Antimicrobial Activities And Action Mechanism Studies Of CPF-C1 And Its Analogues Against Multidrug-resistant Bacteria

Antimicrobial resistance is a growing public health threat of broad concern to numerous countries and multiple sectors.The problem also affects the achievements of modern medicine.However not enough drugs are being developed to solve this problem,creating an urgent need for the development of novel classes of antimicrobials.Gram-positive Staphylococcus aureus and gram-negative E.coli,Pseudomonas aeruginosa,Acinetobacter baumannii are the most common causes of nosocomial infection of bacteria.Antimicrobial peptides(AMPs)are a kind of amphiphilic cationic peptides.As a conserved component of the innate immunity of organisms,AMPs exhibit potent broad antimicrobial spectrum.Unlike traditional antibiotics that have specific molecular targets,AMPs could kill the bacteria by disrupting the membrane and was not affected by the common resistance mechanisms found in bacteria because it is very difficult to change the composition of the cell membrane.Recently,AMPs have attracted much interest because of this special mechanism of action and could be developed as a promising antibiotic candidate.CPF-C1(GFGSLLGKALRLGANVL-NH2)belongs to a family of a-helical cationic AMPs and was first isolated from norepinephrine stimulated skin secretions of the tetraploid frog.CPF-C1 has potent antibacterial activity against standard strains of common Gram-negative bacteria and Gram-positive S.aureus.It has low haemolytic Activity.However,its mode of action and its potential use against multi-drug resistant bacteria have not been reported.In the present study,antimicrobial activity of CPF-C1 against multidrug-resistant bacteria was determined.Our findings indicate that CPF-C1 exhibits potent activity against not only standard strains but also the multidrug-resistant strains that are responsible for most nosocomial infections.CPF-C1 could kill the bacteria by rapidly disrupting the membrane.So CPF-C1 could be developed as an ideal template for the development of antimicrobial agents with high activity.In the further study of biological activity of CPF-C1,we found that the stability of the CPF-C1 is very poor.When incubated with 0.002 ?g/ml of trypsin for 1h,the antimicrobial activity of CPF-C1 had lost.And when incubated in serum,it was very easy to be degraded.The retention rate in serum of CPF-C1 for 4h was only 16.71%.So a series of CPF-C1 analogues were designed and synthesized to improve the stability and increase antimicrobial activities against drug-resistant bacteria by modification.To increases the number of positive charge of CPF-C1,lysines have been introduced.To enhance the hydrophobicity and membrane activity,tryptophans,which are the hydrophobic amino acids,have been introduced.To improve the stability,D-amino acids have been introduced.A series of analogues were designed and synthesised.Their antimicrobial activities against multidrug-resistant bacteria,stability to protease and haemolysis activity were determined.Among these analogues,we successfully screened CPF-8,which has potent antimicrobial activity,better stability and low haemolysis activity.The minimal inhibitory concentrations were 8?g/ml.When incubated with 200 ?g/ml of trypsin for 6h,it also has potent antimicrobial activity.The retention rate in serum of CPF-8 for 24h reached 72.66%.Even if the concentration reaches 512 pg/ml,CPF-8 showed no haemolytic activity.So it can be used as a candidate compound for further in-depth study.Then certain analogues with more potent antimicrobial activities or distinctive characteristics were determined to anti-biofilm activities against S.aureus.Unlike CPF-C1,these analogues demonstrated inhibition activities to biofilm formation at sub-inijibitory concentrations.And some analogues could disperse preformed biofilm at high concentrations.Finally,the modes of action about these analogues have been preliminary studied.These analogues killed bacteria not only by disrupting the membrane but also by binding to DNA,which is not same to CPF-C1.In conclusion,we designed and synthesised a series of analogues of natural AMPs CPF-C1 by chemical modification.We successfully screened CPF-8,which has potent antimicrobial activity,better stability and low haemolysis activity.The study of structure-activity relationship provided the research foundation and theoretical basis for the further modification of AMPs.The study of mechanism provided a beneficial supplement for theory of mechanism of this class of AMPs.