Deciphering the structure and mechanism of action of computer-designed mastoparan peptides.

Mastoparans are cationic peptides with multifunctional pharmacological properties. Mastoparan-R1 and mastoparan-R4 were computationally designed based on native mastoparan-L from wasps and have improved therapeutic potential for the control of bacterial infections. Here, we evaluated whether these peptides maintain their activity against Escherichia coli strains under a range of salt concentrations. We found that mastoparan-R1 and mastoparan-R4 preserved their activity under the conditions tested, including having antibacterial activities at physiological salt concentrations. The overall structure of the peptides was investigated using circular dichroism spectroscopy in a range of solvents. No significant changes in secondary structure were observed (random coil in aqueous solutions and α-helix in hydrophobic and anionic environments). The three-dimensional structures of mastoparan-R1 and mastoparan-R4 were elucidated through nuclear magnetic resonance spectroscopy, revealing amphipathic α-helical segments for Leu3-Ile13 (mastoparan-R1) and Leu3-Ile14 (mastoparan-R4). Possible membrane-association mechanisms for mastoparan-R1 and mastoparan-R4 were investigated through surface plasmon resonance and leakage studies with synthetic POPC and POPC/POPG (4:1) lipid bilayers. Mastoparan-L had the highest affinity for both membrane systems, whereas the two analogs had weaker association, but improved selectivity for lysing anionic membranes. This finding was also supported by molecular dynamics simulations, in which mastoparan-R1 and mastoparan-R4 were found to have greater interactions with bacteria-like membranes compared with model mammalian membranes. Despite having a few differences in their functional and structural profiles, the mastoparan-R1 analog stood out with the highest activity, greater bacteriostatic potential, and selectivity for lysing anionic membranes. This study reinforces the potential of mastoparan-R1 as a drug candidate.

Computer-Aided Design of Mastoparan-like Peptides Enables the Generation of Nontoxic Variants with Extended Antibacterial Properties.

Diverse peptides have been evaluated for their activity against pathogenic microorganisms. Here, five mastoparan variants were designed based on mastoparan-L, among which two (R1 and R4) were selected for in-depth analysis. Mastoparan-L (parent/control), R1, and R4 inhibited susceptible/resistant bacteria at concentrations ranging from 2 to 32 µM, whereas only R1 and R4 eradicated Pseudomonas aeruginosa biofilms at 16 µM. Moreover, the toxic effects of mastoparan-L toward mammalian cells were drastically reduced in both variants. In skin infections, R1 at 64 µM was the most effective variant, reducing P. aeruginosa bacterial counts 1000 times on day 4 post-infection. Structurally, all of the peptides showed varying levels of helicity and structural stability in aqueous and membrane-like conditions, which may affect the different bioactivities observed here. By computationally modifying the physicochemical properties of R1 and R4, we reduced the cytotoxicity and optimized the therapeutic potential of these mastoparan-like peptides both in vitro and in vivo.

Short Cationic Peptide Derived from Archaea with Dual Antibacterial Properties and Anti-Infective Potential.

Bacterial biofilms and associated infections represent one of the biggest challenges in the clinic, and as an alternative to counter bacterial infections, antimicrobial peptides have attracted great attention in the past decade. Here, ten short cationic antimicrobial peptides were generated through a sliding-window strategy on the basis of the 19-amino acid residue peptide, derived from a Pyrobaculum aerophilum ribosomal protein. PaDBS1R6F10 exhibited anti-infective potential as it decreased the bacterial burden in murine Pseudomonas aeruginosa cutaneous infections by more than 1000-fold. Adverse cytotoxic and hemolytic effects were not detected against mammalian cells. The peptide demonstrated structural plasticity in terms of its secondary structure in the different environments tested. PaDBS1R6F10 represents a promising antimicrobial agent against bacteria infections, without harming human cells.

A Computationally Designed Peptide Derived from Escherichia coli as a Potential Drug Template for Antibacterial and Antibiofilm Therapies.

Computer-aided screening of antimicrobial peptides (AMPs) is a promising approach for discovering novel therapies against multidrug-resistant bacterial infections. Here, we functionally and structurally characterized an Escherichia coli-derived AMP (EcDBS1R5) previously designed through pattern identification [α-helical set (KK[ILV](3)[AILV])], followed by sequence optimization. EcDBS1R5 inhibited the growth of Gram-negative and Gram-positive, susceptible and resistant bacterial strains at low doses (2-32 µM), with no cytotoxicity observed against non-cancerous and cancerous cell lines in the concentration range analyzed (<100 µM). Furthermore, EcDBS1R5 (16 µM) acted on Pseudomonas aeruginosa pre-formed biofilms by compromising the viability of biofilm-constituting cells. The in vivo antibacterial potential of EcDBS1R5 was confirmed as the peptide reduced bacterial counts by two-logs 2 days post-infection using a skin scarification mouse model. Structurally, circular dichroism analysis revealed that EcDBS1R5 is unstructured in hydrophilic environments, but has strong helicity in 2,2,2-trifluoroethanol (TFE)/water mixtures (v/v) and sodium dodecyl sulfate (SDS) micelles. The TFE-induced nuclear magnetic resonance structure of EcDBS1R5 was determined and showed an amphipathic helical segment with flexible termini. Moreover, we observed that the amide protons for residues Met2-Ala8, Arg10, Ala13-Ala16, and Trp19 in EcDBS1R5 are protected from the solvent, as their temperature coefficients values are more positive than -4.6 ppb·K-1. In summary, this study reports a novel dual-antibacterial/antibiofilm α-helical peptide with therapeutic potential in vitro and in vivo against clinically relevant bacterial strains.