Structure Design And Activity Evaluation Of Cyclic Peptides Against Acinetobacter Baumannii

BackgroundDue to the widespread use and even abuse of antibiotics,multidrug-resistant(MDR)bacterial infection has become a hot issue threatening global health.Acinetobacter baumannii(AB)is one of the most common pathogens of nosocomial infection.Clinically,about 55% of AB has been resistant to commonly used antibiotics such as ciprofloxacin,imipenem and ceftazidime,which is one of the most serious clinical drug-resistant bacteria.In the face of the increasingly serious problem of AB drug resistance,polymyxin(such as colistin)has become the last effective means for the treatment of drug-resistant AB infection,so it is urgent to develop new antibiotics.Research objectiveBased on the existing research foundation of antimicrobial peptides,Bam A was used as the molecular target,and molecular docking and molecular simulation technology were used to reasonably design and screen antimicrobial cyclic peptides with good safety,high stability and strong antibacterial activity.Through in vivo and in vitro activity evaluation,the foundation for clinical application was laid.Research methodsFirstly,the potential antimicrobial cyclic peptides were obtained by virtual combinatorial design and molecular docking virtual screening with β-barrel protein(Bam A)as the target.The stable conformation of Bama binding peptide was obtained by molecular dynamics simulation,and the candidate molecules were determined according to the binding free energy(MM/GBSA).Secondly,the minimum inhibitory concentration(MIC),dynamic antibacterial analysis and bactericidal kinetics of the candidate antimicrobial cyclic peptides were determined to verify the antibacterial activity of the candidate molecules.Thirdly,CCK8 method,hemolytic toxicity test and salt stability test were used to study the cytotoxicity and stability of antimicrobial cyclic peptide.Then,the murine model of multidrug-resistance Acinetobacter baumannii pneumonia was established to verify the antibacterial activity of antimicrobial cyclic peptide in vivo.Finally,PI uptake and scanning electron microscopy were used to verify the mechanism of antibacterial cyclic peptide.Research resultsIn this study,seven candidate antimicrobial cyclic peptides(hexapeptide: A1-A3,heptapeptide: B1-B2,octapeptide: C1-C2)were obtained,which had good binding affinity to Bam A protein.The results of in vitro antibacterial activity showed that the minimum inhibitory concentration(MIC)of A3 and B2 against multidrug-resistance Acinetobacter baumannii was 6 μg/m L,which was equivalent to the MIC of the positive control imipenem;the killing rate of 7 antimicrobial peptides against drug-resistant AB was equivalent to that of imipenem.The results of safety evaluation in vitro showed that the hemolysis rate of A1,A2,A3,B1,B2 and C2 to sheep red blood cells was less than 4% at256 μg/m L,which proved that they had good safety.At the same time,the antibacterial activity of A1 and B2 in high concentration Na Cl(36 mg/m L)and Ca Cl2(1 mg/m L)salt solution almost did not change,indicating that they had excellent salt stability.In order to further verify the antibacterial activity of cyclic peptide in vivo,the murine model of multidrug-resistance Acinetobacter baumannii pneumonia was successfully constructed.The infected mice were injected intravenously every 12 hours(5 mg/kg).The results showed that after the mice were treated with B2 for 24 h(3 times)and 48 h(5 times),B2 had a significant antibacterial effect,which was comparable to the positive control(imipenem)at the same concentration(5 mg/kg).Finally,PI uptake experiment and scanning electron microscope were used to observe the action mechanism of antimicrobial peptides on AB.The results showed that cyclic peptides B2 may act by destroying the bacterial cell membrane and causing its contents to flow out.ConclusionsIn this study,seven candidate cyclic peptides with good antibacterial activity against drug-resistant AB were designed and screened.Among them,B2 plays an antibacterial role by destroying the AB cell membrane.It has the characteristics of good safety,high stability and good antibacterial activity.It has a good application prospect and is expected to develop into a new type of antibacterial drugs.