Promising approaches to optimize the biological properties of the antimicrobial peptide esculentin-1a(1-21)NH2. amino acids substitution and conjugation to nanoparticles

Antimicrobial peptides (AMPs) represent an interesting class of molecules with expanding biological properties which make them a viable alternative for the development of future antibiotic drugs. However, for this purpose, some limitations must be overcome: (i) the poor biostability due to enzymatic degradation; (ii) the cytotoxicity at concentrations slightly higher than the therapeutic dosages; and (iii) the inefficient delivery to the target site at effective concentrations. Recently, a derivative of the frog skin AMP esculentin-1a, named esculentin-1a(1-21)NH2, [Esc(1-21): GIFSKLAGKKIKNLLISGLKG-NH2] has been found to have a potent activity against the Gram-negative bacterium Pseudomonas aeruginosa; a slightly weaker activity against Gram-positive bacteria and interesting immunomodulatory properties. With the aim to optimize the antimicrobial features of Esc(1-21) and to circumvent the limitations described above, two different approaches were followed: (i) substitutions by non-coded amino acids, i.e., α-aminoisobutyric acid or d-amino acids; and (ii) peptide conjugation to gold nanoparticles. In this mini-review, we summarized the structural and functional properties of the resulting Esc(1-21)-derived compounds. Overall, our data may assist researchers in the rational design and optimization of AMPs for the development of future drugs to fight the worldwide problem of antibiotic resistance

Different approaches to optimize the antimicrobial properties of cationic peptides: substitution by non-coded amino acids and conjugation to nanoparticles

Antimicrobial peptides are small, cationic and amphipathic peptides produced by virtually all living organisms as key components of the innate immune system. Importantly, they hold promise for the development of new broad-spectrum anti-infective agents. These are urgently needed, due to the growing emergence of microorganisms that are resistant to the available therapeutic agents. In recent years, research has focused on the characterization and optimization of these molecules. Albeit thousands of peptides have been isolated and characterized, only a reduced number of them has entered into clinical phases, because of some limitations. Among them: (i) the poor peptide bioavailability, (ii) the toxicity at high doses (iii) the high susceptibility to proteolytic degradation (iv) the inefficient peptide delivery to the target site at high concentration. Nowadays, thanks to advances in biochemical research, computational studies and nanotechnologies it has become possible to overcome some of these problems. Recently, a frog-skin derived AMP, named Esc(1-21), has been investigated for its potent activity against the Gram-negative bacterium Pseudomonas aeruginosa and immunomodulatory properties. However, it showed a weaker activity against Gram-positive bacteria and some cytotoxic effect on mammalian cells. In this thesis work, with the aim to optimize the biological properties of AMPs, e.g. Esc(1-21), three different approaches have been used: - Design and synthesis of an analog of Esc(1-21) carrying three alpha-aminoisobutyric acid (Aib) residues. When inserted into the primary structure of peptides, Aib residues are expected to increase the alpha-helical content of the peptides, due to their strong helicogenicity. Importantly, a stabilized alpha-helical structure is known to be a crucial parameter to improve the AMPs’ activity against Gram-positive bacteria but also to increase their toxicity against mammalian cells. The Aib-containing peptide has been studied for its structural and biological properties and compared with the parent peptide Esc(1-21). - Design and synthesis of a diastereomer of Esc(1-21), named Esc(1-21)-1c, by replacing two L-amino acids in the C-terminal portion, i.e. L-Leu14 and L-Ser17, with the corresponding D-enantiomers. Besides making a peptide more resistant to proteolytic degradation, the insertion of D- amino acids is expected to disrupt the peptide’s alpha-helical content and to reduce its cytotoxicity. The effect(s) of D-amino acids incorporation on the structural/biological properties of the peptide have been investigated. - Design and production of two different types of nanoparticles to protect the AMP from proteolytic degradation and to allow its delivery to the target site at high concentration without altering the antimicrobial properties: (i) inorganic gold nanoparticles (AuNPs) coated with Esc(1-21) and (ii) nano-embedded microparticles for pulmonary delivery of a model cationic antimicrobial peptide, i.e. colistin. As expected, the Aib-analog of Esc(1-21) resulted to be more active against Gram-positive bacteria, but also more cytotoxic against mammalian cells, due to its higher alpha-helical content in the secondary structure. Differently, the insertion of two D-amino acids provoked a disruption of the alpha-helix and, as a consequence, a significant reduction in the peptide’s cytotoxic effect. In addition, the diastereomer resulted to be more active against the biofilm form of P. aeruginosa and more stable in human serum. The conjugation of Esc(1-21) to AuNPs, led to an increase of the antimicrobial activity of the peptide and to a greater stability to proteolytic degradation, without interfering with the mechanism of membrane perturbation. In addition, the conjugation onto AuNPs did not affect the wound-healing properties of the free peptide and AuNPs@Esc(1-21) were not found to be toxic against human keratinocytes. Finally, the engineered biocompatible and biodegradable polymeric microparticles designed as a delivery system for an AMP model, i.e. colistin, resulted to have a prolonged activity against P. aeruginosa biofilm in comparison with the free peptide. This was likely due to their ability to penetrate into bacterial biofilm and to sustain colistin release inside it. The promising results obtained by these different approaches, have made it possible to take a step forward in the optimization of a cationic peptide for the development of potential new antibacterial drugs

