Abstract
All life forms are equipped with rapidly acting, evolutionally conserved components of an innate immune defense system that consists of a group of unique and diverse molecules known as host defense peptides (HDPs). A Systematic and Modular Modification and Deletion (SMMD) approach was followed to analyse the structural requirement of B1CTcu5, a brevinin antibacterial peptide amide identified from the skin secretion of frog Clinotarsus curtipes, India, to show antibacterial activity and to explore the active core region. Seventeen SMMD-B1CTcu5 analogs were designed and synthesised by C and N-terminal amino acid substitution or deletion. Enhancement in cationicity by N-terminal Lys/Arg substitution or hydrophobicity by Trp substitution produced no drastic change in bactericidal nature against selected bacterial strains except S. aureus. But the sequential removal of N-terminal amino acids had a negative effect on bactericidal potency. Analog B1CTcu5-LIAG obtained by the removal of four N-terminal amino acids displayed bactericidal effect comparable to, or in excess of, the parent peptide with reduced hemolytic character. Its higher activity was well correlated with the improved inner membrane permeabilisation capacity. This region may act as the active core of B1CTcu5. Presence of C-terminal disulphide bond was not a necessary condition to display antibacterial activity but helped to promote hemolytic nature. Removal of the C-terminal rana box region drastically reduced antibacterial and hemolytic activity of the peptide, showing that this region is important for membrane targeting. The bactericidal potency of the D-peptide (DB1CTcu5) helped to rule out the stereospecific interaction with the bacterial membrane. Our data suggests that both the C and N-terminal regions are necessary for bactericidal activity, even though the active core region is located near the N-terminal of B1CTcu5. A judicious modification at the N-terminal region may produce a short SMMD analog with enhanced bactericidal activity and low toxicity against eukaryotic cells.
Highlights
Short, cationic, amphiphilic peptides with antimicrobial activity are widely distributed in all life forms including humans which can kill a broad-spectrum of pathogens like bacteria, fungi, parasites, enveloped viruses and even transformed or cancerous cells with ostensibly limited chance to develop resistance [1,2,3,4,5,6,7]
Since increase in cationicity is a robust strategy to improve the antibacterial property of antimicrobial peptides (AMPs), B1CTcu5K and B1CTcu5R was synthesized by substituting the Nterminal Leu with cationic Lys/Arg
Seventeen Systematic and Modular Modification and Deletion (SMMD)-B1CTcu5 analogs were designed and synthesized to study the importance of primary and secondary structure on activity and to identify the pharmacophore region of B1CTcu5. These peptides were designed by swapping predictable factors that govern the antimicrobial and cytotoxic character of a peptide, which include cationicity, hydrophobicity, amphipathicity and helicity
Summary
Cationic, amphiphilic peptides with antimicrobial activity are widely distributed in all life forms including humans which can kill a broad-spectrum of pathogens like bacteria, fungi, parasites, enveloped viruses and even transformed or cancerous cells with ostensibly limited chance to develop resistance [1,2,3,4,5,6,7]. These unique and diverse groups of molecules can be divided into various subgroups based on their amino acid composition and structure. A quantitative analysis of structure-activity relationship and elucidation of the mode of action using SMMD analogs may help to achieve this target
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