Design And Structure-activity Relationships Study Of Antimcirobial Peptides Derived From Tachykinin

The misuse of antibiotics results in the emergence of resistant bacteria,and many traditional antibiotics are becoming increasingly ineffective,which seriously threaten public health.Therefore,alternative antibiotics with novel mechanisms of action are urgently required to overcome resistance.Antimicrobial peptides,as a conserved component of the innate immunity of organisms,exhibit potent broad-spectrum antimicrobial properties.Unlike traditional antibiotics that have specific molecular targets,antimicrobial peptides bind selectively and disrupt rapidly the negatively charged bacterial membranes rather than neutral mammalian cell membranes through a series of physical processes.Recently,antimicrobial peptides have attracted much interest because of this special mechanism of action.Substance P(SP,RPKPQQFFGLM-HN2)and human Hemokinin-1(hHK-1,TGKASQFFGLM-NH2)belong to the tachykinin family of neuropeptides.They participate in modulating many physiological responses,such as pain,immune,cardiovascular system and angiogenesis.Perhaps surprisingly,SP and hHK-1 fall into the same structural class with many antimicrobial peptides.Some reports have showed that both of SP and hHK-1 exhibited antimicrobial activity.Based on the multiple functions of SP and hHK-1,we assume that they can be ideal templates for the development of antimicrobial agents with high activity.SPA(RPKPWQWFWLL-NH2,underlined amino acids are D-enantiomers)is an antagonist of SP.Although the introduction of D-amino acids can disrupt the structure of SPA,we think that the introduction of D-tryptophan and Leucine can be able to increase the interaction of SPA with bacterial membranes,resulting in the enhanced antimicrobial activity of SPA.Our results demonstrated that SPA could inhibit the growth of gram-negative and-positive bacteria,whereas SP did not show any antimicrobial activity at the maximum detectable concentration(128 ?M).More importantly,SPA showed high stability toward to trypsin and serum,respectively.In addition,we also found that SPA also inhibited the inflammation induced by LPS,suggesting that SPA exhibit LPS-neutralizing activity.Our results indicate that introduction of D-amino acids with high hydrophobicity can contribute to improving the antimicrobial activity and enzymatic stability of SP.Our group have studied the multiple physiological activity of hHK-1and its analogues more extensively and intensively.In addition,hHK-1 is more suitable for serving as a template for the design of antimicrobial peptides compared with SP in the structure.Thus,we designed and synthesized a series of analogues of hHK-1.The major structural parameters that affect the activity of antimicrobial peptides include the number of positive charges,hydrophobicity,helicity and amphiphilicity.Our results showed that hHK-1 did not show any antimicrobial activity at the maximum detectable concentration(128?M)like SP.This may be because the number of basic amino acids and hydrophobic amino acids of hHK-1 is too low.To improve the antimicrobial activity of hHK-1,the sequence of LKKW(Leucine-Lysine-Lysine-Tryptophan)or(LKKW)2 was added to the N-terminus of hHK-1 to increase its number of positive charges and hydrophobicity.As expected,the sequence of LKKW obviously enhanced the antimicrobial activity of hHK-1.Especially AH-3,which contains(LKKW)2,exhibited significantly antimicrobial activity and was superior to the classic antimicrobial peptide melittin and MSI-78.More importantly,AH-3 showed almost no hemolytic activity.AH-4,where the amino acid at the position 10 is leucine,showed the highest killing activity to all tested bacteria with a little increase in hemolytic activity at 400?M,which is 200-fold than MIC value.The increased antimicrobial activity and hemolytic activity of AH-4 is probably attributed to the increased helicity and hydrophobicity.Compared with SPA,AH-4 showed significant anti-inflammatory activity,suggesting that AH-4 would be not only used as an antimicrobial agent but also as an anti-inflammatory agent.To further improve the antimicrobial activity of AH-4,we designed a series of analogues by increasing the hydrophobicity and amphiphilicity.Unfortunately,the antimicrobial activity of all analogues was decreased,whereas their hemolytic activity was significantly increased.Among the analogues,AH-10 exhibited the lowest antimicrobial activity but highest hemolytic activity,where the hemolysis rate of AH-10 reached to 70%at 12.5 ?M and was significantly higher than that of classic antimicrobial peptide melittin.This is probably because AH-10 showed the highest hydrophobicity and amphiphilicity among these analogues.Introduction of D-amino acids is a usefull strategy that can decrease the toxicity of antimicrobial peptides and enhance their enzymatic stability.Therefore,we synthesized a series of analogues of AH-10 by introduction of six or eight D-amino acids into the hydrophilic face or the hydrophobic face of AH-10.As expected,introduction of D-amino acids significantly decreased the hemolytic activity and increased the antimicrobial activity and the enzymatic stability of analogues compared with AH-10.Our results indicate that introduction of D-amino acids into the hydrophilic face did not result in significant difference in antimicrobial activity of analogues compared with that into the hydrophobic face.However,introduction of D-amino acids into the hydrophilic face probably contributed to decrease the toxicity and enhance the enzymatic stability of analogues.In this study,we designed and synthesized a series of analogues of tachykinin,and systematically studied their structure and activity relationships.Our results indicate that the change of hydrophobicity,amphiphilicity,number of positive charges and helicity would significantly affect the antimicrobial activity and toxicity of analogues.Although introduction of D-amino acids can disrupt the helical structure of peptides,this type of modification strategy could significantly decrease the hemolytic activity of analogues and enhanced the antimicrobial activity and enzymatic stability of analouges.By screening,we obtained a group of antimicrobial lead compounds with potent application.In short,our study provided a novel idea for developing antimicrobial peptides with multiple functions and some experience for designing and modifying antimicrobial peptides derived from tachykinins.