Mechanism of Action and Relationship Between Structure and Biological Activity of Ctx-Ha: A New Ceratotoxin-like Peptide from Hypsiboas albopunctatus

Abstract

The increase in bacterial resistance to current antibiotics has led to the development of new active molecules. We have isolated the antimicrobial peptide Ctx-Ha from the skin secretion of the frog Hypsiboas albopunctatus. The aim of the present work was to elucidate the mechanism of action of this new antimicrobial peptide. The sequence similarity with Ceratotoxin, the pore size, and the pore-like release of carboxyfluorescein from vesicles indicated that Ctx(Ile21)-Ha has a mechanism of action based on the barrel- stave model. In a second part of this work, we synthesized three analogues to provide information about the relationship between the peptide's structure and its biological activity. Ctx(Ile21)-Ha-VD 16, Ctx(Ile21)- Ha-VD 5,16 and Ctx(Ile21)-Ha-I9K were designed to disrupt the peptide's helical structure and change the hydrophobicity/ hydrophilicity and amphipathicity of the apolar face in order to uncouple the antimicrobial activity of Ctx(Ile21)-Ha from its hemolytic activity. To evaluate the effects of the amino acid substitutions on peptide conformation, secondary structure was accessed using CD measurements. The peptides presented a high amount of α-helical structure in the presence of TFE and LPC. The CD data showed that destruction of the amphipathic α-helix by the replacing isoleucine by lysine is less harmful to the structure than D-amino acid substitutions. Biological tests demonstrated that all peptides have activity. Nevertheless, the peptide Ctx(Ile21)-Ha-I9K showed the highest value of therapeutic index. Our findings suggest that these peptides are potential templates for the development of new antimicrobial drugs. These studies highlight the importance of single amino acid modification as a tool to modulate the biological activity of antimicrobial peptides.

Dynamics and Conformational Studies of TOAC Spin Labeled Analogues of Ctx(Ile21)-Ha Peptide from Hypsiboas albopunctatus

“…This peptide has shown antimicrobial activity against Gram positive and Gram negative bacteria and fungi. In addition, Ctx(Ile 21 )-Ha has shown high amounts of α-helical structure in the presence of TFE and LPC [8]. In this study, three analogues containing the paramagnetic amino acid TOAC strategically inserted in different positions of the sequence (Table I) were designed.…”

“…This peptide – called Ctx(Ile 21 )-Ha (ceratotoxin-like peptide from Hypsiboas albopunctatus ) – presents the following primary structure: Gly-Trp-Leu-Asp-Val-Ala-Lys-Lys-Ile-Gly-Lys-Ala-Ala-Phe-Asn-Val-Ala-Lys-Asn-Phe-(Ile/Leu) [7]. Interestingly, this sequence does not have similarity with other sequences found in amphibians, but it does have homology with the ceratotoxins peptide family, specifically ceratotoxin A [8]. The ceratotoxin peptide family exhibits biological activity against E. coli and other Gram negative bacteria, permeabilizing membranes and forming pores by a “barrel stave” mechanism [9].…”

“…The ceratotoxin peptide family exhibits biological activity against E. coli and other Gram negative bacteria, permeabilizing membranes and forming pores by a “barrel stave” mechanism [9]. Ctx(Ile 21 )-Ha showed biological activity against bacteria and fungi, including Candida [8]. This fungal species is involved in a variety of processes, such as mucocutaneous illnesses and invasive processes.…”

Evaluation of peptides release using a natural rubber latex biomembrane as a carrier

“…In both models, phospholipids are present. The interaction between antimicrobial peptides, such as Hylin a1, and phospholipid vesicles has been previously described (Brogden 2005;Ferreira Cespedes et al 2012;Lorenzón et al 2012). In this interaction, the peptides are initially bound parallel to a lipid bilayer, oriented perpendicular to the membrane (Brogden 2005;Melo et al 2009).…”

Brevinin-2 Drug Family—New Applied Peptide Candidates Against Methicillin-Resistant Staphylococcus aureus and Their Effects on Lys-7 Expression of Innate Immune Pathway DAF-2/DAF-16 in Caenorhabditis elegans

“…The lys-7 from C. elegans were separated on a 15% SDS-polyacrylamide gel. Western blot analysis was performed as previously described [45]. After a sample transfer onto a nitrocellulose membrane, the blot was blocked for 1 h at room temperature with 0.1% Tween-20 and 5% dry milk powder in tris-buffered saline (TBS) (150 mM NaCl, 10 mM KCl, 10 mM Tris-HCl pH 7.6) and then washed with TBS.…”