Design,Synthesis And Biological Research Of Novel Cation-Bridged Stapled Antimicrobial Peptide

Infectious diseases caused by drug-resistant bacteria threaten worldwide public health.The shortage of effective therapeutics for combating increasing drug-resistant bacteria necessitates the development of novel antibiotics with novel mechanisms of action.Antimicrobial peptides are a class of bioactive peptides that widely exist in biological organisms.Different from traditional antibiotics that act on specific cellular targets,most AMPs play a bactericidal role mainly through non-receptor-mediated membrane penetration and lysis.In addition to the direct antimicrobial activities,antimicrobial peptides also have a strong ability of neutralizing endotoxins and regulating immunity.Therefore,antimicrobial peptides have broad prospects for developing new class of anti-bacterial infection drugs.Although antimicrobial peptides have great application as potential antibacterial drugs,their poor biostability resulted from enzymatic degradation in vivo restricted their application as clinical drugs.Cyclization of ?-helix antimicrobial peptides could stabilize the ?-helix secondary structure,enhance protease stability,and improve biological activity.However,the current cyclization protocols could only introduced a staple structure onto the hydrophobic surface of antimicrobial peptides,which lead to enhanced biostability,but as well as increased hemolytic toxicity.To overcome the drawback of current cyclization protocol of antimicrobial peptides,in this Ph D thesis,we developed a new peptide cyclization method by lysine sidechain tethering,which can introduce a cationic bridge onto the hydrophilic surface of antimicrobial peptides.This new cyclization protocol could keep the cationic characteristics of antimicrobial peptides.Then,we employed this novel method for the modification of antimicrobial peptide OH-CM6 and synthesized successfully a series of cation-bridged OH-CM6 analogs.The preliminary biological evaluation of the cationic-bridged stapled OH-CM6 analogs showed that some of these analogs had behaved excellent antibacterial activity,low hemolytic toxicity and high serum stability.Therefore,this research successfully developed a novel peptide cyclization method for antimicrobial peptide modification,which is expected to promote the development of novel antibacterial drugs based on antimicrobial peptide.Focusing on the design and synthesis novel cation-bridged stapled antimicrobial peptides with excellent pharmacological properties,this Ph D thesis generally included the following contents:1.The background of this research was comprehensively summarized,including the current research status of antibacterial drug resistance,the LPS-induced inflammatory response,the classification,mechanism of action,and structure-activity relationship of antimicrobial peptides.Based on these,the purpose,main contents and innovation of this research were generalized.2.The synthesis of cationic-bridged stapled peptide was first explored on short peptides with amphiphilic structure.Exploring the Fukuyama alkylation reaction between the sidechain amino group of lysine,the staple structure was successfully introduced onto the hydrophilic surface of peptide,while the cationic characteristic of peptide was maintained.Next,the novel stapling method was successfully applied to the synthesis of a series of novel cation-stapled peptide analogues of OH-CM6.3.The biological activities of the synthesized cation-bridged antimicrobial peptides were evaluated.By screening the antibacterial activity and serum stability,the structure-activity relationship of cation-bridged antimicrobial peptide was systematically studied.The best insertion site and appropriate linker of staple structure were determined,which lead to the discovery of stapled peptide 12 with enhanced antibacterial activity and improved serum stability.Further exploration of the antibacterial mechanism of the novel cation-bridged peptide found that the cation-bridged peptide 12 was more likely to form pores and damage the cell membrane structure of bacteria.In addition,peptide 12 also had the characteristics of fast sterilization rate,not easy to develop drug resistance and high serum tolerance.The results of anti-inflammatory activity of the novel cation-bridged peptide 12 showed that12 had significant anti-inflammatory regulation effect,which could effectively regulate the expression of IL-6,NO and other inflammatory factors induced by LPS,relieve the symptoms of systemic inflammatory response syndrome(SIRS)mice,and improve the survival rate of mice.The discovery of the novel cation-bridged peptide 12 is an important breakthrough for the clinical application of antimicrobial peptides and provided a new solution for the treatment of multi-drug resistant bacterial infections,which is promising to be developed as novel anti-infectious drugs.In summary,this thesis successfully developed a novel stapling protocol for antimicrobial peptide modification employing the N-alkylation reaction of the ?-amino group of lysine.The new cyclization method could introduce a staple structure onto the hydrophilic surface of antimicrobial peptide while keeping the cationic characteristic.Application of this novel stapling protocol employing antimicrobial peptide OH-CM6 as template,a series of novel cation-bridged stapled antimicrobial peptides was successfully synthesized.Biological screening of these novel stapled antimicrobial peptides provided stapled peptide 12 with high antibacterial activity,excellent anti-inflammatory activity,good serum stability yet low hemolytic toxicity.This research is expected to promote the application of the cationic stapled antimicrobial peptide in the field of anti-infectious drug discovery.