The class of peptides known as cyclotides represent one of the largest family of circular plant proteins, with various plant families each estimated to express over 100,000 different peptide sequences[1]. In addition to their chemical stability, cyclotides have been shown to exhibit a diverse range of bioactivities, most presumably linked to defence mechanisms against herbivores, including immunosuppressive, antimicrobial and cytotoxic activities[2]. Consequently, cyclotides embody a vast exploitable reservoir for drug discovery. Since the original discovery of cyclotides in the African medicinal plant, Oldenlandia affinis, the isolated uterotonic cyclotide, kalata B7, has been identified to interact with the human oxytocin (OT) and arginine-vasopressin (AVP) receptors[3]. Given their diversity, this discovery highlights the potential of cyclotides as tools for G protein-coupled receptor (GPCR) probe design and drug discovery to provide new pharmacophores and stable scaffolds. Hence, we seek to discover new GPCR-active cyclotides from cyclotide-rich plants.
The corticotropin-releasing factor type 1 receptor (CRF1R) plays key roles in many physiological processes, ranging from metabolism to behaviour. Therapeutically, the development of antagonists for this neuropeptide GPCR shows potential for treating anxiety, depression and irritable bowel syndrome[4]. Using a bioactivity-guided fractionation strategy, we sought to discover CRF1R-active cyclotides from the cyclotide-rich plant Carapichea ipecacuanha, known for its use in the herbal medicinal preparation “ipecac”. Following initial screening of the plant extract via luciferase reporter-gene assay, antagonist fractions were chemically analysed. The most effective purified antagonist cyclotide, caripe 8, was chosen for further pharmacological characterisation via cAMP second messenger assays. We show that caripe 8 is a non-competitive cyclotide antagonist of the human CRF1R, offering a new novel lead scaffold for the development of ligands for the CRF1R, and possibly other class B GPCRs. These findings reveal new avenues for novel GPCR ligand discovery and development using these approaches for screening cyclotide plant fractions at GPCRs.