Design,Synthesis And Biological Activity Evaluation Of Phenyl Sulfide-Based Antimicrobial Peptidomimetics

Research BackgroundThe main treatment of pathogenic microbial infections has always been antibiotics.However,antibiotic resistance increases rapidly due to the abuse and misuse of antibiotics.Currently,resistance in almost all antibiotics and the stagnation of a new class of antibiotics pose a serious threat to global public health.Hence,it is extremely important to discover and develop antimicrobial agents with effective antibacterial activity,low toxicity,novel mechanisms of action and undetectably low resistance frequencies.Antimicrobial peptides（AMPs）are widely found in various organisms and are the first line of defense of the host.Compared with traditional antibiotics,AMPs interact with bacterial cell membranes due to their amphiphilic structure,which has the main advantages of fast sterilization,broad antibacterial spectrum and low drug resistance.However,the limited clinical application of AMPs was due to their inherent disadvantages such as their high price,low bioavailability and both in vitro and in vivo instability.By simulating the amphiphilic structure and biological functions of AMPs,small-molecule antimicrobial peptidomimetics can not only maintain the advantages of AMPs,but also be expected to overcome these defects of AMPs.In this paper,we used phenyl sulfide as the parent molecule to design and synthesize a series of amphiphilic antimicrobial peptidomimetics based on phenyl sulfide.The phenyl sulfide is cheap,have excellent drugability and easy modification,which is expected to design a series of small-molecule antibacterial peptidomimetics that can overcome the defects of AMPs and also retain their advantages.Research ObjectiveThe parent scaffold of phenyl sulfide which possessed low cost,excellent drugability and access to modify,were employed to simulate the structural features and biological functions of AMPs.A series of amphiphilic small-molecule antimicrobial peptidomimetics based on phenyl sulfide were designed and synthesized and were conducted to have biological activity evaluation.Research Methods（1）Based on the concept of constructing the amphiphilic structure,phenyl sulfide compounds（2,2’-thiobis（4-tert-octylphenol）,2,2’-thiobis（4-chlorophenol）,or 6,6’-thiobis（2-（tert-butyl）-4-methylphenol））were selected as the starting raw materials to synthesize a series of amphiphilic antimicrobial pephidomimetics by introducing different hydrophilic cationic groups.The chemical structures of the synthesized phenyl sulfide derivatives were characterized by high-resolution mass spectrometry and NMR(¹H NMR and ¹³C NMR).（2）The antibacterial activity of the synthesized phenyl sulfide derivatives was characterized by the minimum inhibitory concentration（MICs）.The hemolytic activities were evaluated in term of HC₅₀values.After the synthesized phenyl sulfide derivatives were evaluated by the values of MICs and HC₅₀,compound T17 was identified as the most promising compound.（3）The most promising compound T17 was selected for further in vitro and in vivo biological activity evaluations,including time-kill kinetics,salt stability assays,antibacterial mechanism studies（SYTOX green assay,LIVE/DEAD Bac Light?bacterial viability assay,TEM assay,BODIPYTM-TR-cadaverine displacement assay）,drug resistance study,in vitro cytotoxicity（CCK-8）,murine corneal infection model.ResultsThe chosen starting materials have hydrophobic groups.Structure-activity relationship of compounds synthesized was studied by introducing different hydrophilic cation groups（alkylamine and basic amino acid）on the phenyl sulfide skeleton,and 23end products were synthesized.We found that amino acid-substituted compounds especially arginine（-OMe as C‐terminus group）displayed more potent biological activity compared with alkylamine substitution.Compound T17 with extremely potent antimicrobial activity（MICs=0.39-1.56μg/m L）,very weak hemolytic activity(HC₅₀>200μg/m L),and low cytotoxicity(CC₅₀>50μg/m L)was selected to be the most promising compound.In addition,its antibacterial activity remained stable in the presence of physiological concentrations of various salts.Compound T17 can rapidly kill Gram-positive bacteria within 0.5 h by membrane-targeting action and avoid the development of antibiotic resistance.More importantly,Compound T17 showed excellent antimicrobial efficacy in a Staphylococcus aureus-induced mouse corneal infection model（P<0.05,significant difference between compound 17 and Control of antimicrobial efficacy）with a comparable efficacy to vancomycin.ConclusionsThrough biomimicking the chemical structure and biological functions of AMPs,a series of cationic amphiphilic small-molecule antimicrobial peptidomimetics based on phenyl sulfide were rationally designed and synthesized.Compound T17 was effective against Gram-positive bacteria in vitro and in vivo and avoided the development of drug resistance.Through studying on this topic,we found that small-molecule antimicrobial peptidomimetics have great research value and advantages,such as high stability,relatively simple synthesis,low production cost,low toxicity and drug resistance,which can effectively overcome the disadvantages of natural AMPs such as instability and high price.In general,the discovery and development of antibacterial agents through the strategies of simulating AMPs is extremely promising,and our design of phenyl sulfide derivatives will provide new ideas for the development of novel effective small molecule antibacterial agents against resistant Gram-positive bacteria.