Study On Screening And Function Of CTR Biased Ligand

Calcitonin receptor(calcitonin Receptor,CTR)is a member of the family class B G protein coupled receptors(GPCR),and widely expressed in various tissues of the human body,such as the kidneys,adrenal glands,intestines and so on.CTR has a variety of functions,including maintaining the static state of muscle stem cells,limiting bone loss and promoting the survival of osteoclasts.Therefore,it’s not surprising that the receptor is an important therapeutic target for treatment of chronic diseases such as osteoporosis and hypercalcemia.At present,The natural CTR peptide agonists of both human calcitonin and salmon calcitonin have been used clinically to treat the osteoporosis and Paget’s disease.Recent studies have also developed calcitonin peptide mimics,including KBP-042,KBP-088 and KBP-089,which not only preserved the high efficacy of salmon calcitonin,but also increased the tolerability in rats.However,due to the lack of the costive effectiveness and potential side effects with unclear mechanism,these peptide based therapy are still limited useds.Alternatively,small compound agonists of CTR with defined signaling mechanism,which may have longer half life in the body and less expense compared to peptide synthesis,could have broader usage than the peptides agonists.Till now,only one small compound agonist,SUN-B8155 was reported to effectively activate CTR in cells.Normally,the activation of a GPCR activate both G protein subtype and arrestin signaling,which independently or cooperatively enable the particular receptor for multiple different physiological functions.A bias ligand of GPCR,which only operate selective G protein subtype or arrestin pathways,may be a better therapy than traditional agonists or antagonists for modern drug development to target GPCRs.Here,we synthesize a series of compounds(S8155-1-9)based on the chemical structure of SUN-B8155 and investigated the biased property using SUN-B8155 as the control.First of all,we construct CTR to pcDNA3.1 containing YFP tags.Then,we detecting GPCR’s second messenger cAMP and through BRET assay,we found that S8155-7 improved the potency of SUN-8155 by approximately 2 fold and displayed Gs biased property.In contrast,at least 5 SUN-8155 derivatives exhibitβ-arrestin2 biased signaling properties and one improve the potency of β-arrestin2 signaling by more than 10 folds.Further cellular studies demonstrated all these compounds inhibited the migration of the breast cancer MCF-7 cell migration via activation of CTR.At the same time,through the BAC to BAC expression system,uesing S8155-7 as the ligand we succeeded recombinanted CTR-G protein complex and did negative dyeing.here,we found that the small molecular bias ligand of CTR for the first time,which laid a foundation for further study of its bias transduction mechanism and changes in structural function,and also provided a new way of thinking for the treatment of related diseases in clinic.