Study On The Design And Antitumor Application Of Neuropeptide Analogs With Antimicrobial Peptide-like Properties

Neuropeptides,majorly secreted from nervous tissues,show multiple biological functions,such as immune regulation,pain regulation,antimicrobial activity and antitumor activity.It is reported that many tumor cells highly express neuropeptide receptors.Neuropeptides can enter cells via receptor-mediated endocytosis after binding their receptors.Therefore,neuropeptides,such as somatostatin,bombesin,substance P and neuropeptide Y,are used to construct tumor targeted delivery system.Antimicrobial peptides,a type of cationic peptides with membrane-lytic activity,show antimicrobial activity,antitumor activity and immune regulation,et al.In addition,more and more studies confirmed that antimicrobial peptides can enter cell like cell-penetrating peptides.We find many neuropeptides are similar to antimicrobial peptides in structure,such as cationic charge,hydrophobicity and secondary structure.Therefore,we developed a series of peptide analogs with antimicrobial peptide-like properties,and studied their application in tumor therapy.Somatostatin（such as octreotide and RC-160）not only display antitumor activity,but also can be used as tumor targeted vectors for delivering imaging agents or antitumor drugs.Interestingly,the structure of RC-160 is similar to many antimicrobial peptides.Based on this type of structure similarity to antimicrobial peptide and tumor targeted activity of RC-160,we developed a novel type of antitumor peptide TPP-RC-160 by conjugating the mitochondria targeted molecule triphenylphosphine（TPP）to the N-terminus of RC-160.Our results demonstrated that TPP-RC-160 displayed some antitumor activity,while RC-160 did not display obviously antitumor activity at the tested concentrations.To further improve the antitumor activity of TPP-RC-160,we synthesized a series of analogs with increased the number of basic amino acids and hydrophobic amino acids by conjugating the N-terminal sequence（D-Lys）-（D-Tyr）-Lys-（D-Tyr）-（D-Lys）of JC-07-69 to the N-terminus of TPP-RC-160.The results demonstrated that the conjugation of TPP is very crucial for antitumor activity,while is not important for antimicrobial activity.In addition,our results also demonstrated that the replacement of D-Tyr with D-Trp could significantly improve the antitumor activity of analogs,with deletion of D-Lys at the position 5 could not influence the antitumor activity of analogs.STA-6 was screened out as the ideal analog.Our results derived from the antimicrobial mechanism study showed that STA-6 could quickly kill bacteria like antimicrobial peptides by disrupting cell membranes.STA-6 could not quickly kill tumor cells by disrupting cell membranes at low concentration（20μM）,at which STA-6 could effectively inhibit tumor cells.However,STA-6 could disrupt tumor cells at concentration of 160μM.These results demonstrated that the death mechanism of STA-6 is different at different tested concentrations.Subsequently,we further studied the killing mechanism of STA-6 at 20μM.Our results demonstrated that STA-6 could enter cells and disrupt mitochondria.However,STA-6 induced cell necrosis but not cell apoptosis mediated by mitochondrial signaling pathway.Our study demonstrated the conjugation of TPP with somatostatin analog is a effective strategy to develop novel antitumor peptides.Although peptide antagonists can not enter cells by receptor-mediated endocytosis,peptide antagonists have attracted substantial attention as targeting molecules because of their high tumor accumulation and antitumor activity compared with agonists.Substance P（SP,RPKPQQFFGLM-NH₂）,a neurokinin-1（NK-1）receptor agonist,is one of the most extensively studied neuropeptides.SPA[Substance P Antagonist,（D-Arg1,D-Trp5,7,9,Leu11）Substance P],a derivative of SP,is an antagonist that inhibits signal transduction and cell proliferation.Our previous study showed that SPA could quickly kill bacteria by disrupting cell membranes like antimicrobial peptides.Because antimicrobial peptides can enter cell like cell penetrating peptides,we inferred that SPA can be used tumor targeted vectors.Our results showed that SPA could indeed enter cells.Our results also showed that SPA could deliver the camptothecin（CPT）into tumor cells and kill the cells.In addition,stearyl-SPA by the attachment of stearic acid to the N-terminus of SPA could compact plasmid DNA into nanoparticles,and could efficiently deliver plasmids into tumor cells.In addition,our results showed that SPA exhibited low toxicity to normal cells and high enzymatic stability.Therefore,SPA can be developed into ideal vectors for tumor therapy.In conclusion,we have developed neuropeptides into antitumor peptides or vectors for tumor therapy based on the structure similarity to antimicrobial peptides.Although only the invitro studies were performed,our work can provide new strategy for developing effective antitumor drugs.