Solid Phase Chemical Synthesis and Structure - Activity Study of Brevinin - 2R and Analogues as Antimicrobial Peptides

Abstract

Background: Brevinin-2R, as 25 amino acids peptide of the skin of Rana ridibunda frog, possesses potent antimicrobial and low hemolytic activity. It has an N-terminal hydrophilic region and a C-terminal loop that is delineated by an intra-disulfide bridge. In our study, Brevinin-2R and its diastereomer as well as its cyclic analogue were synthesized and characterized in order to investigate its structural features and biological implications. Methods: MIC determination is based on the recommended classical method of national comittee for labratory safety standard (NCLSS) and standard by Hancock With some change on cationic peptides. In this study All bacterial strains were obtained from Industrial-Scientific Research center. Results: Both analogues showed lower antimicrobial activities compared to Brevinin-2R. In spite of Brevinin-2R peptide which shows low hemolytic activity, these analogues failed to show any hemolytic activity even at higher concentrations (up to 400 µ g/ml). Based on proteolytic stability measurements,diastereomer and cyclic analogues displayed 90% and 60% residual antimicrobial activity, respectively, while antimicrobial activity of Brevinin-2R was 20%. The CD analysis revealed that amphipathic α-helical conformation of the synthesized peptides is involved in antimicrobial effects. Conclusion: CD studies and HPLC based measurement of retention time using a reverse phase column indicated that the Brevinin-2R can form an amphipathic loop resulting in an enhanced hydrophobicity. The hemolytic activity of Brevinin-2R and its analogues appeared to correlate with the retention time as well as the α-helicity. Accordingly, it seems that the combination of incorporating of D-amino acids into lytic peptides and their cyclization may result in developing new antimicrobial peptides with improved properties for treating infectious diseases.

Keywords

• Brevinin-2
• Rana
• Protein Structure
• Secondary
• Circular Dishroism