Design,Synthesis And Biological Study Of Lysine-stapled Antimicrobial Peptides

Infections caused by multi-drug resistant(MDR)bacteria,especially the ESKAPE pathogens(Enterococcus faecium,Staphylococcus aureus,Klebsiella pneumoniae,Acinetobacter baumannii,Pseudomonas aeruginosa,and Enterobacter species),are growing threat to worldwide public health.Antimicrobial peptides(AMPs),to which bacteria are difficult to develop resistance,emerged as promising next-generation antibiotics to combat MDR bacteria due to its unique membrane-disruptive antibacterial mechanism.However,clinical application of AMPs has been greatly hampered because of its poor in vivo stability.It has been reported that stapled peptides,which restrict the secondary structure of α-helix peptides through side-chain to side-chain cyclization,can significantly improve the in vivo proteolytic stability and drug-like properties of peptides.One of the most well-studied methodologies is all-hydrocarbon stapling technique via ring-closing olefin metathesis,which has been proven to reduce protease susceptibility in vivo.However,the introduction of hydrophobic bridge on the hydrophobic face of amphiphilic antimicrobial peptides lead to the enhanced hemolytic toxicity,thus limiting its application for modification of AMPs.To solve this problem,we proposed a novel lysine-stapling strategy in our previous study.Through the N-alkylation reaction of the ε-amino group of lysine,a “bridge” was successfully introduced onto the hydrophilic side of amphipathic antimicrobial peptides.This strategy enhanced the secondary structure stability of the antimicrobial peptide while ensuring the structural integrity of the hydrophilic side of the antimicrobial peptide.Employing this lysine-stapling strategy,we identified a stapled peptide analogue of antimicrobial peptide OH-CM6,which exhibited stronger antibacterial activity and higher serum stability compared with the linear parent peptide,and low hemolytic toxicity as the linear parent peptide.The results proved that the lysine-stapling strategy has great potential for the development of AMPs-based antibacterial drugs.Thus,this Ph D thesis intends to conduct a more in-depth study on the application of this strategy in the modification of antimicrobial peptides.To explore the applicability of this lysine-stapling strategy and the influence of the stapled structure on the AMPs’ bioactivity,the following studies have been conducted in this Ph D thesis:1.The background of this research,including current status and mechanism of bacterial drug resistance;the classification,physicochemical properties,mechanism of AMPs;chemical modification strategy of peptides as well as stapled peptides were comprehensively reviewed.2.Five linear peptides with different length,charge,and amphipathic properties were screened by lysine-stapling at i,i+4 position.The biological activity of the linear peptides and the stapled peptides were determined and analyzed,which confirmed that the stapling position and the peptide sequence had important influence on biological activity.For antimicrobial peptides with good amphiphilic structures,the lysine-stapling strategy can significantly improve the antibacterial activity and protease stability.On the other hand,the lysine-stapling strategy had limited improvement on the biological activity of antimicrobial peptides with high hydrophobicity.3.Using the amphiphilic cationic antimicrobial peptide BF15-a1 as the template,a series of stapled peptide analogue of BF15-a1 were synthesized by lysine-stapling position scanning with varied alkylating reagents with different hydrophobicity and rigidity.The biological activities of the stapled analogues of BF15-a1 further confirmed the importance of the stapling position selection.At the same time,the rigidity and hydrophobicity of the stapled structure also have a significant impact on the antibacterial activity and hemolytic toxicity of the stapled antimicrobial peptides.4.Based on the research results of the above two parts,a stapled analogue of BF15-a1,LS-BF1,were identified with significantly improved plasma stability and antibacterial activity compared with the linear peptide BF15-a1.LS-BF1 also exhibited excellent broad-spectrum antibacterial activity against clinically identified multidrug-resistant ESKAPE bacteria species.Through the study of its antibacterial mechanism,it was found that the stapled peptide LS-BF1 has the advantages of faster bactericidal activity,stronger membrane damaging ability and less prone to develop drug resistance than the linear peptide.LS-BF1 exhibited significant in vivo antibacterial activity in the treatment of mouse MRSA infection model,and at the same time,LS-BF1 did not show obvious toxicity under the condition of continuous high-dose administration,showing good in vivo safety.In conclusion,in this thesis,the lysine-stapling strategy was applied to α-helical AMPs with different physicochemical properties.The scope of application of this strategy in the modification of antimicrobial peptides was explored and the influence of peptide sequence,stapling position and staple structure on the biological activities of stapled antimicrobial peptides were investigated.The biological activity screening of synthesized stapled antimicrobial peptides highlighted the stapled peptide LS-BF1 with broad-spectrum antibacterial activity,significantly enhanced protease stability and low hemolytic toxicity.The mouse model confirmed that LS-BF1 has excellent antibacterial activity and safety in vivo,indicating that it is a good candidate of antibacterial drug with potential clinical application prospects.Through the research of this thesis,it is confirmed that the lysine-stapling strategy has important application value in the development of new antimicrobial drugs based on antimicrobial peptides,and is expected to promote the development of new antibiotics based on antimicrobial peptides.