Synthesis,Structure And Function Of Novel α4/4-Conotoxins

α-Conotoxin is a kind of polypeptides in the venom of tropical conus,which target nicotinic acetylcholine receptors（nAChRs）.Nicotinic acetylcholine receptors are a class of ligand-gated ion channels with many subtypes,which have important physiological function and clinical significance.The structure of each subtype is very similar,but the physiological and pharmacological functions are quite different.It is the target of many complex diseases and therapeutic drugs,including neuralgia（chronic pain）,addiction,Parkinson’s disease,depression,anxiety,schizophrenia,dementia,deafness,cancer,obesity,aging,wrinkles and so on.Therefore,it is urgent to find specific ligand compounds that can distinguish different receptor subtypes.α-Conotoxin has unprecedented selectivity for each subtype of nAChRs.It can be developed as a valuable molecular probe to study the structure and function of the subtype with specifical block,as well as the pathogenesis of related diseases,also be directly developed as an innovative drug for the treatment of certain diseases.In this paper,theα4/4-conotoxins and their mutants from Conus lividus and Conus vexillum in the China South Sea were synthesized,and identified.Besides,the relationships of their structures and functions were analyzed.A total of 52α-conotoxin peptides with natural conformation were synthesized by Fmoc solid-phase peptide synthesis,and then measured two-step oxidation folding after purification.Each subtype of nAChRs was recombined and expressed in Xenopus oocytes,and the target of the synthesized peptide was screened and identified by electrophysiological measurement.For the peptides with target binding activity,the spatial structure and the molecular mechanism of interaction with receptors were further studied.The results are summarized as follows.A novelα4/4-conotoxin LvIB and its 4 mutant peptides（analogs）were successfully synthesized.The wild type LvIB was cloned from Conus lividus.The binding activities of the above five synthetic peptides to all neurotypic subtypes of rat nAChRs and muscle subtypes of mouse nAChRs were screened.It was found that the analog 2（Amidated[Q1G,ΔR14]LvIB）was an antagonist at ratα7 n ACh R(IC₅₀:97 n M;K_D:44 n M).Analog 2 was more potent than the other 4 peptide analogs,and the recovery rate was slow.The NMR structure of the analog 2 was analyzed.Furthermore,the activity of analog 2 against humanα7 n ACh R was tested.It showed that the activity of analog 2 on the humanα7 n ACh R was weak,and its IC₅₀was as high as 1570 n M,which was 16-fold lower than at ratα7 n ACh R.In order to study the great difference in species activity between rat and humanα7n ACh R of amidated[Q1G,ΔR14]LvIB,the point mutation of different amino acids in rat and humanα7 n ACh R subunits was carried out,that is,the amino acid at a certain site of ratα7 n ACh R subunit was mutated to the corresponding amino acid at the site of human subunit,and all heterotopic receptor mutants of rat and humanα7 extracellular ligand binding domain were prepared.The activity of analog 2 to all receptor mutants were detected,and found that three key amino acid sites on ratα7 n ACh R subunit,that is the combined influence of Gln141,Asn184 and Lys186 determined the species specificity ofα7 nAChRs,and it is different from previous study.The molecular modelling was measured between analog 2 and rat/humanα7 n ACh R.The results showed that the interaction sites between analogue 2 and ratα7 n ACh R is a new receptor binding site.Thus,the analog 2（Amidated[Q1G,ΔR14]LvIB）was a new probe for ratα7 n ACh R,also provides a lead compound and research basis for the design of humanα7 n ACh R specific blockers and the development of new drugs.Another newα4/4-conotoxin Lv IC and its 2 analog peptides were synthesized.The wild type Lv IC was also cloned from Conus lividus.The results of target screening showed that the analog[D1G,ΔQ14]Lv IC was a highly selective and strong antagonist ofα6/α3β4n ACh R with an IC₅₀of 24 n M,and had little activity against other n ACh R subtypes.Therefore,the[D1G,ΔQ14]Lv IC was a rare and valuable new specific molecular probe forα6/α3β4 n ACh R.In order to further study which amino acids on[D1G,ΔQ14]Lv IC interact withα6/α3β4 n ACh R,alanine scanning mutation was carried out,and 23 mutant peptides were synthesized.The activity of all alanine scan mutant peptides atα6/α3β4 n ACh R were determined.The results showed that the Asn5,Pro6 and His12 were the key amino acid sites,and the mutation would lead to the significant decrease or even complete loss of the activity.The activity and selectivity of[N9A]analogue atα6/α3β4 n ACh R did not change significantly.The[G10A]analogue was more effective than the wild type,but it also blockedα3β4 n ACh R.The[G10V],[G10L]and[G10I]mutants with aliphatic side chains were similar to[G10A]mutants.However,the double mutant[N9A,G10A]maintained the activity ofα6/α3β4 n ACh R and eliminated the additional activity ofα3β4 n ACh R caused by Ala10 mutation.The molecular modelling was measured between[D1G,ΔQ14]Lv IC andα6/α3β4 n ACh R.The key amino acids and ligand receptor interactions between the peptide and receptor were analyzed.These results reveal the mechanism of[D1G,ΔQ14]Lv IC targetingα6/α3β4 n ACh R,and layed a theoretical foundation for the development of[D1G,ΔQ14]Lv IC as a new probe and a therapeutic drug for diseases related to this receptor.Two newα4/4-conotoxin precursor sequences named Lv M1 and Ve M9 respectivily,were cloned from Conus lividus and conus vexillum.The 6 analog peptides were synthesized and screened,in results that the analogue Lv M1L1 was a highly selective and strong antagonist of rat and humanα6/α3β2 nAChRs(IC₅₀:18.9 n M and 9.7 n M,respectivily)with similar activity and no species difference.Lv M1L1 had no activity against other nAChRs subtypes containingβ2.Lv M1L1 was the firstα-conotoxin cloned from C.vexillum with similar blocking effect on rat and humanα6/α3β2 nAChRs.It was a new molecular probe forα6/α3β2 nAChRs.The systematically structure-function study of Lv M1L1 provided a new idea for the research of intermolecular ligand-receptor binding mechanism.In additional,the head-to-tail cyclization of selectiveα3β4 n ACh R blocker[S9A]TxID was also measured in this article.Six analogues with different length linkers were designed,synthesized and identified.Among them,c[S9A]TxID-6 is the most effective analog,which maintained the original activity and had higher stability to protease K than the wild type peptide[S9A]TxID.In this paper,we screened and identified several newα4/4-conotoxins with pharmacological activities from two common Conus species（C.lividus and C.vexillum）in the South China Sea,the results provided new insights into the binding mechanism of ligands targeting receptors,and also provided references for the design of lead compounds targeting human beings.