Chemical Modification Of ?~*-conotoxins To Improve Their Activity And Stability

Conotoxins(CTxs)are a diverse group of evolved venom peptides used for prey capture or defense.Each species of cone snails produces in excess of 1000 conopeptides,with those pharmacologically characterized targeting a diverse range of membrane proteins typically with high potency and specificity.The majority of conopeptides inhibit voltage-or ligand-gated ion channels.The conopeptides are categorized into various superfamilies and classified by their structural and functional features,such as cysteine pattern and pharmacology.The largest family of conopeptides are competitive antagonists of nicotinic acetylcholine receptors(nAChRs),especially for ?-CTxs.Previous researches have shown the nAChRs are implicated in the pathophysiology of a number of disease states including pain,epilepsy,Parkinson's disease,and addiction et al.Accordingly,CTxs inhibiting nAChRs have been proved to be valuable tools for identifying the composition and roles of nAChR subtypes and some of them have become either promising drug leads or novel pharmaceutics.However,one potential drawback of CTxs as drugs is that they may suffer the generic problems of peptides in vivo,such as poor absorption,susceptibility to proteolysis,and short biological half-lives.In this study,three strategies for improving the stability or activity of CTxs has been successfully applied in four conotoxins.?-CTx RgIA and ?O-CTx GeXIVA were redesigned using D-amino acid substitution.?-CTx LvIA was cyclized and Met-11 of ?-CTx TxID was mutated to other similar residues.?-CTx RgIA is a selective and potent competitive antagonist of rat ?9?10 nAChR,but it is much less potent on towards human versus rat a9?10 nAChR.Furthermore,RgIA is more susceptible to proteolytic degradation due to containing four arginine residues.Therefore,we used the D-amino acid scan method to identify critical stereocenters of RgIA and discover more stable analogues enhancing its bioavailability.The activity of each variant was investigated against rat and human ?9?10 nAChRs,which were expressed in Xenopus oocytes.Experimental assays showed that 14 out of 15 analogues had a substantial reduction in potency towards rat ?9?10 nAChR.Noticeably,RgIA[13r]retained full biological activity compared with RgIA.Meanwhile,two other analogues RgIA[7,9,11,13-r]and RgIA[7,9,11-r#],of which L-Args were substituted with D-Args,had significantly increased potency at the towards human ?9?10 nAChR,although these analogues showed decreased their activities at the against rat ?9?10 nAChR.Additionally,these three analogues exhibited high resistance against enzymatic degradation in human serum and simulated intestinal fluid(SIF).?O-CTx GeXIVA was cloned from Conus generalis in our lab which is an antagonist of ?9?10 nAChRs and has therapeutic potential for the treatment of neuropathic pain.In contrast to other well-structured ?-conotoxins,?O-GeXIVA has eight arginines and a flexible structure which lead to being degraded by proteases easily.In order to improve the stability of GeXIVA,we synthesized eight GeXIVA analogues which replaced the C-and N-terminal flanking residues or Arg residues with their D-amino acid at counterparts.Our data showed that all D-amino acid substitutions exhibited similar potency at ?9?10 nAChR and other subtypes.Significantly,GeXIVA[15-18r]analogs exhibited about 2.5-fold more potency for the ?9?10 nAChR compared with the original peptide.Furthermore,GeXIVA[all-r]and GeXIVA[1t,28v,all-r]exhibited high resistance against enzymatic degradation in human serum,and the half-life in human serum is extended from 30 min to 8-12 hours.Especially for GeXIVA[1t,28v,all-r],it even existed in SIF for 90 min.Together,these findings suggest that suitable D-amino acid substitution in GeXIVA retain original activity and enhance stability which may improve potential therapeutic utility.a-CTx LvIA was cloned from Conus lividus,which was found the high potency on?3?2 nAChR.In addition,it has the ability to discriminate between similar neuronal ?3?2 and ?6/?3?2?3 nAChRs.Here,we improve the stability of LvIA by backbone cyclization using linkers head-to-tail chemical ligation method.Cyclization with a six-residue linker preserve the activity of ?3?2 nAChR and improves stability in human serum.The results provide insights to further improve the therapeutic properties of LvIA and confirm that cyclization is an applicable strategy to improve the stability of peptides.?-CTx TxID was discovered from Conus textile by gene cloning in our lab,which has 4/6 inter-cysteine loop spacing and selectively inhibits ?3?4 nAChR.However,TxID is susceptible to modification due to it containing a methionine(Met)residue that easily forms methionine sulfoxide(MetO)in an oxidative environment.In this study,we investigated how Met-11 and its derivatives affect the activity of TxID.The results showed most TxID analogues had substantially decreased activities on ?3?4 nAChR with more than 10-fold potency loss and 5 of them demonstrated no inhibition on ?3?4 nAChR.However,one mutant,[M11I]TxID,displayed potent inhibition at ?3?4 nAChR with an IC50 of 69 nM,which only exhibited 3.8-fold less compared with TxID.The results indicate replacement of Met with a hydrophobic moderate-sized lie in TxID is an alternative strategy to reduce the impact of Met oxidation,which may help to redesign CTxs containing methionine residue.In summary,the relatively small size of conotoxins makes them very amenable to chemical synthesis.This opens the possibility of improving stability through synthetic chemical modifications.Our findings suggest that the substitution of D-amino acids,cyclization and amino acid mutations affect the structure and function of peptides and may facilitate the development of more stable analogues to increase conotoxins' therapeutic potential.