Design,Synthesis Of Elabela Analogues And Their Activity Against Acute Kidney Injury

Purpose: There are 1.7 million deaths associated with acute kidney injury(AKI)annually and the incidence of AKI is increasing.Currently,renal replacement therapy is the major clinical treatment for AKI,and no effective drugs for AKI therapy have been applied in clinical.Therefore,it is urgent to find effective drugs to combat current difficulties in clinical treatment of AKI.Elabela(ELA)is an endogenous ligand for angiotensin receptor-like protein J receptor(APJ).The mature form of ELA,32-residue ELA(E32),plays important role in regulation of a variety of physiological processes.In previous study,a furin-cleaved11-residue fragment of ELA(ELA[22-32],E11)have been reported to protect against renal ischemia-reperfusion(I/R)injury.However,the poor plasma stability and weak APJ-binding ability of E11 limited its further application.Therefore,we aims to improve the plasma stability and APJ binding ability of E11 by chemical modification,so as to obtain effective drug candidates against AKI.Method: In section two,five analogues of E11: K(pal)-E11,Pal-E11,PEG6-E11,PEG～40-E11 and Pal-dimer-E11(Dimer-E11)were obtained by C-terminal amidation,and N-terminal palmitic acid(Pal)modification or polyethylene glycol(PEG)modification.E11 analogues were confirmed by mass spectrometry and their purity was determined by RP-HPLC.In section three,the binding ability of E11 analogues to APJ was determined by ligand competitive binding assay.The activation ability of APJ were determined by c AMP assay.The half-lives of E11 analogues,as well as the binding rates to mice plasma protein of E11 analogues,were measured by peptides and plasma co-incubation in vitro.The secondary structures of E11 analogues were studied by using circular dichroism spectra(CD)and fourier transform infrared spectra(FTIR).The effects of E11 analogues were assessed in hypoxia/reperfusion-injured and cisplatin-injured renal tubular cells.In section four,we investigated pre-injury reno-protective effects of E11 analogues on a renal I/R-injury mouse model.The half-life of Pal-E11 in vivo was studied by pharmacokinetic experiments in mice.The post-injury therapeutic effects of Pal-E11 in renal I/R-injury mouse model were investigated as well.Result: Compared with E11,the binding ability of the analogues to APJ was significantly enhanced,and they(except Dimer-E11)could effectively activate APJ.The half-lives of the E11 analogues were prolonged in human and mice plasma.The binding rates to mice plasma protein of E11 analogues were higher than that of E11.E11 analogues showed protective effects on renal tubular cells carryying hypoxia/reperfusion-injury and cisplatin-injury in vitro.Comparing to E11,E11 analogues showed better pre-injury reno-protective effects in AKI mouse models(except Dimer-E11);Pal-E11 showed the strongest pre-injury reno-protective effects.Pharmacokinetic experiments in mice showed that the half-life of Pal-E11 in vivo was also significantly longer than that of E11.Pal-E11 showed good post-injury therapeutic effects within 24 hours after the occurrence of AKI,suggesting its potential to be applied for AKI prevention and treatment.Conclusion: In summary,we designed and synthesized a set of E11 analogues with improved plasma stability and APJ-binding ability via rational chemical modification.E11 analogues showed protective effects on renal tubular cells with hypoxia/reperfusion-injury and cisplatin-injury in vitro.In a renal I/R-injury mouse model,E11 analogues also showed better reno-protective effects than E11 in animal models of AKI,among which Pal-E11 showed the strongest pre-injury reno-protective effects.Moreover,Pal-E11 showed good post-injury therapeutic effects within 24 hours after the occurrence of AKI,showing a promising potential for clinical transformation,thus it is expected to be a drug candidate for the prevention and treatment of AKI.Innovation point: E11 analogues(except Dimer-E11)in the present study showed better reno-protective effects than E11 in vivo.These results indicated Pal-E11 as a drug candidate for the prevention and treatment of AKI,which is expected to improve the current situation that the clinical treatment of AKI mainly relies on renal replacement therapy.In addition,we designed a biotin-labeled ligand competitive binding assay to study the binding ability of E11 analogues to APJ,avoiding the possible radioactive pollution caused by radioactive ligand competition assay,and providing a reference for the future study on the binding ability of ligand to APJ.