Structural Optimization And In Vitro And In Vivo Activity And Toxicity Study Of Natural Antimicrobial Peptide Jelleine-1

In recent years,as the resistance of Gram-negative bacteria to traditional antibiotics has gradually increased,posing a greater threat to human health and life,there is an urgent need to develop new antibiotics to combat the evolving drug-resistant bacteria.Antimicrobial peptides（AMPs）,as important components of innate immunity,are widely found in microorganisms,insects,amphibians,mammals,and plants.Antimicrobial peptides have a unique mechanism of non-specific cell membrane targeting,which is not easy to produce tolerance,and has broad-spectrum antibacterial activity,which is expected to become a new generation of antimicrobial agents against drug-resistant bacteria.However,its high synthesis cost,limited antibacterial activity,poor stability and side effects limit the clinical application of antimicrobial peptides.Jelleine-1（Pro-Phe-Lys-Leu-Ser-Leu-His-Leu-NH₂）was originally isolated from royal jelly and contains only 8 natural amino acids.It is a typical amphiphilic antimicrobial peptide with broad-spectrum antibacterial activity and low toxicity,especially jelleine-1 shows some selectivity to Gram-negative bacteria.In order to improve its antibacterial activity,15analogs were designed and synthesized,and their antibacterial activity and toxicity in vivo and in vitro were studied in the present study.Our results showed that fine tuning of the cationic charge,polarity,and basicity of the sequence through amino acids substitution at position 3,5,7 and maintaining position 1,4,6,8 unchanged could improve the bioactivity of jelleine-1 significantly.Meanwhile,we also found that the substitution of phenylalanine by tryptophan also could improve the antimicrobial activity of jelleine-1.Among all the analogs,analog 15,which is enriched in arginine and leucine,showed the most potent antimicrobial activity against both gram-negative and gram-positive bacteria,especially to multi-drug resistant Pseudomonas aeruginosa in vivo and in vitro.In addition,the results of outer and inner membrane experiments showed that analog 15was consistent with the parental peptide mechanism,mainly killing bacteria through destruction of membranes mechanism.Analog 15 also showed negligible toxicity,which was certified by MTT,hemolysis,blood assay,and biochemical analysis.In conclusion,the amino acid substitution strategy was used to obtain the analogue 15,which is rich in arginine and leucine.Its antibacterial activity is significantly higher than that of jelleine-1,while maintaining the advantage of low toxicity of jelleine-1,which has potent potential in the fight against multi-drug resistant Gram-negative infection.