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 (Ki) of 1 µM and a potency (EC50) of 8 µM. 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.

Plant-Derived Cyclotides Modulate κ-Opioid Receptor Signaling.

Cyclotides are plant-derived disulfide-rich peptides comprising a cyclic cystine knot, which confers remarkable stability against thermal, proteolytic, and chemical degradation. They represent an emerging class of G protein-coupled receptor (GPCR) ligands. In this study, utilizing a screening approach of plant extracts and pharmacological analysis we identified cyclotides from Carapichea ipecacuanha to be ligands of the κ-opioid receptor (KOR), an attractive target for developing analgesics with reduced side effects and therapeutics for multiple sclerosis (MS). This prompted us to verify whether [T20K]kalata B1, a cyclotide in clinical development for the treatment of MS, is able to modulate KOR signaling. T20K bound to and fully activated KOR in the low µM range. We then explored the ability of T20K to allosterically modulate KOR. Co-incubation of T20K with KOR ligands resulted in positive allosteric modulation in functional cAMP assays by altering either the efficacy of dynorphin A1-13 or the potency and efficacy of U50,488 (a selective KOR agonist), respectively. In addition, T20K increased the basal response upon cotreatment with U50,488. In the bioluminescence resonance energy transfer assay T20K negatively modulated the efficacy of U50,488. This study identifies cyclotides capable of modulating KOR and highlights the potential of plant-derived peptides as an opportunity to develop cyclotide-based KOR modulators.