Design,Synthesis And Antibacterial Evaluation Of Membrane Active Capsaicin Derivatives

BackgroundAntibiotic resistance is becoming a“global public health problem”.The main reason for the emergence of antibiotic resistance is not only the misuse and abuse of antibiotics,but also the discovery and development of new antibiotics significantly lags behind the speed of bacterial resistance.Therefore,most international organizations have formulated appropriate policies to encourage the research and development of antibiotics in academia and the pharmaceutical industry,and the development of noval antibacterial agents to combat multidrug resistant bacteria has become an urgent priority.“Membrane disruption”is thought to be the main mechanism responsible for the antimicrobial activity of most antimicrobial peptides,which have a unique ability to overcome or reduce the occurrence of bacterial resistance.In recent years,CAMPs have been widely recognized as one of the most promising alternatives to antibiotics due to their rapid killing kinetics,broad spectrum antimicrobial activity,low resistance rate,and unique mode of action.Compared with natural antimicrobial peptides,small molecular peptidomimetics have many significant advantages,including flexible chemical modification framework,reduced toxicity,enhanced antibacterial activity,higher in vitro and in vivo stability,and better pharmacokinetic properties.The development of novel membrane-targeted small molecule peptidomimetics against drug-resistant bacteria using the strategie of mimicing the structure and function of antimicrobial peptides has attracted extensive attention from researchers,and will become an important research frontier.MethodsIn this study,by simulating the structural and functional characteristics of CAMP,a series of cationic amphiphilic small molecular antimicrobial peptides were designed and synthesized from nonivamide and capsaicine,by introducing cationic moieties such as basic amino acids,alkyl amines and guanidinium groups,and balancing its amphiphilic property by adjusting the length of hydrophobic chain.By comparing their antibacterial and hemolytic activities,and conducting structure-activity relationship analysis,the candidate antibacterial agent CP51 with excellent broad-spectrum antibacterial activity was finally selected.Then,further conducted biological evaluation of compound CP51 by in vitro cytotoxicity,temporal bactericidal kinetics,salt ion stability,drug resistance,as well as evaluation of its antibacterial efficacy in vivo local application through mouse bacterial keratitis model,and examination of corneal toxicity by fluorescein sodium staining.The in vitro biological evaluation of candidate compound CP51 was evaluated by cytotoxicity,temporal bactericidal kinetics,salt ions stability and drug resistance studies.Its in vivo antibacterial efficacy was evaluated by topical application in murine bacterial keratitis model,and its corneal toxicity was evaluated by sodium fluorescein staining.Finally,the preliminary antibacterial mechanism was investigated,including SYTOX Green,N-phenyl-1-naphthylamine uptake,Di SC₃（5）,BODIPY?-TR cadenamine displacement,LTA/LPS competitive binding,and anti-biofilm activity experiments.Results1.In this study,a total of 26 nonivamide and capsaicin derivatives with novel structures were synthesized for the structure-activity relationship study.Among them,CP51,which is modified with hexanoguanidine groups on both sides of the benzene ring ortho of nonivamide,was identified as a candidate compound.2.In vitro antibacterial activity studies found that CP51 has a broad spectrum antibacterial activity.It’s MICs ranged from 0.39 to 0.78μg/m L against Gram-positive bacteria,including Staphylococcus aureus ATCC 29213,methicillin-resistant Staphylococcus aureus NCTC 10442,and methicillin-resistant Staphylococcus aureus N315.And the MICs for Gram-negative bacteria were 1.56～6.25μg/m L,Pseudomonas aeruginosa ATCC 9027,Acinetobacter baumannii ATCC 17978,Acinetobacter baumannii R2899,Klebsiella pneumoniae ATCC 10031,Klebsiella pneumoniae ATCC 14581,and Escherichia coli ATCC 25922 were included.3.Through further in vitro biological evaluation found that it had poor hemolytic activity(HC₅₀=134.8±4.6μg/m L),low cytotoxicity(CC₅₀>25μg/m L),rapid bactericidal property,high salt tolerance in vitro and low resistance frequency.4.In vivo antibacterial efficacy studies found that,it showed comparable therapeutic efficacy to vancomycin and tobramycin in the murine model of bacterial keratitis infected with Staphylococcus aureus and Pseudomonas aeruginosa,indicating its large potential application value in the treatment of bacterial keratitis.5.In the preliminary antibacterial mechanism study,compound CP51 was observed to have strong membrane affinity and permeability,which can kill bacteria by membrane destruction in addition to strong anti-biofilm activity.ConclusionIn this study,the candidate compound CP51 was obtained from using nonivamide and capsaicin as raw materials,simulating CAMP,adjusting charge/hydrophobic balance and optimizing system structure.It has high efficiency,low toxicity,rapid sterilization and strong anti-biofilm activity.In addition,it can effectively avoid the generation of drug resistance by killing bacteria through membrane destruction mechanism.More importantly,It showed great potential for clinical application in mouse models of bacterial keratitis induced by Staphylococcus aureus or Pseudomonas aeruginosa.Therefore,this study provides a promising design strategy to overcome antibiotic resistance,and provides new ideas and compound basis for the development of small molecular antimicrobial peptidomimetics.