Benzyl stapled modification and anticancer activity of antimicrobial peptide A4K14-Citropin 1.1.

A4K14-Citropin 1.1 (GLFAVIKKVASVIKGL-NH2) is a derived antimicrobial peptide (AMP) with a more stable α-helical structure at the C-terminal compared to prototype Citropin 1.1 which was obtained from glandular skin secretions of Australian freetail lizards. In a previous report, A4K14-Citropin 1.1 has been considered as an anti-cancer lead compound. However, linear peptides are difficult to maintain stable secondary structure, resulted in poor pharmacokinetic properties. In this study, we designed and synthesized a series of benzyl-stapled derivatives of A4K14-Citropin 1.1. And their physical and chemical properties, as well as biological activity, were both explored. The result showed that AC-CCSP-2-o and AC-CCSP-3-o exhibited a higher degree of helicity and greater anti-cancer activity compared with the prototype peptide. Besides, there was no significant difference in the hemolytic effect between the stapled peptides and the prototype peptide. AC-CCSP-2-o and AC-CCSP-3-o could serve as promising anti-cancer lead compounds for the novel anti-cancer drug development.

Counter-ion effect on antistaphylococcal activity and cytotoxicity of selected antimicrobial peptides.

In view of an appreciable increase in resistance of Staphylococcus aureus to the conventional antibiotics, it is desired to develop new effective drugs. Antimicrobial peptides (AMPs) seem to be attractive candidates. In general, AMPs samples used for in vitro studies consist of a peptide, counter-ion, and water. The presence of the counter-ion could be significant as it affects peptide secondary structure and biological activity. The purpose of this study was to estimate the impact of counter-ion on antistaphylococcal activity of selected AMPs (CAMEL, citropin 1.1, LL-37, pexiganan, temporin A). To do this, three kinds of salts were prepared, namely, acetates, hydrochlorides, and trifluoroacetates. In addition, the hemolytic activity against human red blood cells (hRBCs) and cytotoxicity (HaCaT) were determined. The results indicate that there is a substantial difference between different salts, but the pattern is not consistent for the peptides. In general, the antistaphylococcal activity decreased in the order: CAMEL > temporin A > pexiganan > citropin 1.1 â‰« LL-37. The highest selectivity indexes were determined for CAMEL hydrochloride, pexiganan acetate, and temporin A trifluoroacetate. This study shows how important is to take into account the kind of counter-ions when designing novel peptide-based antimicrobials.

Comparison of reversible membrane destabilisation induced by antimicrobial peptides derived from Australian frogs.

The membrane destabilising properties of the antimicrobial peptides (AMP) aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1, have been studied by dual polarisation interferometry (DPI). The overall process of peptide induced membrane destabilisation was examined by the changes in bilayer order as a function of membrane-bound peptide mass per unit area and revealed three different modes of action. Aurein 1.2 was the only peptide that significantly destabilised the neutral membrane (DMPC), while all four peptides induced destabilisation of the negatively charged membrane (DMPC/DMPG). On DMPC, citropin 1.1, maculatin 1.1 and caerin 1.1 bound irreversibly at low concentrations but caused a reversible drop in the bilayer order. In contrast to DMPC/DMPG, these three peptides caused a mass drop at the higher concentrations, which may correspond to insertion and bilayer expansion. The critical level of bound peptide necessary to induce membrane destabilisation (peptide:lipid ratio) was determined and correlated with peptide structure. As the most lytic peptide, aurein 1.2 adsorbed strongly prior to dissolution of the bilayer. In contrast, the binding of citropin 1.1, maculatin 1.1 and caerin 1.1 needed to reach a critical level prior to insertion into the membrane and incremental expansion and disruption. Our results demonstrate that sequential events can be monitored in real-time under fluidic conditions to elucidate the complex molecular mechanism of AMP action. In particular, the analysis of birefringence in real time allows the description of a detailed mechanistic model of the impact of peptides on the membrane bilayer order. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Design, Synthesis, and Antitumor Activities Study of Stapled A4K14-Citropin 1.1 Peptides.

A4K14-citropin 1.1 is a structurally optimized derivative derived from amphibians' skin secreta peptide Citropin, which exhibits broad biological activities. However, the application of A4K14-citropin 1.1 as a cancer therapeutic is restricted by its structural flexibility. In this study, a series of all-hydrocarbon stapled peptides derivatives of A4K14-citropin 1.1 were designed and synthesized, and their chemical and biological characteristics were also investigated. Among them, A4K14-citropin 1.1-Sp1 and A4K14-citropin 1.1-Sp4 displayed improved helicity levels, greater protease stability, and increased antitumor activity compared with the original peptide, which establishes them as promising lead compounds for novel cancer therapeutics development. These results revealed the important influence of all-hydrocarbon stapling side chain on the secondary structure, hydrolase stability, and biological activity of A4K14-citropin 1.1.

