An N-capping asparagine-lysine-proline (NKP) motif contributes to a hybrid flexible/stable multifunctional peptide scaffold.

Structural diversity drives multiple biological activities and mechanisms of action in linear peptides. Here we describe an unusual N-capping asparagine-lysine-proline (NKP) motif that confers a hybrid multifunctional scaffold to a computationally designed peptide (PaDBS1R7). PaDBS1R7 has a shorter α-helix segment than other computationally designed peptides of similar sequence but with key residue substitutions. Although this motif acts as an α-helix breaker in PaDBS1R7, the Asn5 presents exclusive N-capping effects, forming a belt to establish hydrogen bonds for an amphipathic α-helix stabilization. The combination of these different structural profiles was described as a coil/N-cap/α-helix scaffold, which was also observed in diverse computational peptide mutants. Biological studies revealed that all peptides displayed antibacterial activities. However, only PaDBS1R7 displayed anticancer properties, eradicated Pseudomonas aeruginosa biofilms, decreased bacterial counts by 100-1000-fold in vivo, reduced lipopolysaccharide-induced macrophages stress, and stimulated fibroblast migration for wound healing. This study extends our understanding of an N-capping NKP motif to engineering hybrid multifunctional peptide drug candidates with potent anti-infective and immunomodulatory properties.

Snake Venom Cathelicidins as Natural Antimicrobial Peptides.

Bioactive small molecules isolated from animals, plants, fungi and bacteria, including natural antimicrobial peptides, have shown great therapeutic potential worldwide. Among these peptides, snake venom cathelicidins are being widely exploited, because the variation in the composition of the venom reflects a range of biological activities that may be of biotechnological interest. Cathelicidins are short, cationic, and amphipathic molecules. They play an important role in host defense against microbial infections. We are currently facing a strong limitation on pharmacological interventions for infection control, which has become increasingly complex due to the lack of effective therapeutic options. In this review, we will focus on natural snake venom cathelicidins as promising candidates for the development of new antibacterial agents to fight antibiotic-resistant bacteria. We will highlight their antibacterial and antibiofilm activities, mechanism of action, and modulation of the innate immune response.

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.

Computer-Aided Design of Antimicrobial Peptides: Are We Generating Effective Drug Candidates?

Antimicrobial peptides (AMPs), especially antibacterial peptides, have been widely investigated as potential alternatives to antibiotic-based therapies. Indeed, naturally occurring and synthetic AMPs have shown promising results against a series of clinically relevant bacteria. Even so, this class of antimicrobials has continuously failed clinical trials at some point, highlighting the importance of AMP optimization. In this context, the computer-aided design of AMPs has put together crucial information on chemical parameters and bioactivities in AMP sequences, thus providing modes of prediction to evaluate the antibacterial potential of a candidate sequence before synthesis. Quantitative structure-activity relationship (QSAR) computational models, for instance, have greatly contributed to AMP sequence optimization aimed at improved biological activities. In addition to machine-learning methods, the de novo design, linguistic model, pattern insertion methods, and genetic algorithms, have shown the potential to boost the automated design of AMPs. However, how successful have these approaches been in generating effective antibacterial drug candidates? Bearing this in mind, this review will focus on the main computational strategies that have generated AMPs with promising activities against pathogenic bacteria, as well as anti-infective potential in different animal models, including sepsis and cutaneous infections. Moreover, we will point out recent studies on the computer-aided design of antibiofilm peptides. As expected from automated design strategies, diverse candidate sequences with different structural arrangements have been generated and deposited in databases. We will, therefore, also discuss the structural diversity that has been engendered.

Comparative transcriptome analyses of magainin I-susceptible and -resistant Escherichia coli strains.

Antimicrobial peptides (AMPs) have attracted considerable attention because of their multiple and complex mechanisms of action toward resistant bacteria. However, reports have increasingly highlighted how bacteria can escape AMP administration. Here, the molecular mechanisms involved in Escherichia coli resistance to magainin I were investigated through comparative transcriptomics. Sub-inhibitory concentrations of magainin I were used to generate four experimental groups, including magainin I-susceptible E. coli, in the absence (C) and presence of magainin I (CM); and magainin I-resistant E. coli in the absence (R) and presence of magainin I (RM). The total RNA from each sample was extracted; cDNA libraries were constructed and further submitted for Illumina MiSeq sequencing. After RNA-seq data pre-processing and functional annotation, a total of 103 differentially expressed genes (DEGs) were identified, mainly related to bacterial metabolism. Moreover, down-regulation of cell motility and chaperone-related genes was observed in CM and RM, whereas cell communication, acid tolerance and multidrug efflux pump genes (ABC transporter, major facilitator and resistance-nodulation cell division superfamilies) were up-regulated in these same groups. DEGs from the C and R groups are related to basal levels of expression of homeostasis-related genes compared to CM and RM, suggesting that the presence of magainin I is required to change the transcriptomics panel in both C and R E. coli strains. These findings show the complexity of E. coli resistance to magainin I through the rearrangement of several metabolic pathways involved in bacterial physiology and drug response, also providing information on the development of novel antimicrobial strategies targeting resistance-related transcripts and proteins herein described.

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.

An acidic model pro-peptide affects the secondary structure, membrane interactions and antimicrobial activity of a crotalicidin fragment.

In order to study how acidic pro-peptides inhibit the antimicrobial activity of antimicrobial peptides, we introduce a simple model system, consisting of a 19 amino-acid long antimicrobial peptide, and an N-terminally attached, 10 amino-acid long acidic model pro-peptide. The antimicrobial peptide is a fragment of the crotalicidin peptide, a member of the cathelidin family, from rattlesnake venom. The model pro-peptide is a deca (glutamic acid). Attachment of the model pro-peptide only leads to a moderately large reduction in the binding to- and induced leakage of model liposomes, while the antimicrobial activity of the crotalicidin fragment is completely inhibited by attaching the model pro-peptide. Attaching the pro-peptide induces a conformational change to a more helical conformation, while there are no signs of intra- or intermolecular peptide complexation. We conclude that inhibition of antimicrobial activity by the model pro-peptide might be related to a conformational change induced by the pro-peptide domain, and that additional effects beyond induced changes in membrane activity must also be involved.

The Structure/Function Relationship in Antimicrobial Peptides: What Can we Obtain From Structural Data?

Antimicrobial peptides (AMPs) have been widely isolated from most organisms in nature. This class of antimicrobials may undergo changes in their sequence for improved physicochemical properties, including charge, hydrophobicity, and hydrophobic moment. It is known that such properties may be directly associated with AMPs' structural arrangements and, consequently, could interfere in their modes of action against microorganisms. In this scenario, biophysical methodologies, such as nuclear magnetic resonance spectroscopy, X-ray crystallography, and cryo-electron microscopy, allied to in silico approaches, including molecular modeling, docking, and dynamics nowadays represent an enormous first step for the structural elucidation of AMPs, leading to further structure-function annotation. In this context, this chapter will focus on the main atomic-level experimental and computational tools used for the structural elucidation of AMPs that have assisted in the investigation of their functions.