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Antivirulence strategies aim to target pathogenicity factors while bypassing the pressure on the bacterium to develop resistance. The MgtC membrane protein has been proposed as an attractive target that is involved in the ability of several major bacterial pathogens, including Pseudomonas aeruginosa , to survive inside macrophages. In liquid culture, P. aeruginosa MgtC acts negatively on biofilm formation. However, a putative link between these two functions of MgtC in P. aeruginosa has not been experimentally addressed. In the present study, we first investigated the contribution of exopolysaccharides (EPS) in the intramacrophage survival defect and biofilm increase of mgtC mutant. Within infected macrophages, expression of EPS genes psl and alg was increased in a P. aeruginosa mgtC mutant strain comparatively to wild-type strain. However, the intramacrophage survival defect of mgtC mutant was not rescued upon introduction of psl or alg mutation, suggesting that MgtC intramacrophage role is unrelated to EPS production, whereas the increased biofilm formation of mgtC mutant was partially suppressed by introduction of psl mutation. We aimed to develop an antivirulence strategy targeting MgtC, by taking advantage of a natural antagonistic peptide, MgtR. Heterologous expression of mgtR in P. aeruginosa PAO1 was shown to reduce its ability to survive within macrophages. We investigated for the first time the biological effect of a synthetic MgtR peptide on P. aeruginosa . Exogenously added synthetic MgtR peptide lowered the intramacrophage survival of wild-type P. aeruginosa PAO1, thus mimicking the phenotype of an mgtC mutant as well as the effect of endogenously produced MgtR peptide. In correlation with this finding, addition of MgtR peptide to bacterial culture strongly reduced MgtC protein level, without reducing bacterial growth or viability, thus differing from classical antimicrobial peptides. On the other hand, the addition of exogenous MgtR peptide did not affect significantly biofilm formation, indicating an action toward EPS-independent phenotype rather than EPS-related phenotype. Cumulatively, our results show an antivirulence action of synthetic MgtR peptide, which may be more potent against acute infection, and provide a proof of concept for further exploitation of anti- Pseudomonas strategies.

During the last decade, the potential of peptides for drug delivery into cells has been highlighted by the discovery of several cell-penetrating peptides (CPPs). CPPs are very efficient in delivering various molecules into cells. However, except in some specific cases, their lack of cell specificity remains the major drawback for their clinical development. At the same time, various peptides with specific binding activity for a given cell line (cell-targeting peptides) have also been reported in the literature. One of the goals of the next years will be to optimize the tissue and cell delivery of therapeutic molecules by means of peptides which combine both targeting and internalization advantages. In this review, we describe the main strategies that are currently in use or likely to be employed in the near future to associate both targeting and delivery properties.

During the last decade, cell penetrating peptides (CPP) have been extensively used to mediate the cellular delivery of non-permeant biomolecules, including oligonucleotides (ONs). A covalent linkage between the CPP and the transported ON is required to mediate efficient cell internalization, and a disulfide bridge between the CPP and the ON has been shown to induce the most potent biological response. In this paper, we describe the activation. In a one step process of the sulfhydryl function from a synthon commercially available for ON synthesis. In addition, since the highly cationic nature of currently used CPP caused serious precipitation problems during the coupling step, we further improved the method by adsorbing the crude activated ON on an anion exchange matrix prior to specific peptide coupling.

Glioblastoma multiform (GBM) is considered the deadliest brain cancer. Conventional therapies are followed by poor patient survival outcomes, so novel and more efficacious therapeutic strategies are imperative to tackle this scourge. Gene therapy has emerged as an exciting and innovative tool in cancer therapy. Its combination with chemotherapy has significantly improved therapeutic outcomes. In line with this, our team has developed temozolomide–transferrin (Tf) peptide (WRAP5)/p53 gene nanometric complexes that were revealed to be biocompatible with non-cancerous cells and in a zebrafish model and were able to efficiently target and internalize into SNB19 and U373 glioma cell lines. The transfection of these cells, mediated by the formulated peptide-drug/gene complexes, resulted in p53 expression. The combined action of the anticancer drug with p53 supplementation in cancer cells enhances cytotoxicity, which was correlated to apoptosis activation through quantification of caspase-3 activity. In addition, increased caspase-9 levels revealed that the intrinsic or mitochondrial pathway of apoptosis was implicated. This assumption was further evidenced by the presence, in glioma cells, of Bax protein overexpression—a core regulator of this apoptotic pathway. Our findings demonstrated the great potential of peptide TMZ/p53 co-delivery complexes for cellular transfection, p53 expression, and apoptosis induction, holding promising therapeutic value toward glioblastoma. [ABSTRACT FROM AUTHOR]

