title: Hydrocarbon stapled B chain analogues of relaxin-3 retain biological activity.
abstract: Relaxin-3 or insulin-like peptide 7 (INSL7) is the most recently discovered relaxin/insulin-like family peptide. Mature relaxin-3 consists of an A chain and a B chain held by disulphide bonds. According to structure activity relationship studies, the relaxin-3 B chain is more important in binding and activating the receptor. RXFP3 (also known as Relaxin-3 receptor 1, GPCR 135, somatostatin- and angiotensin- like peptide receptor or SALPR) was identified as the cognate receptor for relaxin-3 by expression profiles and binding studies. Recent studies imply roles of this system in mediating stress and anxiety, feeding, metabolism and cognition. Stapling of peptides is a technique used to develop peptide drugs for otherwise undruggable targets. The main advantages of stapling include, increased activity due to reduced proteolysis, increased affinity to receptors and increased cell permeability. Stable agonists and antagonists of RXFP3 are crucial for understanding the physiological significance of this system. So far, agonists and antagonists of RXFP3 are peptides. In this study, for the first time, we have introduced stapling of the relaxin-3 B chain at 14th and 18th positions (14s18) and 18th and 22nd position (18s22). These stapled peptides showed greater helicity than the unstapled relaxin-3 B chain in circular dichroism analysis. Both stapled peptides bound RXFP3 and activated RXFP3 as observed in an inhibition of forskolin-induced cAMP assay and a ERK1/2 activation assay, although with different potencies. Therefore, we conclude that stapling of the relaxin3 B chain does not compromise its ability to activate RXFP3 and is a promising method for developing stable peptide agonists and antagonists of RXFP3 to aid relaxin-3/RXFP3 research.
authors: Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Neurobiology and Ageing Programme, Life Sciences Institute, National University of Singapore, Singapore; Singapore Institute for Neurotechnology (SINAPSE), National University of Singapore, Singapore.
authors: Tharindunee
authors: T
authors: Jayakody
authors: Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore.
authors: Subhi
authors: S
authors: Marwari
authors: Singapore Eye Research Institute, Singapore; Ophthalmology and Visual Sciences Academic Clinical Program, Duke-NUS Medical School, Singapore.
authors: Rajamani
authors: R
authors: Lakshminarayanan
authors: Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors: Francis Chee Kuan
authors: FC
authors: Tan
authors: Institute of Chemical and Engineering Sciences, Agency for Science, Technology and Research (A*STAR), Singapore.
authors: Charles William
authors: CW
authors: Johannes
authors: Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore.
authors: Brian William
authors: BW
authors: Dymock
authors: Department of Medicinal Chemistry, Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore.
authors: Anders
authors: A
authors: Poulsen
authors: Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors: Deron Raymond
authors: DR
authors: Herr
authors: Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Neurobiology and Ageing Programme, Life Sciences Institute, National University of Singapore, Singapore; Singapore Institute for Neurotechnology (SINAPSE), National University of Singapore, Singapore. Electronic address: gavin_dawe@nuhs.edu.sg.
authors: Gavin Stewart
authors: GS
authors: Dawe
mesh_terms: D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis; D057809:HEK293 Cells; D006801:Humans; D006838:Hydrocarbons / Q000737:chemistry; D020935:MAP Kinase Signaling System / Q000187:drug effects; D010455:Peptides / Q000737:chemistry / Q000235:genetics*; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012065:Relaxin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D006838:Hydrocarbons; D010455:Peptides; C480238:RLN3 protein, human; C410092:RXFP3 protein, human; D043562:Receptors, G-Protein-Coupled; D005576:Colforsin; D012065:Relaxin; D000242:Cyclic AMP
keywords: ERK1/2; INSL7; RXFP3; Relaxin-3; Stapled peptide; cAMP