Studies On Structure-Function Relationship And Protease-Resistant Activity Of Imperfectly Amphiphilic α-Helical Peptides

With the global burden of antimicrobial resistant infections rising at an alarming pace,the development of a new generation of antibacterial drug is extremely urgent.Antimicrobial peptides（AMPs）as an important defensive line for the organism immune systems,have the great potential to be the alternative to antibiotics due to broad-spectrum antimicrobial activity,rapid sterilization efficiency and unique physical membrane binding and disruption mechanism.（1）Structure-function relationship is the theoretical basis of antimicrobial peptide design,and the perfectly or imperfectly amphipathic structure is always the bone of contention in structure-function relationship research on AMPs.In this study,we designed an imperfectly amphipathic palindromic structure R_n（XRXXXRX）R_n（n=1,2;X=L,I,F,W）based on a centrosymmetric α-helical template.A series of imperfectly amphipathic α-helical peptides was synthesized and analyzed for their antibacterial and antifungal properties.The corresponding perfectly amphipathic peptides with the same amino acid composition but with a different amino acid distribution were also designed to investigate the effect of perfect/imperfect amphipathicity on the biological activity.The main results were summarized as follows:1)Biological activity determination indicated that imperfectly amphiphilic peptides showed greater antimicrobial activity against most of the microbes tested,indicating that imperfect amphipathicity was necessary to facilitate the formation of membrane pores and increased the antimicrobial activity of peptides.However,most of the imperfectly amphipathic peptides had similar hemolytic activity and cytotoxicity as their perfect counterparts,suggesting that the toxicity of the short peptides was independent of the type of amphipathicity,which was a greatly different result from in previous studies.It is noteworthy that hydrophobicity and cationicity had a relatively greater effect on the antifungal activity of the short peptides than the antibacterial activity,thus high cationicity and moderate hydrophobicity were necessary for antifungal peptides.Of the short peptides,13 had the greatest activity(GM_all=2.2μM)and selectivity(SI_all=116.36)against fungi and bacteria,specifically against drug-resistant fungi,with rapid sterilization efficiency,low likelihood of resistance development and high salt and serum tolerance.2)Antimicrobial mechanism investigation showed that 13 exerted its fungicidal and bactericidal action by targeting the cell membrane surfaces,damaging the membrane integrity,binding the intracellular target DNA and inducing the formation of intracellular ROS,eventual leading to cell death.（2）Poor proteolytic resistance is an urgent problem to be solved in the clinical application of AMPs,yet common solutions,such as complicated chemical modifications and utilization of Damino acids,greatly increase the difficulty and cost of producing AMPs.In this study,we successfully designed a novel peptide structure unit（XYPX）_n（X represents I,L and V;Y represents R and K）with high resistance to trypsin/chymotrypsin by systematic amino acid arrangement,and a set of peptides was synthesized and explored for their structure-function relationships,and a peptide with high cell selectivity and high protease resistance was screened out among them.The main results were summarized as follows:1)Biological activity determination indicated that diverse lengths and subtypes of hydrophobic/cationic amino acids had a considerable influence on the cell selectivity of the peptides.The peptides with seven repeat units showed the best bactericidal activities among their respective series,and any further increase in the number of repeat units instead compromised their bactericidal activities.Furthermore,replacement of Lys with Arg,which has a higher cationic degree served to broaden the bactericidal spectrum and increase the sterilization rate of the peptides.Among these peptides,R7 I had the highest average selectivity index(GM_SI=99.07) for gram-negative bacteria,although its bactericidal activity was not the best(GM_{MBC:gram-negative bacteria}=4.97μM),which indicated that an appropriate equilibrium between cationicity and hydrophobicity was important for the selectivity of the peptides.On the contrary,R7 I had weak activity against most of the tested gram-positive strains,showing the characteristics of narrow-spectrum bactericidal activity.Not only that,R7 I showed good salt tolerance and serum stability in vitro,and a low likelihood of resistance development,meanwhile it also had rapid sterilization ability,which could eliminate >95% of the bacterial cells within 30 min.Importantly,R7 I still maintained excellent activity in a mouse peritonitis model.Compared with the control group,R7 I could eliminated >99.99% bacterial cells in the peritoneal cavity and showed no detectable hepatic or renal toxicity in mice at intraperitoneal doses of up to 40 mg /kg（the highest tested dose）every 12 hours for 3 days.These results indicated that R7 I possessed excellent biocompatibility and safety.2)After incubation with trypsin and chymotrypsin,R7 I kept the effective bactericidal activity,and its secondary structure and molecular weight also had no significant change,suggesting that R7 I had excellent resistance to trypsin and chymotrypsin.However,high concentration of pepsin had a slight inhibitory effect on the bactericidal activity of R7 I,and after pepsin treatment,the helicity of R7 I decreased,but its molecular weight had no change.Surprisingly,R7 I had great resistance to proteinase K,which is a serine protease with broad-spectrum cleavage activity and preferentially cleaves at aliphatic and aromatic amino acid residues in position P1.3)Antimicrobial mechanism investigation showed that R7 I initially interacted with LPS via electrostatic adsorption.With the aggregation of peptide molecules,the hydrophobic core of R7 I inserted into the outer membrane phospholipid layer,and it destroyed and traversed the outer membrane and thin peptidoglycan layer by the transmembrane electrical potential.Subsequently,sufficient peptide molecules reached the cytoplasmic membrane surface and interacted with the phospholipid bilayer,causing membrane potential disturbances and membrane disruption（pore formation）,which led to the loss of the intracellular content and eventual bacterial cell lysis and death.R7I as a new class of antimicrobial agents broke through the bottleneck of clinical applications of AMPs（poor salt/serum/proteolytic resistance in vitro,high toxicity and inactivation in vivo）and had the potential capability to solve the lack of new antimicrobial agents for treating multidrugresistant gram-negative ESKAPE pathogens.In summary,this study started from the structure-function relationship of α-helical peptides,and elucidated the effect of perfect/imperfect amphipathicity on the biological activity of AMPs.Because of the broad-spectrum antimicrobial activity and great stability,the screened peptide 13 had a good application prospect in clinical and animal husbandry.Subsequently,we have established a novel imperfectly amphipathic anti-protease hydrolytic structure unit,and explained the effect of diverse structure parameters on the cell selectivity and the proteolytic resistance of AMPs,which provided a new idea for the development of anti-protease hydrolytic peptides.The selected peptide R7 I with the highly efficient antimicrobial activity in vivo and high protease resistance,has broken through the key obstacles in the application of AMPs.These findings laid the foundation for using AMPs in animal industry as new feed additives.