Design,Synthesis And Activity Study Of Short Peptide Mutants Based On αO-Conotoxin GeXIVA

Nicotinic acetylcholine receptors(nAChRs)are a class of transmembrane receptor proteins that respond to the binding of the neurotransmitter acetylcholine.The muscle-type nAChRs mainly exist in the neuromuscular junctions and are mainly divided into adult(α1β1δε)and fetal(α1β1δγ)subtypes.It is found that muscle-type nAChRs are associated with rhabdomyosarcoma,pediatric soft tissue tumors,myasthenia gravis and other diseases.In addition,muscle nAChR subtype is also associated with skin anti-wrinkle,for example SYN(?)-AKE is an anti-wrinkle cosmetic product that inhibits α1β1δε nAChR.Conotoxins(conopeptides,CTxs)are a large group of neuropeptides found in the venom of Conus carnivorous marine molluscs.They have a small molecular weight and arerich in disulfide bonds,usually consisting of 7-46 amino acids.αO-CTx GeXIVA is a novel conotoxin found in our laboratory from C.generalis native to Hainan.It consists of 28 amino acids and contains two pairs of disulfide bonds.GeXIVA is a blocker of α1β1δεnAChR with an IC50 of 394 nmol/L.The high molecular weight and long-chain of amino acids of GeXIVA limited its application as a cosmetic peptide.Therefore,we designed a batch of short peptides with 10 amino acids or less based on the active region of GeXIVA to test activities on muscle nAChR subtype and anti-wrinkle function.In addition,the crude venom of C.Betulinus was extracted.Its fractions were purified,in which a few natural conotoxin fractions were found to block different subtypes of nAChRs by electrophysiology technology of two-electrode voltage clamp.In this study,we used molecular docking technique to acquire the active center information of GeXIVA on muscle nAChR.A series of short peptides were designed and synthesized based on the active center of GeXIVA.Their effect on α1β1δε nAChR expressed in xenopus oocytes were detected by two-electrode voltage clamp technique.Then the anti-wrinkle activity of the most active short peptide was examined on mice aging model.In addition,native toxin fractions were isolated and purified from C.betulinus crude venom by high performance liquid chromatography.Various subtypes of nAChRs expressed in xenopus oocytes were used as drug targets.Activities of each toxin fraction on different nAChR subtypes were assayed using electrophysiology technology of two-electrode voltage clamp.The experimental results showed that GeXIVA intermediate a-helical structure(R10-Y19)was a key segment that binded to α1/δ.Based on this,53 GeXIVA short peptides with five batches(GeMs)were designed and synthesized.Among them,GeM-D5A,GeM-9aa-1 and GeM-D5A#had inhibition effect on α1β1δε nAChR.Their IC50 were 8.038 μmol/L,4.697 μmol/L and 2.742 μmol/L,respectively.According to the anti-wrinkle active investigation,GeM-D5A had anti-wrinkle activity.Compared with the control group,GeM-D5A made the skin structure and collagen fibers more compact in the mouse aging model.The mouse skin structure was improved.What’s more,16 natural toxin fractions were isolated from C.betulinus venom and tested on different nAChR subtypes.Three fractions,CBL-2、CBL-3 and CBL-4,had strong blocking effect onα1β1δε nAChR.Two fractions,CBL-4 and CBL-5 were potent to block α3β2 nAChR.One fraction,CBL-12,had inhibition on α4β2 nAChR.In this study,three short peptides that had strong inhibition on α1β1δε nAChR were discovered.Among them,the GeM-D5A containing only 10 amino acids had a certain anti-wrinkle activity.Not only could this study lay the foundation for the exploitation of anti-wrinkle cosmetic peptide,but also create new ideas for the study of conotoxins.Five toxin fractions were discovered to be active on α3β2,α4β2 and α1β1δε nAChR subtypes respectively.The results would provide the basis for the development of molecular probes and new drugs targeting different nAChR subtypes in the future.CBL-2,CBL-3 and CBL-4 may be used in anti-wrinkle cosmetic peptided evelopment by easy smearing way.