Mário R. Felício

New frontiers for anti-biofilm drug development

Pathogenic microbial biofilm, a consortium of microbial cells protected by a self-produced polymer matrix, is considered a worldwide challenge due to the inherent antibiotic resistance conferred by its lifestyle. Living, as it does, in a community of microbial organisms in a clinical situation, makes it responsible for severe and dangerous cases of infection. Combating this organisation of cells usually requires high antibiotic doses for a prolonged time, and these approaches often fail, contributing to infection persistence. In addition to therapeutic limitations, biofilms can be a source of infections when they grow in medical devices. The challenge imposed by biofilms has mobilised researchers in the entire world to prospect or develop alternatives to control biofilms. In this context, this review summarises the new frontiers that could be used in clinical circumstances in order to prevent or eliminate pathogenic biofilms.

Peptides with Dual Antimicrobial and Anticancer Activities

In recent years, the number of people suffering from cancer and multi-resistant infections has increased, such that both diseases are already seen as current and future major causes of death. Moreover, chronic infections are one of the main causes of cancer, due to the instability in the immune system that allows cancer cells to proliferate. Likewise, the physical debility associated with cancer or with anticancer therapy itself often paves the way for opportunistic infections. It is urgent to develop new therapeutic methods, with higher efficiency and lower side effects. Antimicrobial peptides (AMPs) are found in the innate immune system of a wide range of organisms. Identified as the most promising alternative to conventional molecules used nowadays against infections, some of them have been shown to have dual activity, both as antimicrobial and anticancer peptides (ACPs). Highly cationic and amphipathic, they have demonstrated efficacy against both conditions, with the number of nature-driven or synthetically designed peptides increasing year by year. With similar properties, AMPs that can also act as ACPs are viewed as future chemotherapeutic drugs, with the advantage of low propensity to resistance, which started this paradigm in the pharmaceutical market. These peptides have already been described as molecules presenting killing mechanisms at the membrane level, but also acting toward intracellular targets, which increases their success compartively to one-target specific drugs. This review will approach the desirable characteristics of small peptides that demonstrated dual activity against microbial infections and cancer, as well as the peptides engaged in clinical trials.

A polyalanine peptide derived from polar fish with anti-infectious activities

Due to the growing concern about antibiotic-resistant microbial infections, increasing support has been given to new drug discovery programs. A promising alternative to counter bacterial infections includes the antimicrobial peptides (AMPs), which have emerged as model molecules for rational design strategies. Here we focused on the study of Pa-MAP 1.9, a rationally designed AMP derived from the polar fish Pleuronectes americanus. Pa-MAP 1.9 was active against Gram-negative planktonic bacteria and biofilms, without being cytotoxic to mammalian cells. By using AFM, leakage assays, CD spectroscopy and in silico tools, we found that Pa-MAP 1.9 may be acting both on intracellular targets and on the bacterial surface, also being more efficient at interacting with anionic LUVs mimicking Gram-negative bacterial surface, where this peptide adopts α-helical conformations, than cholesterol-enriched LUVs mimicking mammalian cells. Thus, as bacteria present varied physiological features that favor antibiotic-resistance, Pa-MAP 1.9 could be a promising candidate in the development of tools against infections caused by pathogenic bacteria.

Structural and functional evaluation of the palindromic alanine-rich antimicrobial peptide Pa-MAP2.

Recently, several peptides have been studied regarding the defence process against pathogenic microorganisms, which are able to act against different targets, with the purpose of developing novel bioactive compounds. The present work focuses on the structural and functional evaluation of the palindromic antimicrobial peptide Pa-MAP2, designed based on the peptide Pa-MAP from Pleuronectes americanus. For a better structural understanding, molecular modelling analyses were carried out, together with molecular dynamics and circular dichroism, in different media. Antibacterial activity against Gram-negative and positive bacteria was evaluated, as well as cytotoxicity against human erythrocytes, RAW 264.7, Vero and L6 cells. In silico docking experiments, lipid vesicle studies, and atomic force microscopy (AFM) imaging were carried out to explore the activity of the peptide. In vivo studies on infected mice were also done. The palindromic primary sequence favoured an α-helix structure that was pH dependent, only present on alkaline environment, with dynamic N- and C-terminals that are stabilized in anionic media. Pa-MAP2 only showed activity against Gram-negative bacteria, with a MIC of 3.2 μM, and without any cytotoxic effect. In silico, lipid vesicles and AFM studies confirm the preference for anionic lipids (POPG, POPS, DPPE, DPPG and LPS), with the positively charged lysine residues being essential for the initial electrostatic interaction. In vivo studies showed that Pa-MAP2 increases to 100% the survival rate of mice infected with Escherichia coli. Data here reported indicated that palindromic Pa-MAP2 could be an alternative candidate for use in therapeutics against Gram-negative bacterial infections.

