A Preliminary Study On The Structure-activity Relationship And Mechanism Of Antimicrobial Peptides Of The Brevinin-2 Family

Antimicrobial peptides(Antimicrobial peptides,AMPs)existing in the innate immune system are a class of low molecular-weight polypeptides with antibacterial activities.Due to the growing problem of pathogenic microorganisms resistant to conventional antibiotics,AMPs that have a unique mechanism of action show a wide range of applications.Brevinin-2CE,a natural antimicrobial peptide purified from the skin secretions of Chinese brown frog,Rana chensinensis,contains 37 amino acid residues and its primary structure is GLLSVFKGVLKTAGKNVAKNVAGSLLDQLKCKISGGC.Brevinin-2Ka,a natural antimicrobial peptide purified from the skin secretions of Rana kukunoris,contains 33 amino acid residues and its primary structure is GLLSVFKGVLKTAGKHVAGSLLHQLKCKISGGC.Both of them have the typical characteristics of Brevinin-2 family,and they can inhibit the common gram-positive bacteria and gram-negative bacteria.In this study,we designed and synthesized a series of analogs by modifying the sequence length,disulfide bonds,the number of charges,hydrophobic and amphiphilic characteristics based on Brevinin-2CE and Brevinin-2Ka.And we had detected the activities of antimicrobial peptides on bacterium,red cells and eukaryotic mammalian cells.Combined with the results,we analyzed the structure-activity relationships of Brevinin-2 family.The main results are as follows.B2CE and B2Ka have the similar modification:B2CE-nonDS,B2CE-C31,37S and B2Ka-nonDS?B2Ka-C27,33S are designed by not adding disulfide bonds or replace cysteine by serine in the synthesis of AMPs;B2CE-N26,B2Ka-N26 and B2-N15 were obtained by truncating sequences;respectively using B2CE-N26 and B2Ka-N26 as a template,we designed B2CE-N26-V5K,B2CE-N26-G14K,B2CE-N26-V5KG14K and B2Ka-N26-V5K,B2Ka-N26-G14K,B2Ka-N26-V5KG14K by replacing single or double amino acids,both of which have the properties of increasing charge number and amphiphilic,decreased hydrophobicity;according to the helical wheel,we designed B2CE-N26-N16WA18KG23K and B2Ka-N26-H16WA18KG19K;in order to estimate the different effect of histidine and lysine in the sequence,we acquired B2Ka-N26-G14KH-K.Analyzing the activities of AMPs by detecting the minimum inhibitory concentration(MIC).Compared to B2CE and B2Ka,antimicrobial activities of the peptides that missing disulfide bonds have no changes,indicating that disulfide bonds do not play a role in the process of inhibition of antimicrobial peptides;the shorter sequences of AMPs,the lower antibacterial activities;the antimicrobial activities of B2CE-N26-V5K is 1-8 times than B2CE-N26,while B2Ka-N26-V5KG14K has decreased observably,proving that increasing the number of charges and amphiphilic,decreasing hydrophobicity appropriately will help improve the antibacterial activities;B2CE-N26-N16WA18KG23K and B2Ka-N26-H16WA18KG19K have obvious broad spectrum,indicating that the amphiphilic is an important factor in the inhibition of AMPs;antibacterial activity of B2Ka-N26-G14KH-K has increased 2-6 fold,including that lysine instructions will help improve antibacterial activity.The results of hemolysis activities are showed.The hemolytic activity of B2CE-C31,37S is 2 times than B2CE,indicating that disulfide bonds enhanced damages on erythrocyte;the shorter sequences of AMPs,the lower sensitivity of red blood cells(RBC);in the transformation B2CE-N26 and B2Ka,B2CE-N26-N16WA18KG23K and B2Ka-N26-H 16WA18KG19K showed strong hemolytic activity,indicating that amphiphilic is an important factor.We detect the cytotoxicities of antimicrobial peptides on tumor cells and normal cells using the method of MTT.Compared to B2CE and B2Ka,the anti-tumor activities of the peptide missing disulfide bond is slightly lower,indicating that disulfide bonds is important to the AMPs;the shorter sequences of AMPs,the lower anti-tumor activities;the anticancer activity of peptides based on B2CE-N26 and B2Ka-N26 decreased,while B2Ka-N26-H16WA18KG19K anticancer activity was significantly increased,proving that moderately reducing the number of charges,hydrophobicity and enhancing amphiphilic are beneficial to antimicrobial peptides;anticancer activity of B2Ka-N26-G14KH-K is 4-5 times than B2Ka-N26-G14K,lysine instructions help to improve the anti-cancer activity.To clarify the mechanism of action,we examined the mechanism on cytoplasmic membrane of bacterium.The results of Atomic Force Microscope(AFM)and bacterial toxicity testing indicate that membrane were disrupted by the antimicrobial peptide.And bacteria were disintegrated into small fragments and led to the death.The topic is the first time to study the structure-activity relationships based on antimicrobial peptide of brevinin-2 family.Finally,we obtained the new antimicrobial peptides that have applications.