Stability Modification And Activity Study Of Antimicrobial Peptide Polybia-MPI

In the past few decades,due to extensive use and abuse of conventional antibiotics,the antibiotic resistance became to be an emerging worldwide threat to human health,so it is urgent to develop new therapeutic agents to cope with antibiotic resistance.Antibacterial peptides(AMPs),also termed as Host Defence Peptides,were considered the first line of defense against invading pathogens in most literatures,with direct antimicrobial activity even against multidrug-resistant bacteria.Polybia-MPI,a naturally occurred antimicrobial peptide isolated from the venom of the social wasp Polybia paulista in 2005,has potent antimicrobial activity against Gram-positive and Gram-negative bacterial but no hemolysis to erythrocytes.Its chemical modification,antimicrobial activity and membrane-lytic mechanism were studied intensively in our previous work as well as the anticancer activity,but its further application was limited for their poor in vivo stability.In this work,unnatural amino acids Sar,D-Ala and corresponding D-amino acids were used to replace every single position of the polybia-MPI respectively,and 40 analogs were synthesized by Fmoc-tactical solid phase peptide synthesis.The serum stability result shows that the 1st,2nd,3rd and 11 th positions in polybia-MPI sequence were enzyme-labile and the serum stability was apparently improved after replacement of these sites with unnatural amino acids.The antimicrobial activity against Gram-positive bacteria and Garm-negative bacteria of all 40 analogs were screened,and their hemolytic side effect and cytoxicity were also tested.The MIC value and hemolysis rate shows that analog Sar-2,D-Ala-2 and D-Ala-8 exhibited 2-4 time-increased antibacterial activity against E.coli(ATCC 25922)than polybia-MPI,accompanied with lower(6,12,12 times respectively)hemolytic activity to mouse red blood cells,and lower cytotoxicity to mouse monocyte macrophage(RAW 264.7).The membrane-lytic mechanism of these three analogs were clarified by endomembrane infiltration,PI uptake assay,and scanning electron microscopy,as polybia-MPI itself.In order to investigate the possibility of drug-resistance induction of these three analogs,20 days drug resistance-inducing test were runned comparing with Kanamycin Sulfate.The data shows that the MIC value of Kanamycin Sulfate was 16-fold high at the end of the test,but the MIC value of analog D-Ala-8 unchanged,and the MIC value of Sar-2 only doubled,indicating that the polybia-MPI analog was not easy to induce drug resistance like conventional antibiotics under the same condition.In addition,the in vivo antibiotial activity of these three analogs was tested,and the results implied that analog Sar-2 and D-Ala-2 possessed high antibacterial activity than parent peptide.According to our result mentioned above,the 40 polybia-MPI analogs were developed and screened,and analog Sar-2 presented higher antibacterial activity,significantly improved serum stability,lower toxicity,and inferior bacterial resistance,have great potential to be new antibacterial drug candidate.Moreover,this work also provided a new strategy for the development of more stable peptide drugs.