Design,Efficient Synthesis And Evaluation Of Anticancer Activity Of Spider Toxin Peptide LVTX-8 Derivatives

Cancer is the main cause of human death worldwide,and it is also a major challenge for current drug treatment.Among them,cytotoxic peptides extracted from animals are the focus of anticancer research in recent years.Amphiphilicity with 25 amino acid residues isolated from spider Lycosa vittata α-helical LVTX-8 has a good killing effect on different lung cancer cells.However,as an L-type peptide,it was easy to be hydrolyzed by protease,resulting in its poor stability and short half-life,which limited its wide application.Therefore,this study is based on the cytotoxic peptide LVTX-8 to modify the structure.Design derived peptides which have better anticancer effects and can overcome protease hydrolysis and improve stability.Finally,we were studying the mechanism of LVTX-8 at the cellular level.This has important reference significance for broadening the design and modification of peptide drugs and improving the stability.This study introduced the design and synthesis process of LVTX-8 and its 10 derived peptides,and explored the anticancer cell activity,stability and subcellular imaging experiments of synthetic peptides.1.Solid phase peptide synthesis showed that the synthesis of peptides by two different solid phase peptide condensation systems,using two different solid phase peptide condensation systems,HCTU/DIEA or DIC/Oxyma,under different reaction temperature conditions(30℃ or 50℃)were able to yield the target product LVTX-8 with the sequence IWLTALKFLGKNLGKHLAKQQLSKL and were verified separately,demonstrating that the molecular weights were correct and the yields could both reach over 90%,but the rate of peptide synthesis at 50℃ was significantly higher than that at 30℃.2.The results of computer simulation and CD spectrum showed that,except 828,829 and 831,the other peptides were α-helical structure.3.The results of cytotoxicity tests MTT and CCK8 showed that the seven derived peptides had good antitumor activity on suspension cells,but had poor antitumor effect on adherent cells,which was better than or comparable to that of natural LVTX-8.It is worth mentioning that 827,which does not contain five glutamate conjugates,exhibits stronger antitumor effects than MTX alone.4.The time kill curve assay,serum stability and hemolytic activity experiments showed that compared with the L-type lead peptide LVTX-8,both N-acetyl and C-hydrazide modified LVTX-8(825),and MTXGFLG-LVTX-8(827)possessed more durable anticancer efficiency,higher proteolytic stability,as well as lower hemolysis.5.Fluorescence imaging experiments have shown for the first time that LVTX-8 can rapidly destroy the integrity of cell membranes,internalize into cancer cells,act on mitochondrial membranes,reduce mitochondrial membrane potential,destroy mitochondrial functions,and induce cell death.Taken together,this study established a high efficient solid phase synthesis method for peptides.LVTX-8 and its derived peptides can be quickly and effectively obtained by using SPPS and DIC/Oxyma condensation system at 50℃.Through structural modification of LVTX-8,derivatives 825 and 827 were obtained with higher stability,and the mechanism of action of LVTX-8 at the subcellular level was explored for the first time.It provided an important reference basis for the development and characterization evaluation of peptide drugs in the future.