Despite the great progress over the past few decades in both the diagnosis and treatment of a great variety of human cancers, glioblastoma remains the most lethal brain tumor. In recent years, cancer gene therapy focused on non-viral vectors which emerged as a promising approach to glioblastoma treatment. Transferrin (Tf) easily penetrates brain cells of the blood–brain barrier, and its receptor is highly expressed in this barrier and glioblastoma cells. Therefore, the development of delivery systems containing Tf appears as a reliable strategy to improve their brain cells targeting ability and cellular uptake. In this work, a cell-penetrating peptide (WRAP5), bearing a Tf-targeting sequence, has been exploited to condense tumor suppressor p53-encoding plasmid DNA (pDNA) for the development of nanocomplexes. To increase the functionality of developed nanocomplexes, the drug Temozolomide (TMZ) was also incorporated into the formulations. The physicochemical properties of peptide/pDNA complexes were revealed to be dependent on the nitrogen to phosphate groups ratio and can be optimized to promote efficient cellular internalization. A confocal microscopy study showed the capacity of developed complexes for efficient glioblastoma cell transfection and consequent pDNA delivery into the nucleus, where efficient gene expression took place, followed by p53 protein production. Of promise, these peptide/pDNA complexes induced a significant decrease in the viability of glioblastoma cells. The set of data reported significantly support further in vitro research to evaluate the therapeutic potential of developed complexes against glioblastoma. [ABSTRACT FROM AUTHOR]

Therapeutic peptides have regained interest as they can address unmet medical needs and can be an excellent complement to pharmaceutic small molecules and other macromolecular therapeutics. Over the past decades, correctors and potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel causing cystic fibrosis (CF) when mutated, were developed to reduce the symptoms of the patients. In this context, we have previously designed a CFTR-stabilizing iCAL36 peptide able to further increase the CFTR amount in epithelial cells, thereby resulting in a higher CFTR activity. In the present study, optimization of the peptidyl inhibitor was performed by coupling five different cell-penetrating peptides (CPP), which are Tat, dTat, TatRI (retro-inverso), MPG, and Penetratin. Screening of the internalization properties of these CPP-iCAL36 peptides under different conditions (with or without serum or endocytosis inhibitors, etc.) was performed to select TatRI as the optimal CPP for iCAL36 delivery. More importantly, using this TatRI-iCAL36 peptide, we were able to reveal for the first time an additive increase in the CFTR amount in the presence of VX-445/VX-809 compared to VX-445/VX-809 treatment alone. This finding is a significant contribution to the development of CFTR-stabilizing peptides in addition to currently used treatments (small-molecule correctors or potentiators) for CF patients. [ABSTRACT FROM AUTHOR]

Together with the nucleus, the mitochondrion has its own genome. Mutations in mitochondrial DNA are responsible for a variety of disorders, including neurodegenerative diseases and cancer. Current therapeutic approaches are not effective. In this sense, mitochondrial gene therapy emerges as a valuable and promising therapeutic tool. To accomplish this goal, the design/development of a mitochondrial-specific gene delivery system is imperative. In this work, we explored the ability of novel polymer- and peptide-based systems for mitochondrial targeting, gene delivery, and protein expression, performing a comparison between them to reveal the most adequate system for mitochondrial gene therapy. Therefore, we synthesized a novel mitochondria-targeting polymer (polyethylenimine–dequalinium) to load and complex a mitochondrial-gene-based plasmid. The polymeric complexes exhibited physicochemical properties and cytotoxic profiles dependent on the nitrogen-to-phosphate-group ratio (N/P). A fluorescence confocal microscopy study revealed the mitochondrial targeting specificity of polymeric complexes. Moreover, transfection mediated by polymer and peptide delivery systems led to gene expression in mitochondria. Additionally, the mitochondrial protein was produced. A comparative study between polymeric and peptide/plasmid DNA complexes showed the great capacity of peptides to complex pDNA at lower N/P ratios, forming smaller particles bearing a positive charge, with repercussions on their capacity for cellular transfection, mitochondria targeting and, ultimately, gene delivery and protein expression. This report is a significant contribution to the implementation of mitochondrial gene therapy, instigating further research on the development of peptide-based delivery systems towards clinical translation. [ABSTRACT FROM AUTHOR]

Short peptides can show high affinity for specific receptors overexpressed on tumor cells. Some of these are already used in cancerology as diagnostic tools and others are in clinical trials for therapeutic applications. Therefore, peptides exhibit great potential as a diagnostic tool but also as an alternative or an additional antitumoral approach upon the covalent attachment of a therapeutic moiety such as a radionuclide or a cytotoxic drug. The chemistry offers flexibility to graft onto the targeting-peptide either fluorine or iodine directly, or metallic radionuclides through appropriate chelating agent. Since short peptides are straightforward to synthesize, there is an opportunity to further improve existing peptides or to design new ones for clinical applications. However, several considerations have to be taken into account to optimize the recognition properties of the targeting-peptide to its receptor, to improve its stability in the biological fluids and its residence in the body, or to increase its overall therapeutic effect. In this review, we highlight the different aspects which need to be considered for the development of an efficient peptide receptor-mediated radionuclide therapy in different neoplasms. [ABSTRACT FROM AUTHOR]

