Molecular Design Of Nav1.7 Spider Toxin

Voltage-gated sodium ion channels(VGSC or Navs)are important ion channels for nerves,muscles and other excitable tissues to generate action potentials,and they participate in the metabolic processes of most cells.The 9 subtypes of sodium channels(NaV1.1-Nav1.9)have different tissue distributions.Nav1.7 is mainly distributed in peripheral neurons,in the DRG,sympathetic ganglia,Schwann cells and Neuroendocrine cells are selectively expressed on the cell membrane and preferentially expressed in pain fibers of the human peripheral nervous system.In animals with inflammatory pain patterns,Nav 1.7 expression levels were found to be significantly up-regulated.Moreover,studies have shown that when the SCN9 A gene has an invalid mutation,the loss of Nav1.7 function will cause congenital painlessness(CIP).Therefore,Nav1.7 is one of the most potential targets for analgesic drug development.Spiders are the largest group of poisonous animals.Their venom is rich in polypeptides,and some of them specifically inhibit the activity of sodium channel subtype Nav1.7,providing potential drug lead molecules for pain treatment.According to the amino acid sequence and cysteine ??position,the peptides acting on Nav1.7 are divided into different NaV regulator families,named NaSpTx Family1-12,of which NaSpTx Family 1 is widely concerned because of its excellent selectivity and high efficiency.The peptides in this family are generally composed of33-35 amino acid residues with three disulfide bonds,forming an inhibitor cystine knot(ICK)motif.This "knot" converts the ICK peptide into an ultra-stable miniature protein with high chemical,thermal and biological stability,which can resist extreme pH,organic solvents,high temperatures and proteases.In this study,molecular docking technology was used to simulate the combination of two NaSpTx Family 1 peptide toxins(HWTX-IV(PDB ID: 1MB6)and HNTX-III(PDB ID: 2jtb))that acted on Nav1.7.The best complex obtained usesvirtual alanine scanning mutations to predict the key residues of HWTX-IV and HNTX-III binding to Nav1,7.Comparing the prediction results with the experimental results,it was found that the accuracy of the key residues of the software on Nav1.7for two peptide toxins(HWTX-IV and HNTX-III)was 57.14%(HWTX-IV predicted7 key residues,Four of them are consistent with the experimental results)and 70.00%(HNTX-III predicts 10 key residues,seven of which are consistent with the experimental results).The prediction results have a very high accuracy and good reliability.A new Nav1.7 peptide toxin μ-TRTX-Ca2a(Ca2a)was isolated from the venom of Cyriopagopus albostriatus.It contains 35 amino acids and 3 pairs of disulfide bonds,and its molecular weight is 3905.08 Da(Nav1.7 IC50 = 98 nM)belongs to NaSpTx Family1.After successful modeling,molecular docking technology was used to simulate the combination of Ca2 a and Nav1.7,and then the virtual alanine scanning mutation method was used to predict and found the 3,4,5,15,18,27,30,32 and 33 positions.After the residue is mutated to alanine,the energy of the complex system decreases(that is,the binding affinity decreases).It is speculated that these amino acids are the key amino acids for the interaction between poly Ca2 a and Nav1.7.The predicted results were compared with the key residues verified by the HWTX-IV experiment,and it was found that the amino acids at positions K18,K27,W30,K32,and Y33 were consistent with the key residues of HWTX-IV.These five Ca2 a mutants and Ca2 a were synthesized by chemical solid phase.After successful renaturation,the activity was detected by whole-cell patch clamp.It was found that the activity of 5mutants on Nav1.7 was reduced(IC50> 1μM).The prediction result was successful.In summary,this study used computer-aided design methods,through model building,molecular docking,virtual alanine scanning and other techniques,successfully predicted the key amino acid residues of NaSpTx Family1 Ca2 a and Nav1.7 interaction,and through experiments The amino acids at positions K27,W30,K32 and Y33 were verified as key amino acid residues.This provides some clues for understanding the mechanism of action between Nav1.7 and peptide inhibitors,and at the same time,it also makes a reasonable attempt to optimize the modification methodfor Nav1.7 analgesic peptides.