Antimicrobial peptides for novel antiviral strategies in the current post-COVID-19 pandemic

The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted how urgent and necessary the discovery of new antiviral compounds is for novel therapeutic approaches. Among the various classes of molecules with antiviral activity, antimicrobial peptides (AMPs) of innate immunity are among the most promising ones, mainly due to their different mechanisms of action against viruses and additional biological properties. In this review, the main physicochemical characteristics of AMPs are described, with particular interest toward peptides derived from amphibian skin. Living in aquatic and terrestrial environments, amphibians are one of the richest sources of AMPs with different primary and secondary structures. Besides describing the various antiviral activities of these peptides and the underlying mechanism, this review aims at emphasizing the high potential of these small molecules for the development of new antiviral agents that likely reduce the selection of resistant strains

Antimicrobial, cytotoxic and insulin‐releasing activities of the amphibian host‐defense peptide ocellatin‐3N and its L‐lysine‐substituted analogs

The host-defense peptide ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH2), first isolated from the Caribbean frog Leptodactylus nesiotus, inhibited growth of clinically relevant Gram-positive and Gram-negative bacteria as well as a strain of the major emerging yeast pathogen Candida parapsilosis. Increasing cationicity while maintaining amphipathicity by the substitution Asp(4)-> Lys increased potency against the microorganisms by between 4- and 16-fold (MIC <= 3 mu M) compared with the naturally occurring peptide. The substitution Ala(18)-> Lys and the double substitution Asp(4)-> Lys and Ala(18)-> Lys had less effects on potency. The [D4K] analog also showed 2.5- to 4-fold greater cytotoxic potency against non-small-cell lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells (LC50 values in the range of 12-20 mu M) compared with ocellatin-3N but was less hemolytic to mouse erythrocytes. However, the peptide showed no selectivity for tumor-derived cells [LC50 = 20 mu M for human umbilical vein endothelial cells (HUVECs)]. Ocellatin-3N and [D4K]ocellatin-3N stimulated the release of insulin from BRIN-BD11 clonal beta-cells at concentrations >= 1 nM, and [A18K]ocellatin-3N, at concentrations >= 0.1 nM. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 mu M, indicating that plasma membrane integrity had been preserved. The three peptides produced an increase in intracellular [Ca2+] in BRIN-BD11 cells when incubated at a concentration of 1 mu M. In view of its high insulinotropic potency and relatively low hemolytic activity, the [A18K] ocellatin analog may represent a template for the design of agents with therapeutic potential for the treatment of patients with type 2 diabetes

