Study Of Solid Phase Synthesis Of Antifungal Peptide CGA-N46 And Its Derivatives Of Bioinformatics Analysis And Biological Activity

The widespread use of antibiotics has led to the rapid emergence of antibiotic-resistant bacteria and fungi. Compared with traditional antibiotics, antimicrobial peptides(AMPs) have attracted considerable consideration for their broad-spectrumantimicrobial activity and less possibility to cuase microbial resistance. in the research of biological medicine. CGA-N46 is a novel antifungal peptide containing 46 amino acid residues. In this study, CGA-N46 and its derivatives were analyzed by bioinformatics software. The potential antifungal derived fragments were designed and prepared by solid phase synthesis. Their biological activities in vitro and in vivo were studied. This results lay a theoretical basis for the development of CGA-N46 derivatives to be a novel antifungal peptides.The secondary structures and physicochemical properties of CGA-N46 and its derivatives were analyzed using bioinformatics tools. A series of derived fragments with high stability and long half-life were designed and named as CGA-N15, CGA-N16, CGA-N12 and CGA-N8. NPS@ software analysis of CGA-N46 and its derivatives showed their secondary structure were α-helix. Helical Wheel analysis of CGA-N46 and its derivatives showed that hydrophilic residues and hydrophobic residues of CGA-N46 and its derivatives scattered randomly, and there were no typical hydrophilic face and hydrophobic face. CGA-N46 derivatives were prepared by solid phase peptide synthesis. The results of circular dichroism(CD) confirmed that CGA-N46 and its derived peptides displayed α-helical structure in an aqueous solution and 30 mM sodium dodecyl sulfate, but α-helical contents decreased in hydrophobic lipid vesicles. CGA-N15, CGA-N16, CGA-N12 and CGA-N8 had higher antifungal activities. Yeasts exhibited the various greatest sensitivity for different peptides. C. krusei(MIC, 0.37 mmol·L-1) for CGA-N46, C. tropicalis(MIC, 0.073 mmol·L-1) for CGA-N15, C. glabrata(MIC, 0.28 mmol·L-1) for CGA-N16, C. tropicalis(MIC, 0.075 mmol·L-1) for CGA-N12, and C. krusei and C. albicans(MIC, 0.24 mM L-1) for CGA-N8. The results of the hemolysis of CGA-N46 test by spectrophotometry showed that hemolytic activity decreased with the order of CGA-N15, CGA-N16, CGA-N8 and CGA-N12, their hemolytic concentrations were shown as follow : 0.031 mmol·L-1, 0.056 mmol·L-1, 0.27 mmol·L-1 and 0.39 mmol·L-1. The results of cytotoxicity on the primary mouse kidney cell showed that CGA-N15 has the strongest cytotoxicity at a concentration of 2mg·mL-1, and the mouse kidney cell survival rate was only 7.8%. While CGA-N16, CGA-N12 and CGA-N8 had no effect on the growth of normal mouse kidney cell at a concentration of 2mg·mL-1.The interaction between CGA-N12 and bovine serum albumin(BSA) was measure by CD. The results showed that CGA-N12 can reduce the proportion of α-helix structure in BSA, indicating that CGA-N12 could interact with BSA and affect its conformation.The result provided directions to explore the mechanism of action of CGA-N12.The treatment of CGA-N12 on the mice deep-infected with Candida tropicalis could significantly increase the survival rate of the deep infected mice, increase the thymus index and spleen index and decrease the quantity of Candida tropicalis strains in the infected-mice kiney and has good therapeutic effect on the Candida tropicalis-infected mice.In one conclusion, after the bioinformatic analysis and solid phase synthesis, on CGA-N46 derivatives, named CGA-N12, with high anti-fungal activity and biosafty was obtained. CGA-N12 has a strong anti-Candida activity,low hemolytic activity and low cell toxicity.It also have treatment on the Candida tropicalis-infected mice. It is a safe and effective antimicrobial peptides, with the potential development of a new antifungal agents.