Structure-Activity Relationship and Mode of Action of a Frog Secreted Antibacterial Peptide B1CTcu5 Using Synthetically and Modularly Modified or Deleted (SMMD) Peptides

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.

Identification and characterization of novel host defense peptides from the skin secretion of the fungoid frog, Hydrophylax bahuvistara (Anura: Ranidae)

“…Strong inhibitory activity against MRSA and VRE may be a proof of the competence of HDPs against the multidrug‐resistant pathogens. These observations were in good agreement with the earlier studies on the peptides identified from Clinotarsus curtipes , Rana rugosa , and Glandirana emeljanovi …”

“…It was previously reported that C‐terminal amidation increases peptide activity and increases its hemolytic effect, which is inconsistent with our results. These peptides exhibit hemolytic activity similar to amidated brevinin1 isolated from C. curtipes , another endemic frog species of Western Ghats, where the percentage hemolysis is below 40% . Results of MTT assay (Figure ) revealed that both amidated and acidic brevinin1 peptides were highly toxic to the Hep 3B cancer cell line and less toxic to normal HEK cells (similar to the positive control, silymarin, a potential anticancer drug).…”

“…To access the cytolytic activity of both the acidic and amidated peptides, MTT assay was carried out on tumorous Hep 3B cell lines and normal human embryonic kidney (HEK) cell lines. Hep 3B and HEK cells were seeded in 96‐well plate at a concentration of 5000 cells per well and treated with varying concentrations of the peptides (2–100 μg/ml).…”

Modification Targeting the “Rana Box” Motif of a Novel Nigrocin Peptide From Hylarana latouchii Enhances and Broadens Its Potency Against Multiple Bacteria

“…This similarity of AMPs derived from different species indicates that they are likely to have an evolutionary origin, the significance of this characteristic structure on potency of AMPs against microbes remains to be elucidated, though. Previous paper reported that deletion of “Rana box” radically abolished the antimicrobial activity of AMP, while others dismissed its function, as the elimination or replacement of cysteine residue did not affect potency of AMPs (Kwon et al, 1998; Vignal et al, 1998; Abraham et al, 2015). …”

“…To illustrate, study on brevinin 1E amide suggested that the disulphide bridge was pivotal for the α-helical structure but not significant for its antimicrobial potency, as activity was unaffected even when cysteine residues are substituted by serine (Kwon et al, 1998). In contrast, the elimination of the heptapeptide motif in B1CTcu5, an AMP isolated from Clinotarsus curtipes , prominently reduced antibiotic and hemolytic activity of it (Abraham et al, 2015). Nigrocin is a representative anuran AMP family, and after sequence alignment between nigrocin-HL and other members of nigrocin family reported previously, a highly conserved heptapeptide motif “CGLXGLC” named “Rana box” at C-terminus was confirmed in the nigrocin family (Park et al, 2001; Conlon et al, 2006; Li et al, 2007; Che et al, 2008; Iwakoshi-Ukena et al, 2011b).…”