Design,Synthesis And Antitumor Activity Of Acid-sensitive Anticancer Combinatorial Peptides

Cancer is the main source of morbidity and mortality in the world.Although significant progress has been made in the research and development of anti-cancer drugs,cancer is still one of the main causes of death in the world.At present,traditional chemotherapy is still one of the main strategies for tumor treatment.In the research and development of anti-cancer drugs,anticancer peptides(ACPs),biologically active peptides with anti-tumor activity,have better advantages in enhancing the effect of chemotherapeutic drugs and reducing multi-drug resistance due to their cationic,amphiphilic structural characteristics and multiple anti-cancer mechanisms.Nevertheless,the research and application of anti-cancer peptides still face many challenges,such as stability,selectivity,toxicity and so forth.To explore how to better improve the selectivity of anti-cancer peptides and reduce toxicity and side effects has become our main focus.Due to the abnormal metabolism of tumor cells,a major difference between many solid tumors and normal tissues is that tumors have a more acidic extracellular environment.Therefore,this specific acidic environment of tumor tissue can be used as an effective target to improve the selectivity of anticancer peptides.The imidazole group in histidine has a p Ka value of about 6.5,so in an acidic environment,it will protonate and be positively charged,while in a physiological environment it will be slightly negatively charged.Based on this feature,a pH targeting peptide containing histidine can be designed.LK(LKKLLKLLKKLLKL)is a positively charged α-helical peptide with strong anti-tumor activity,but lacks selectivity.In order to improve the anti-tumor selectivity of LK,targeting the acidic microenvironment of the tumor,specific anion shielding peptide LE were conjugated to LK through disulfide bonds to construct acid-sensitive anticancer combinatorial peptides.At normal pH,due to the electrostatic attraction of LK and the anion shielding peptide,it is closed,and the activity of the parent peptide LK is shielded;under the acidic condition of the tumor,due to the charge conversion of histidine and glutamic acid,the electrostatic repulsion of two molecules could be generated.The molecule is opened,the parent peptide is dissociated from the anion shielding peptide,the activity is activated,and then it exerts an anti-tumor effect.In this study,three anion shielding peptides LE-1,LE-2,LE-3 are designed and synthesized,and the combinatorial peptides LK-LE-1,LK-LE-2 and LK-LE-3 are finally obtained through disulfide bond conjugation reaction.The secondary structure,acid-sensitive anti-tumor activity,toxicity,stability and related mechanisms of the newly designed and synthesized combinatorial peptides were studied.The results showed that in compared with LK,the secondary structure of the combinatorial peptides did not change significantly,all peptides show a standard α-helical structure.Compared with the parent peptide LK,all the combinatorial peptides show obvious pH-dependent antitumor activity,which effectively improves the selectivity of LK.Lactate dehydrogenase(LDH)release,PI staining,and scanning electron microscopy experiments showed that the combinatorial peptides and LK both exert anti-tumor activity through membrane rupture mechanism.The results of hemolysis experiments showed that the hemolysis rate of the combinatorial peptides is 7-14 times lower than that of LK,which significantly improves the safety of LK.And the serum stability experiment showed that the stability of the designed combinatorial peptides in the serum is significantly higher than that of the parent peptide LK.Through a series of experimental studies,this work proves that the acid-sensitive anti-cancer combinatorial peptides derived from the anion shielding peptide LE conjugated to to LK through disulfide bonds has successfully improved the anti-tumor selectivity,safety and stability of LK.The design of this new acid-sensitive anti-cancer combination peptides provides a new idea to improve the selectivity of anti-cancer peptides.