Psd1 Effects on Candida albicans Planktonic Cells and Biofilms

Candida albicans is an important human pathogen, causing opportunistic infections. The adhesion of planktonic cells to a substrate is the first step for biofilm development. The antimicrobial peptide (AMP) Psd1 is a defensin isolated from Pisum sativum seeds. We tested the effects of this AMP on C. albicans biofilms and planktonic cells, comparing its activity with amphotericin B and fluconazole. Three C. albicans variants were studied, one of them a mutant deficient in glucosylceramide synthase, conferring resistance to Psd1 antifungal action. Atomic force microscopy (AFM) was used to assess morphological and biomechanical changes on fungal cells. Surface alterations, with membrane disruption and leakage of cellular contents, were observed. Cytometry assays and confocal microscopy imaging showed that Psd1 causes cell death, in a time and concentration-dependent manner. These results demonstrate Psd1 pleiotropic action against a relevant fungal human pathogen, suggesting its use as natural antimycotic agent.

Application of Light Scattering Techniques to Nanoparticle Characterization and Development

Over the years, the scientific importance of nanoparticles for biomedical applications has increased. The high stability and biocompatibility, together with the low toxicity of the nanoparticles developed lead to their use as targeted drug delivery systems, bioimaging systems, and biosensors. The wide range of nanoparticles size, from 10 nm to 1 μm, as well as their optical properties, allow them to be studied using microscopy and spectroscopy techniques. In order to be effectively used, the physicochemical properties of nanoparticle formulations need to be taken into account, namely, particle size, surface charge distribution, surface derivatization and/or loading capacity, and related interactions. These properties need to be optimized considering the final nanoparticle intended biodistribution and target. In this review, we cover light scattering based techniques, namely dynamic light scattering and zeta-potential, used for the physicochemical characterization of nanoparticles. Dynamic light scattering is used to measure nanoparticles size, but also to evaluate their stability over time in suspension, at different pH and temperature conditions. Zeta-potential is used to characterize nanoparticles surface charge, obtaining information about their stability and surface interaction with other molecules. In this review, we focus on nanoparticle characterization and application in infection, cancer and cardiovascular diseases.

Fast and potent bactericidal membrane lytic activity of PaDBS1R1, a novel cationic antimicrobial peptide

Antimicrobial peptides (AMPs) are promising candidates for the development of future antibiotics. In an attempt to increase the efficacy of therapeutic AMPs, computer-based design methods appear as a reliable strategy. In this study, we evaluated the antimicrobial efficiency and mechanism of action of a novel designed AMP named PaDBS1R1, previously designed by means of the Joker algorithm, using a fragment of the ribosomal protein L39E from the archaeon Pyrobaculum aerophilum as a template. PaDBS1R1 displayed low micromolar broad-spectrum antimicrobial activity against Gram-negative (MIC of 1.5 μM) and Gram-positive (MIC of 3 μM) bacteria, including carbapenem-resistant Klebsiella pneumoniae (MIC of 6.25 μM) and methicillin-resistant Staphylococcus aureus (MIC of 12.5 μM), without cytotoxicity towards HEK-293 cells. In addition, membrane permeabilization and depolarization assays, combined with time-kill studies and FEG-SEM imaging, indicated a fast membrane permeation and further leakage of intracellular content. Biophysical studies with lipid vesicles show a preference of PaDBS1R1 for Gram-negative bacteria-like membranes. We investigated the three-dimensional structure of PaDBS1R1 by CD and NMR analyses. Our results suggest that PaDBS1R1 adopts an amphipathic α-helix upon interacting with hydrophobic environments, after an initial electrostatic interaction with negative charges, suggesting a membrane lytic effect. This study reveals that PaDBS1R1 has potential application in antibiotic therapy.

Corrigendum: A polyalanine peptide derived from polar fish with anti-infectious activities.

Scientific Reports 6: Article number: 2138510.1038/srep21385; published online: February 26 2016; updated: July 20 2016 In this Article, there is a typographical error in the Results section. “Pa-MAP 1.9 (NH2-LAAKLTKAATKLTAALTKLAAALT-COOH) was designed …” should read: “Pa-MAP 1.9 (NH2-LAAKLTKAATKLTAALTKLAAALTAAAT-COOH) was designed …”