Isolation,Synthesis And Activity Of New Conotoxins From The Venoms Of Conus Marmoreus And Conus Leopardus From South China Sea

Conotoxins are a kind of neurotoxic peptides secreted by venom duct of marine mollusk cone snails for predation and defense.They have become an important source of new drug development because of their diversified structures,high selectivity,potent activity and low side effects.They have shown excellent application prospects in the treatment of various tough diseases such as pain,addiction,psychosis and epilepsy,etc.There are more than 60 species of cone snails in the South China Sea,which is an important strategic resource for China to become a strong country through marine technology and medicinal development.Hence,the cone snail resource is of great research and development value.α4β2 n ACh R was found to be a key target for drug development for addiction,alzheimer’s disease,anxiety disorder,depression,ADHD,etc.Drug development based on the blocking effect of α4β2n ACh R is of great research significance.In order to deeply explore the rich biological resources of cone snails to discover new blockers against α4β2 n ACh R from the cone snail venom,we studied the isolation,identification,synthesis and activity detection of conotoxins from the venoms of Conus marmoreus and Conus leopardus from South China Sea.Firstly,the venom ducts from snail specimen of five species of cone snails(Conus marmoreus,Conus leopardus,Conus litteratus,Conus imperialis,Conus virgo)were dissected for venom extraction.Then the five venom samples were fractionated to obtain respective components to test their blocking activity against α4β2 and α3β2 nAChRs.The results illustrated:(1)Most of the venom components of Conus leopardus showed higher blocking activity against α4β2 n ACh R than against α3β2 n ACh R.(2)Most of the venom components of Conus marmoreus,Conus litteratus,Conus imperialis and Conus virgo showed similar blocking activity against between α4β2 and α3β2 nAChRs.Based on the activity results and the venom amount,we chose the venom of Conus litteratus(the best blocking activity)and venom of Conus marmoreus(the largest venom amount)to conduct the subsequent isolation process.Based on the blocking activity results of α4β2 and α3β2 nAChRs,isolation,sequencing,synthesis and activity detection of the conotoxins from crude venoms of Conus marmoreus and Conus leopardus were conducted.The purification process led to the isolation of 36 conopeptides.Amino acid sequences of 16 conopeptides were identified by combined analysis of MAl DI-TOF-MS/MS and Edman degradation.Twelve sequences therein were new.Eight conopeptides were synthesized by Fmoc solid phase peptide synthesis and oxidative folding,and their blocking activities for several n ACh R subtypes(α1β1、α3β2、α3β4、α4β2)were determined.The result showed that conotoxins H-3,H-14 and H-32 could block α4β2 n ACh R with about(20 ± 8.5)% blocking rate at the concentration of 100 μM,which meant they showed no blocking effect against α4β2 n ACh R.Their activities against other types of receptors are remained to be further studied.This project studied on the isolation,sequencing,synthesis and activity detection of the conotoxins from crude venoms of Conus marmoreus and Conus leopardus,based on the activity screening of blocking activity against α4β2 and α3β2 nAChRs of venom fractions of five cone snail species.Structures and activities of conotoxins from venom of Conus marmoreus were investigated.The result of this study lay a foundation and provide references for the discovery of novel α4β2 n ACh R blockers and the further exploitation and utilization of cone snail venom resource in the South China Sea.