Study Of The Synthesis And Anti-tumor Activity Of Aza-staple Peptides Based On PDI

Cancer is a serious threat to human health and has become the second "killer" after cardiovascular disease.The development of cancer therapy and cancer drugs is urgent.Studies have found that 50% of tumors have mutations in the p53 gene,and it has also been found that the function of p53 is inhibited by its negative regulatory protein in tumors expressing wild-type p53 gene.MDM2 and its homologous protein MDMX are two negative regulatory proteins of p53,which inhibit the activity of p53 through different mechanisms.MDM2 can inhibit the activity of p53 through a variety of ways:1)As a member of the ubiquitin family,MDM2 accelerates the degradation of p53 by promoting the ubiquitination process of p53;2)The N-terminal binding of p53 inhibits the transcriptional activity of p53;3)Deliver p53 from the nucleus to the cytoplasm.MDMX combines with p53 to form an inactive p53-MDMX complex,so that inhibits the transactivation of p53,causing p53 to lose the activity of inhibiting tumor growth.Therefore,the development of drugs that can effectively inhibit the binding of p53 with MDM2 and MDMX is an important entry point for the treatment of p53pathway-related tumors.However,due to the subtle differences in the structure of MDM2 and MDMX,it is proposed to develop inhibitors that can simultaneously inhibit the activity of MDM2/MDMX protein.According to the crystal structure,p53 binds to MDM2 and MDMX through protein-protein interaction(PPI).The N-terminus of p53 is inserted into the hydrophobic pocket of MDM2 and MDMX.It is worth noting that when binding,there are 3 amino acids in the p53 sequence.Residues(Phe-19,Trp-23,Leu-26)play a key role.Therefore,mimicking the protein-protein interaction between p53 and MDM2/MDMX is an important basis for the development of MDM2/MDMX protein-related inhibitors.Current target inhibitors for MDM2 and MDMX include non-peptide small molecule inhibitors and peptide inhibitors.Due to the small molecular volume of small molecule drugs,it is difficult to exert effects through PPI,and most of the small molecule inhibitors currently developed can only bind to MDM2,and there is little ability to bind to MDMX,which makes its inhibitory activity difficult to achieve the desired effect.Peptide drugs have become a hot area for tumor drug development due to their wide range of activities,clear specific targets,and low systemic toxicity.With the development of proteomics and genomics,researchers have developed a keen interest in alternative therapies for active peptides and peptide drugs.With the development of phage display technology,the peptide PDI,which has a dual inhibitory effect on MDM2 and MDMX,had been found,opening the way for p53-MDM2/MDMX dual target drug development.Peptides also have some inherent shortcomings,such as the poor stability of proteolytic enzymes and poor membrane permeability,which limit the potential of peptide drugs for clinical transformation.Therefore,researchers have carried out medicinal chemical modifications to active peptides,such as introducing unnatural amino acids,D-amino acids,azaamino acids,etc.,to avoid the natural recognition by proteolytic enzymes,enhance the resistance of the peptides to proteases,and improve the stability of the peptides;By cyclizing the peptide,such as introducing a staple structure,the rigidity of the peptide structure can be enhanced,and the membrane permeability of the peptide can be improved;the strategy of introducing a penetrating peptide can also be used to enhance the cell penetrating ability.In this research,PDI,which has a dual targeting effect on MDM2/MDMX,was selected as the template peptide for chemical modification.Firstly,the formation ofα-helical and the key amino acid residues of PDI,which is important for PDI’s activity,were considered.Then,aza amino acids substitutions were carried out at the positions of i/i +4 and i/i +7 of PDI,and the side chains of aza amino acids were alkylated with different alcohols with terminal alkenyl group.Finally,the staple structure was formed through olefin metathesis reacstion between two azaamino acid residues.In total,three aza-stapled peptide analogues of PDI were successfully synthesized,namely,ZH-SPDI-48,ZH-SPDI-59 and ZH-SPDI-411.The synthesized peptide analogues were separated and purified,and their secondary structures were characterized by circular dichroism.It was found that the α-helicity of the three peptide analogues decreased to different degrees.Then,the antitumor activity of azazeptide was evaluated.By studying the binding affinity of azapeptide analogs with MDM2 and MDMX proteins,it was found that the binding affinity of azapeptide analogs based on PDI was improved,and the binding ability of i/i + 7 position modified azapeptide(ZH-SPDI-411)had the best binding affinity.Exploring the intracellular localization of the aza-staple peptide by laser confocal,the cell membrane permeability of ZH-SPDI-411 is significantly better than that of the template peptide PDI.The ability of aza-staple peptide analogues to inhibit tumor cell proliferation in different tumor cells was screened,and its inhibitory activity was consistent with the results of the binding affinity of MDM2/MDMX protein,and the anti-tumor activity was higher than that of the template peptide PDI,of which the activity of ZH-SPDI-411 increased most obviously,verifying the feasibility of the aza-staple peptide strategy.Finally,the cytotoxicity of aza staple peptide was explored in renal cortex cells(293T),and the results showed that its cytotoxicity did not increase significantly.The transformation of aza and staple strategies helps to improve the stability,biological activity and cell membrane permeability of the peptide,and further proves that the degree of α-helixity of peptide is not deciding effector to its anti-tumor activity.In conclusion,we have successfully developed a novel peptide modification strategy and demonstrated its feasibility in MDM2/MDMX dual-target inhibitor PDI.This method can be widely used in the modification of other active peptides,laying a foundation for the construction of peptide library.At the same time,this strategy can significantly improve the anti-tumor activity of PDI,which is expected to be selected as the lead compound of peptide anti-tumor,and contribute to the development of peptide drugs.