Comparing activity, toxicity and model membrane interactions of Jelleine-I and Trp/Arg analogs: analysis of peptide aggregation.

Increasing resistance in antibiotic and chemotherapeutic treatments has been pushing studies of design and evaluation of bioactive peptides. Designing relies on different approaches from minimalist sequences and endogenous peptides modifications to computational libraries. Evaluation relies on microbiological tests. Aiming a deeper understanding, we chose the octapeptide Jelleine-I (JI) for its selective and low toxicity profile, designed small modifications combining the substitutions of Phe by Trp and Lys/His by Arg and tested the antimicrobial and anticancer activity on melanoma cells. Biophysical methods identified environment-dependent modulation of aggregation, but critical aggregation concentrations of JI and analogs in buffer show that peptides start membrane interactions as monomers. The presence of model membranes increases or reduces the partial aggregation of peptides. Compared to JI, analog JIF2WR shows the lowest tendency to aggregation on bacterial model membranes. JI and analogs are lytic to model membranes. Their composition-dependent performance indicates preference for the higher charged anionic bilayers in line with their superior performance toward Staphylococcus aureus and Streptococcus pneumoniae. JIF2WR presented the higher partitioning, higher lytic activity and lower aggregated contents. Despite these increased membranolytic activities, JIF2WR exhibited comparable antimicrobial activity in relation to JI at the expenses of some loss in selectivity. We found that the substitution Phe/Trp (JIF2W) tends to decrease antimicrobial but to increase anticancer activity and aggregation on model membranes and the toxicity toward human cells. However, the concomitant substitution Lys/His by Arg (JIF2WR) modulates some of these tendencies, increasing both the antimicrobial and the anticancer activity while decreasing the aggregation tendency.

Membrane targeting peptides toward antileishmanial activity: Design, structural determination and mechanism of interaction.

BACKGROUND: Leishmaniasis threatens poor areas population worldwide, requiring new drugs less prone to resistance development. Antimicrobial peptides with antileishmanial activity are considered among fulfilling alternatives, but not much is known about the mode of action of membrane-targeting peptides, considering promastigote and infected macrophage membranes. In a previous work, structural features of very active known peptides were prospected using molecular dynamics simulations. METHODS: Combining sequences of these peptides, analogs were designed. The structure of analog DecP-11 was validated by NMR. In vitro bioassays determined the peptide cytotoxicity toward mammalian cells, IC50 values on promastigotes and amastigotes, and membranolytic activity compared to Decoralin, one of the parent peptides. With biophysical methods, the mechanism of interaction with membrane mimetic systems was investigated. RESULTS: The designed peptide exhibits potent cytolytic and membrane permeabilizing activities, and decreased antileishmanial activity compared to the parent peptide. Interactions with lipid bilayers mimicking those of promastigotes, infected macrophage and mammalian cells showed that these peptides strongly bind to vesicles with intense lytic activity at low concentrations. Additionally, circular dichroism and light scattering experiments showed changes in the secondary structure of peptides and in vesicle size, depending on vesicles compositions. Altogether they suggest that DecP-11 antileishmanial activity is impaired by the aggregation and that aminophospholipids are probably involved. CONCLUSIONS: DecP-11 potent cytolytic and membranolytic activities with lack of selectivity toward promastigote model membranes warrant further structural studies to improve selectivity. GENERAL SIGNIFICANCE: Strong interactions of peptides with aminophospholipids, abundant in parasite membranes, potentially lead to aggregated forms impairing activity.

Polydim-I antimicrobial activity against MDR bacteria and its model membrane interaction.

The rapid spread of multi-drug resistant pathogens represents a serious threat to public health, considering factors such as high mortality rates, treatment restrictions and high prevalence of multi-drug resistant bacteria in the hospital environment. Antimicrobial peptides (AMPs) may exhibit powerful antimicrobial activity against different and diverse microorganisms, also presenting the advantage of absence or low toxicity towards animal cells. In this study, the evaluation of the antimicrobial activity against multi-drug resistant bacteria of a recently described AMP from wasp, Polydim-I, was performed. Polydim-I presented activity against standard strains (non-carriers of multi-resistant genes) that are susceptible to commercial antimicrobials, and also against multi-drug resistant strains at concentrations bellow 1µg/ml (0.41 µM). This is a rather low concentration among those reported for AMPs. At this concentration we found out that Polydim-I inhibits almost 100% of the tested pathogens growth, while with the ATCC strains the minimum inhibitory concentration (MIC100) is 400 times higher. Also, in relation to in vitro activity of conventional drugs against multi-drug resistant bacteria strains, Polydim-I is almost 10 times more efficient and with broader spectrum. Cationic AMPs are known as multi-target compounds and specially for targeting the phospholipid matrix of bacterial membranes. Exploring the interactions of Polydim-I with lipid bilayers, we have confirmed that this interaction is involved in the mechanism of action. Circular dichroism experiments showed that Polydim-I undergoes a conformational transition from random coil to a mostly helical conformation in the presence of membrane mimetic environments. Zeta potential measurements confirmed the binding and partial charge neutralization of anionic asolectin vesicles, and also suggested a possible aggregation of peptide molecules. FTIR experiments confirmed that some peptide aggregation occurs, which is minimized in the presence of strongly anionic micelles of sodium dodecyl sulfate. Also, Polydim-I induced channel-like structures formation to asolectin lipid bilayers, as demonstrated in the electrophysiology experiments. We suggest that cationic Polydim-I targets the membrane lipids due to electrostatic attraction, partially accumulates, neutralizing the opposite charges and induces pore formation. Similar mechanism of action has already been suggested for other peptides from wasp venoms, especially mastoparans.