Gold-nanoparticles coated with the antimicrobial peptide esculentin-1a(1-21)NH2 as a reliable strategy for antipseudomonal drugs

Naturally occurring antimicrobial peptides (AMPs) hold promise as future therapeutics against multidrug resistant microorganisms. Recently, we have discovered that a derivative of the frog skin AMP esculentin-1a, Esc(1-21), is highly potent against both free living and biofilm forms of the bacterial pathogen Pseudomonas aeruginosa. However, bringing AMPs into clinics requires to overcome their low stability, high toxicity and inefficient delivery to the target site at high concentrations. Importantly, peptide conjugation to gold nanoparticles (AuNPs), which are among the most applied inorganic nanocarriers in biomedical sciences, represents a valuable strategy to solve these problems. Here we report that covalent conjugation of Esc(1-21) to soluble AuNPs AuNPs@Esc(1-21)] via a poly(ethylene glycol) linker increased by ~15-fold the activity of the free peptide against the motile and sessile forms of P. aeruginosa without being toxic to human keratinocytes. Furthermore, AuNPs@Esc(1-21) resulted to be significantly more resistant to proteolytic digestion and to disintegrate the bacterial membrane at very low concentration (5 nM). Finally, we demonstrated for the first time the capability of peptide-coated AuNPs to display a wound healing activity on a keratinocytes monolayer. Overall, these findings suggest that our engineered AuNPs can serve as attractive novel biological-derived material for topical treatment of epithelial infections and healing of the injured tissue. Statement of Significance Despite conjugation of AMPs to AuNPs represents a worthwhile solution to face some limitations for their development as new therapeutics, only a very limited number of studies is available on peptide-coated AuNPs. Importantly, this is the first report showing that a covalent binding of a linear AMP via a poly(ethylene glycol) linker to AuNPs highly enhances antipseudomonal activity, preserving the same mode of action of the free peptide, without being harmful. Furthermore, AuNPs@Esc(1-21) are expected to accelerate recovery of an injured skin layer. All together, these findings suggest our peptide-coated AuNPs as attractive novel nanoscale formulation to treat bacterial infections and to heal the injured tissue

KDEON WK-11: A short antipseudomonal peptide with promising potential

The plight of antimicrobial resistance continues to limit the availability of antibiotic treatment effective in combating resistant bacterial infections. Despite efforts made to rectify this issue and minimise its effects on both patients and the wider community, progress in this area remains minimal. Here, we de-novo designed a peptide named KDEON WK-11, building on previous work establishing effective residues and structures active in distinguished antimicrobial peptides such as lactoferrin. We assessed its antimicrobial activity against an array of bacterial strains and identified its most potent effect, against Pseudomonas aeruginosa with an MIC value of 3.12 mu M, lower than its counterparts developed with similar residues and chain lengths. We then determined its anti-biofilm properties, potential mechanism of action and in vitro cytotoxicity. We identified that KDEON WK-11 had a broad range of antimicrobial activity and specific capabilities to fight Pseudomonas aeruginosa with low in vitro cytotoxicity and promising potential to express anti-lipopolysaccharide qualities, which could be exploited to expand its properties into an anti-sepsis agent

Rapid Assessment of Susceptibility of Bacteria and Erythrocytes to Antimicrobial Peptides by Single-Cell Impedance Cytometry