Differential activity of lytic α-helical peptides on lactobacilli and lactobacilli-derived liposomes.

Eukaryotic antimicrobial peptides (AMPs) interact with plasma membrane of bacteria, fungi and eukaryotic parasites. Noteworthy, Lactobacillus delbrueckii subsp. lactis (CIDCA 133) and L. delbrueckii subsp. bulgaricus (CIDCA 331) show different susceptibility to human beta-defensins (ß-sheet peptides). In the present work we extended the study to α-helical peptides from anuran amphibian (Aurein 1.2, Citropin 1.1 and Maculatin 1.1). We studied the effect on whole bacteria and liposomes formulated with bacterial lipids through growth kinetics, flow cytometry, leakage of liposome content and studies of peptide insertion in lipid monolayers. Growth of strain CIDCA 331 was dramatically inhibited in the presence of all three peptides and minimal inhibitory concentrations were lower than those for strain CIDCA 133. Flow cytometry revealed that AMPs lead to the permeabilization of bacteria. In addition, CIDCA 331-derived liposomes showed high susceptibility, leading to content leakage and structural disruption. Accordingly, peptide insertion in lipid monolayers demonstrated spontaneous interaction of AMPs with CIDCA 331 lipids. In contrast, lipids monolayers from strain CIDCA 133 were less susceptible. Summarizing we demonstrate that the high resistance of the probiotic strain CIDCA 133 to AMPs extends to α helix peptides Aurein, Citropin and Maculatin. This behavior could be ascribed in part to differences in membrane composition. These findings, along with the previously demonstrated resistance to ß defensins from human origin, suggest that strain CIDCA 133 is well adapted to host innate immune effectors from both mammals and amphibians thus indicating conserved mechanisms of interaction with key components of the innate immune system.

Citropin 1.1 Trifluoroacetate to Chloride Counter-Ion Exchange in HCl-Saturated Organic Solutions: An Alternative Approach.

In view of the increasing interest in peptides in various market sectors, a stronger emphasis on topics related to their production has been seen. Fmoc-based solid phase peptide synthesis, although being fast and efficient, provides final products with significant amounts of trifluoroacetate ions in the form of either a counter-ion or an unbound impurity. Because of the proven toxicity towards cells and peptide activity inhibition, ion exchange to more biocompatible one is purposeful. Additionally, as most of the currently used counter-ion exchange techniques are time-consuming and burdened by peptide yield reduction risk, development of a new approach is still a sensible solution. In this study, we examined the potential of peptide counter-ion exchange using non-aqueous organic solvents saturated with HCl. Counter-ion exchange of a model peptide, citropin 1.1 (GLFDVIKKVASVIGGL-NH2), for each solvent was conducted through incubation with subsequent evaporation under reduced pressure, dissolution in water and lyophilization. Each exchange was performed four times and compared to a reference method-lyophilization of the peptide from an 0.1 M HCl solution. The results showed superior counter-ion exchange efficiency for most of the organic solutions in relation to the reference method. Moreover, HCl-saturated acetonitrile and tert-butanol provided a satisfying exchange level after just one repetition. Thus, those two organic solvents can be potentially introduced into routine peptide counter-ion exchange.

A Novel Delivery System for the Controlled Release~of Antimicrobial Peptides: Citropin 1.1 and Temporin A.

Antimicrobial peptides (AMPs) are prospective therapeutic options for treating multiple-strain infections. However, clinical and commercial development of AMPs has some limitations due to their limited stability, low bioavailability, and potential hemotoxicity. The purpose of this study was to develop new polymeric carriers as highly controlled release devices for amphibian peptides citropin 1.1 (CIT) and temporin A (TEMP). The release rate of the active pharmaceutical ingredients (APIs) was strongly dependent on the API characteristics and the matrix microstructure. In the current work, we investigated the effect of the polymer microstructure on in vitro release kinetics of AMPs. Non-contact laser profilometry, scanning electron microscopy (SEM), and differential scanning calorimetry (DSC) were used to determine the structural changes during matrix degradation. Moreover, geno- and cytotoxicity of the synthesized new carriers were evaluated. The in vitro release study of AMPs from the obtained non-toxic matrices shows that peptides were released with near-zero-order kinetics. The peptide "burst release" effect was not observed. New devices have reached the therapeutic concentration of AMPs within 24 h and maintained it for 28 days. Hence, our results suggest that these polymeric devices could be potentially used as therapeutic options for the treatment of local infections.