Translocation through the plasma membrane is a major limiting step for the cellular delivery of macromolecules. A␣promising strategy to overcome this problem consists in the chemical conjugation (or fusion) to cell penetrating peptides (CPP) derived from proteins able to cross the plasma membrane. A large number of different cargo molecules such as oligonucleotides, peptides, peptide nucleic acids, proteins or even nanoparticles have been internalized in cells by this strategy. One of these translocating peptides was derived from the HIV-1 Tat protein. The mechanisms by which CPP enter cells remain unknown. Recently, convincing biochemical and genetic findings has established that the full-length Tat protein was internalized in cells via the ubiquitous heparan sulfate (HS) proteoglycans. We demonstrate here that the short Tat CPP is taken up by a route that does not involve the HS proteoglycans. [ABSTRACT FROM AUTHOR]

Fluorescein labeling of peptides and proteins is required for numerous biophysical or biological experiments such as fluorescence microscopy, fluorescence resonance energy transfer (FRET) or fluorescence imaging. The commonly used strategy relied on the coupling of the dye reagent followed by a gel filtration to recover the labeled molecule. Here we report a simplified method for the labeling of peptides and proteins on a cysteine residue and their purification. The method is based on the precipitation of peptides and proteins in acetone, fluorescein maleimide being soluble in this solvent. The excess of dye is fully eliminated after a couple of acetone washes and the precipitated peptide or protein is readily recovered. [Copyright &y& Elsevier]

Due to the antibiotic resistance crisis, novel therapeutic strategies need to be developed against bacterial pathogens. Hydrophobic bacterial peptides (small proteins under 50 amino acids) have emerged as regulatory molecules that can interact with bacterial membrane proteins to modulate their activity and/or stability. Among them, the Salmonella MgtR peptide promotes the degradation of MgtC, a virulence factor involved in Salmonella intramacrophage replication, thus providing the basis for an antivirulence strategy. We demonstrate here that endogenous overproduction of MgtR reduced Salmonella replication inside macrophages and lowered MgtC protein level, whereas a peptide variant of MgtR (MgtR-S17I), which does not interact with MgtC, had no effect. We then used synthetic peptides to evaluate their action upon exogenous addition. Unexpectedly, upon addition of synthetic peptides, both MgtR and its variant MgtR-S17I reduced Salmonella intramacrophage replication and lowered MgtC and MgtB protein levels, suggesting a different mechanism of action of exogenously added peptides versus endogenously produced peptides. The synthetic peptides did not act by reducing bacterial viability. We next tested their effect on various recombinant proteins produced in Escherichia coli and showed that the level of several inner membrane proteins was strongly reduced upon addition of both peptides, whereas cytoplasmic or outer membrane proteins remained unaffected. Moreover, the α-helical structure of synthetic MgtR is important for its biological activity, whereas helix-helix interacting motif is dispensable. Cumulatively, these results provide perspectives for new antivirulence strategies with the use of peptides that act by reducing the level of inner membrane proteins, including virulence factors. [ABSTRACT FROM AUTHOR]

Abstract: Peptides are emerging as attractive drug delivery tools. The HIV Tat-derived peptide is a small basic peptide that has been successfully shown to deliver a large variety of cargoes, from small particles to proteins, peptides and nucleic acids. The ‘transduction domain’ or region conveying the cell penetrating properties appears to be confined to a small (9 amino acids) stretch of basic amino acids, with the sequence RKKRRQRRR [S. Ruben, A. Perkins, R. Purcell, K. Joung, R. Sia, R. Burghoff, W.A. Haseltine, C.A. Rosen, Structural and functional characterization of human immunodeficiency virus tat protein, J. Virol. 63 (1989) 1–8; S. Fawell, J. Seery, Y. Daikh, C. Moore, L.L. Chen, B. Pepinsky, J. Barsoum, Tat-mediated delivery of heterologous proteins into cells, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 664–668; E. Vives, P. Brodin, B. Lebleu, A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus, J. Biol. Chem. 272 (1997) 16010–16017; S. Futaki, T. Suzuki, W. Ohashi, T. Yagami, S. Tanaka, K. Ueda, Y. Sugiura, Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery, J. Biol. Chem. 276 (2001) 5836–5840.]. The mechanism by which the Tat peptide adheres to, and crosses, the plasma membrane of cells is currently a topic of heated discussion in the literature, with varied findings being reported. This review aims to bring together some of those findings. Peptide interactions at the cell surface, and possible mechanisms of entry, will be discussed together with the effects of modifying the basic sequence and attaching a cargo. [Copyright &y& Elsevier]