Antimicrobial peptides (AMPs) represent a promising classof compoundsto fight antibiotic-resistant infections. In most cases, they killbacteria by making their membrane permeable and therefore exhibitlow propensity to induce bacterial resistance. In addition, they areoften selective, killing bacteria at concentrations lower than thoseat which they are toxic to the host. However, clinical applicationsof AMPs are hindered by a limited understanding of their interactionswith bacteria and human cells. Standard susceptibility testing methodsare based on the analysis of the growth of a bacterial populationand therefore require several hours. Moreover, different assays arerequired to assess the toxicity to host cells. In this work, we proposethe use of microfluidic impedance cytometry to explore the actionof AMPs on both bacteria and host cells in a rapid manner and withsingle-cell resolution. Impedance measurements are particularly well-suitedto detect the effects of AMPs on bacteria, due to the fact that themechanism of action involves perturbation of the permeability of cellmembranes. We show that the electrical signatures of Bacillus megaterium cells and human red blood cells(RBCs) reflect the action of a representative antimicrobial peptide,DNS-PMAP23. In particular, the impedance phase at high frequency (e.g.,11 or 20 MHz) is a reliable label-free metric for monitoring DNS-PMAP23bactericidal activity and toxicity to RBCs. The impedance-based characterizationis validated by comparison with standard antibacterial activity assaysand absorbance-based hemolytic activity assays. Furthermore, we demonstratethe applicability of the technique to a mixed sample of B. megaterium cells and RBCs, which paves the wayto study AMP selectivity for bacterial versus eukaryotic cells inthe presence of both cell types

Pulmonary Safety Profile of Esc Peptides and Esc-Peptide-Loaded Poly(lactide-co-glycolide) Nanoparticles: A Promising Therapeutic Approach for Local Treatment of Lung Infectious Diseases

In recent years, we have discovered Esc(1-21) and its diastereomer (Esc peptides) as valuable candidates for the treatment of Pseudomonas lung infection, especially in patients with cystic fibrosis (CF). Furthermore, engineered poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) were revealed to be a promising pulmonary delivery system of antimicrobial peptides. However, the "ad hoc" development of novel therapeutics requires consideration of their stability, tolerability, and safety. Hence, by means of electrophysiology experiments and preclinical studies on healthy mice, we demonstrated that neither Esc peptides or Esc-peptide-loaded PLGA NPs significantly affect the integrity of the lung epithelium, nor change the global gene expression profile of lungs of treated animals compared to those of vehicle-treated animals. Noteworthy, the Esc diastereomer endowed with the highest antimicrobial activity did not provoke any pulmonary pro-inflammatory response, even at a concentration 15-fold higher than the efficacy dosage 24 h after administration in the free or encapsulated form. The therapeutic index was ≥70, and the peptide was found to remain available in the bronchoalveolar lavage of mice, after two days of incubation. Overall, these studies should open an avenue for a new up-and-coming pharmacological approach, likely based on inhalable peptide-loaded NPs, to address CF lung disease

Novel Peptides with Dual Properties for Treating Pseudomonas aeruginosa Keratitis::Antibacterial and Corneal Wound Healing

The corneal epithelium is a layer in the anterior part of eye that contributes to light refraction onto the retina and to the ocular immune defense. Although an intact corneal epithelium is an excellent barrier against microbial pathogens and injuries, corneal abrasions can lead to devastating eye infections. Among them, Pseudomonas aeruginosa-associated keratitis often results in severe deterioration of the corneal tissue and even blindness. Hence, the discovery of new drugs able not only to eradicate ocular infections, which are often resistant to antibiotics, but also to elicit corneal wound repair is highly demanded. Recently, we demonstrated the potent antipseudomonal activity of two peptides, Esc(1-21) and its diastereomer Esc(1-21)-1c. In this study, by means of a mouse model of P. aeruginosa keratitis and an in vivo corneal debridement wound, we discovered the efficacy of these peptides, particularly Esc(1-21)-1c, to cure keratitis and to promote corneal wound healing. This latter property was also supported by in vitro cell scratch and ELISA assays. Overall, the current study highlights Esc peptides as novel ophthalmic agents for treating corneal infection and injury, being able to display a dual function, antimicrobial and wound healing, rarely identified in a single peptide at the same micromolar concentration range

A novel in vitro wound healing assay to evaluate cell migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 μm). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events