Study On Cyclization And Stability Of α-Conotoxin TxIB

As competitive antagonists of nicotinic acetylcholine receptors,α-conotoxins are composed of 12～20 amino acid residues and can be used as molecular probes for studying the function and structure of n ACh Rs.In addition,α-conotoxins have great potential in the development of drugs for the treatment of small cell lung cancer,depression,Parkinson’s disease,addiction,neuralgia and other diseases.α-Conotoxin TxIB is a novel α-4/7 conotoxin cloned from Conus textile containing 16 amino acids and two disulfide bonds.It specifically inhibits α6/α3β2β3(α6β2*)n ACh Rs,which has the potential to be developed as a lead for the treatment of diseases such as nicotine addiction.As a peptide,TxIB suffers from poor stability,low bioavailability,etc.Therefore,testing the stability of TxIB can provide guidance for subsequent research.In this study,the three isomers of TxIB were first synthesized by Fmoc solid-phase synthesis and two-step oxidation.Then,we measured the stability of TxIB in simulated gastric fluid(SGF),simulated intestinal fluid(SIF),reduced glutathione(GSH)and human serum by RP-UPLC.In addition,the stability of TxIB under the influence of physical factors such as light,temperature,and humidity was also investigated.The results showed that TxIB was relatively stable in SGF and human serum,but unstable in SIF.Meanwhile,disulfide bonds partially rearranged in GSH.According to the results of the previous study,we selected backbone cyclization strategy to modify TxIB.In addition,we tested the biological activity and serum stability of the mutants.In this experiment,four linkers consisting of 4 to 7 amino acids were designed based on the distance between the N-and C-termini of TxIB,and then four cyclic analogs were synthesized by head-to-tail chemical synthesis(HCL).Then,the cyclic peptides were oxidized by a two-step oxidation protocol to form two pairs of disulfide bonds,and purified by preparative RP-HPLC.RP-UPLC was used to determine the purity of the peptides,and mass spectrometry was used to identify them.The inhibition of four cyclic mutants on α6/α3β2β3 n ACh Rs and their stability in human serum were tested by electrophysiological assays and RP-HPLC,respectively.The results showed that all cyclized analogs had similar potency on α6/α3β2β3 n ACh Rs compared to the wild-type TxIB.The inhibition of TxIB was about 42%,while the inhibition of the four cyclic peptides were about 35%,42%,48%,and 41%,respectively.Stability results showed that less than 20% of wild-type TxIB remained,while about 50% of the c TxIB-7 mutant remained after 48 h in serum.These results indicated that cyclization could effectively maintain the activity of TxIB while improving the stability,which is conducive to its future development and application.