the CB2 R 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 CB2 R. This study introduces a macrocyclic peptide phytocannabinoid, which served as template for the development of synthetic CB2 R peptide modulators. These findings offer opportunities for future peptide-based probe and drug development at cannabinoid receptors.