Structural Optimization And Light-responsive PEGylated Prodrug Based On Antimicrobial Peptide Gramicidin S

Bacterial resistances are increasing while the development of novel antibiotics is declining.In Europe,approximately 25000 people die every year due to antibiotic-resistant infections.In addition to a tight control of the use of the existing antibiotics,there is an urgent need for novel anti-infection agents that possessing alternative mechanism of action to combat antimicrobial resistance.Delightfully,among the new emerging categories of potential antibiotics,antimicrobial peptides（AMPs）are considered to be one of the most promising alternatives to classic antibiotics.Many AMPs are known to display an extraordinarily broad range of antimicrobial activities covering both Gram-positive and Gram-negative bacteria as well as viruses,fungi and tumors.More importantly,it is common knowledge that AMPs kill microorganism through the disruption of lipid bilayers of cell membrane,which makes it very difficult for microorganisms to develop resistance.Unfortunately,the majority of naturally occurring AMPs aren’t suitable for directly clinical applications due to severe hemolytic and cytotoxic effects.Gramicidin S（GS）is an amphiphilic cyclodecapeptide which adopts a rigid C₂-symmetricβ-hairpin structure with the primary sequence cyclo-（Pro-Val-Orn-Leu-^DPhe）₂.There is a general agreement that GS kills bacteria by the disruption of membrane bilayer,though its precise mechanism of action is still debated.Unfortunately,GS is not selective toward the lipid bilayer which causes severe toxicity towards human erythrocyte.In this work,both methods including structural optimization and prodrug design were employed to obtain non-toxic GS analogues.1.Structural optimization based on gramicidin SExtensive studies have shown that seemingly subtle changes at theβ-turn region could have a dramatic impact on antimicrobial activity,suggesting that this region may be a hot spot for modulating GS structure and activity.We have previously shown that when introduced into short peptides,β,γ-Di AAs are capable to induce stable C₉ or C₁₂ intra-molecular H-bonds which finally stabilize well-defined conformations.Therefore,β,γ-Di AAs appear very promising to stabilize theβ-turn region of GS and in the same time to maintain its global amphiphilic character.In this regard,we present a molecular approach where the native GSβ-turn region is replaced by syntheticβ,γ-diamino acids（β,γ-Di AAs）.Fourβ,γ-Di AA diastereomers were employed to mimicβ-turn structure to afford GS analogues GS3-6 that exhibited diminished hemolytic activity.A comparative structural study demonstrates that the（βR,γS）-Di AA displays the most stableβ-turn mimic.To further improve the therapeutic index（e.g.high antibacterial activity and low hemolytic activity）and to extend the molecular diversity,GS5 and GS6 were used as structural scaffolds to introduce additional hydrophobic or hydrophilic groups.We show that GS6K,GS6F and GS display comparable antibacterial activity while GS6K and GS6F possess significantly decreased toxicity.Moreover,antibacterial mechanism studies suggest that GS6K kills bacteria mainly through the disruption of membrane.2.Light-responsive PEGylated prodrug based on gramicidin SPEGylated peptides are known to display reduced renal clearance,enzymatic degradation and immunogenicity while increased solubility.However,PEGylation often results to significant decrease of biological activities of a drug,especially for small peptides.We report a concise while efficient prodrug system in which the GS was conjugated with photo-releasable m PEG linkage.The resulting prodrug c GS-m PEG₂₀₀₀ exhibited negligible hemolysis and cytotoxicity while increased proteolytic stability.Upon irradiation,GS was spatiotemporally released to display antimicrobial activity that was as potent as natural product.In conclusion,we have synthesized a series of GS analogues and light-responsive PEGylated prodrugs.Their secondary conformation,antibacterial activity,hemolytic activity,cytotoxicity as well as antibacterial mechanism were studied.We have shown that analogue GS6K is a superior mimic of GS while prodrug c GS-m PEG₂₀₀₀ can be spatiotemporally released to reduce hemolytic effect.