Filomena Guida

A non–complement-fixing antibody to β2 glycoprotein I as a novel therapy for antiphospholipid syndrome

A single-chain fragment variable (scFv) recognizing β2-glycoprotein 1 (β2GPI) from humans and other species was isolated from a human phage display library and engineered to contain an IgG1 hinge-CH2-CH3 domain. The scFv-Fc directed against β2GPI domain I-induced thrombosis and fetal loss, thus mimicking the effect of antibodies from patients with antiphospholipid syndrome (APS). Complement is involved in the biological effect of anti-β2GPI scFv-Fc, as demonstrated by its ability to promote in vitro and in vivo complement deposition and the failure to induce vascular thrombosis in C6-deficient rats and fetal loss in C5-depleted mice. A critical role for complement was also supported by the inability of the CH2-deleted scFv-Fc to cause vessel occlusion and pregnancy failure. This antibody prevented the pathological effects of anti-β2GPI antibodies from APS patients and displaced β2GPI-bound patient antibodies. The CH2-deleted antibody represents an innovative approach potentially useful to treat APS patients refractory to standard therapy.

Antibacterial and anti-biofilm effects of cathelicidin peptides against pathogens isolated from cystic fibrosis patients

Six different cathelicidin-derived peptides were compared to tobramycin for antibacterial and anti-biofilm effects against S. aureus, P. aeruginosa, and S. maltophilia strains isolated from cystic fibrosis patients. Overall, SMAP-29, BMAP-28, and BMAP-27 showed relevant antibacterial activity (MIC(50) 4-8μg/ml), and in some cases higher than tobramycin. In contrast, indolicidin, LL-37, and Bac7(1-35) showed no significant antimicrobial activity (MIC(50)>32μg/ml). Killing kinetics experiments showed that in contrast to tobramycin the active cathelicidin peptides exert a rapid bactericidal activity regardless of the species tested. All three peptides significantly reduced biofilm formation by S. maltophilia and P. aeruginosa strains at 1/2× MIC, although at a lower extent than tobramycin. In addition, BMAP-28, as well as tobramycin, was also active against S. aureus biofilm formation. Preformed biofilms were significantly affected by bactericidal SMAP-29, BMAP-27 and BMAP-28 concentrations, although at a lesser extent than tobramycin. Overall, our results indicate the potential of some cathelicidin-derived peptides for the development of novel therapeutic agents for cystic fibrosis lung disease.

Native oligomerization determines the mode of action and biological activities of human cathelicidin LL-37.

LL-37 is a multifunctional component of innate immunity, with a membrane-directed antimicrobial activity and receptor-mediated pleiotropic effects on host cells. Sequence variations in its primate orthologues suggest that two types of functional features have evolved; human LL-37-like peptides form amphipathic helical structures and self-assemble under physiological conditions, whereas rhesus RL-37-like peptides only adopt this structure in the presence of bacterial membranes. The first type of peptide has a lower and more medium-sensitive antimicrobial activity than the second type, but an increased capacity to stimulate host cells. Oligomerization strongly affects the mode of interaction with biological membranes and, consequently, both cytotoxicity and receptor-mediated activities. In the present study we explored the effects of LL-37 self-association by using obligate disulfide-linked dimers with either parallel or antiparallel orientations. These had an increased propensity to form stacked helices in bulk solution and when in contact with either anionic or neutral model membranes. The antimicrobial activity against Gram-positive or Gram-negative bacteria, as well as the cytotoxic effects on host cells, strongly depended on the type of dimerization. To investigate the extent of native oligomerization we replaced Phe5 with the photoactive residue Bpa (p-benzoyl-L-phenylalanine), which, upon UV irradiation, enabled covalent cross-linking and allowed us to assess the extent of oligomerization in both physiological solution and in model membranes.

Effect of Size and N-Terminal Residue Characteristics on Bacterial Cell Penetration and Antibacterial Activity of the Proline-Rich Peptide Bac7

Bac7 is a proline-rich antimicrobial peptide, selective for Gram-negative bacteria, which acts intracellularly after membrane translocation. Progressively shortened fragments of Bac7 allowed determining the minimal sequence required for entry and antimicrobial activity as a 16-residue, N-terminal fragment, while further shortening led to a marked decrease in both functions. Furthermore, two N-terminal arginine residues were required for efficient translocation and activity. Analogues in which these residues were omitted, or where the side chain steric or physicochemical characteristics were systematically altered, were tested on different Escherichia coli strains, including a mutant with a destabilized outer membrane and one lacking the relevant SbmA membrane transport protein. H-bonding capacity, stereochemistry, and charge, in that order, played a determining role for efficient transit through both the outer and cytoplasmic membranes. Our studies allowed building a more detailed model for the mode-of-action of Bac7, and confirming its potential as an anti-infective agent, also suggesting it may be a vehicle for internalization of other antibiotic cargo.

Comparative activity and mechanism of action of three types of bovine antimicrobial peptides against pathogenic Prototheca spp.

The yeast‐like algae of the genus Prototheca are ubiquitous saprophytes causing infections in immunocompromised patients and granulomatous mastitis in cattle. Few available therapies and the rapid spread of resistant strains worldwide support the need for novel drugs against protothecosis. Host defence antimicrobial peptides inactivate a wide array of pathogens and are a rich source of leads, with the advantage of being largely unaffected by microbial resistance mechanisms. Three structurally diverse bovine peptides [BMAP‐28, Bac5 and lingual antimicrobial peptide (LAP)] have thus been tested for their capacity to inactivate Prototheca spp. In minimum inhibitory concentration (MIC) assays, they were all effective in the micromolar range against clinical mastitis isolates as well as a Prototheca wickerhamii reference strain. BMAP‐28 sterilized Prototheca cultures within 30–60 min at its MIC, induced cell permeabilization with near 100% release of cellular adenosine triphosphate and resulted in extensive surface blebbing and release of intracellular material as observed by scanning electron microscopy. Bac5 and LAP inactivated Prototheca following 3–6 h incubation at fourfold their MIC and did not result in detectable surface damage despite 70–90% killing, suggesting they act via non‐lytic mechanisms. In circular dichroism studies, the conformation of BMAP‐28, but not that of Bac5 or LAP, was affected by interaction with liposomes mimicking algal membranes. Our results indicate that BMAP‐28, Bac5 and LAP kill Prototheca with distinct potencies, killing kinetics, and modes of action and may be appropriate for protothecal mastitis treatment. In addition, the ability of Bac5 and LAP to act via non‐lytic mechanisms may be exploited for the development of target‐selective drugs. Copyright

Identification of antimicrobial peptides from teleosts and anurans in expressed sequence tag databases using conserved signal sequences

The problem of multidrug resistance requires the efficient and accurate identification of new classes of antimicrobial agents. Endogenous antimicrobial peptides produced by most organisms are a promising source of such molecules. We have exploited the high conservation of signal sequences in teleost and anuran antimicrobial peptides to search cDNA (expressed sequence tag) databases for likely candidates. Subject sequences were then analysed for the presence of potential antimicrobial peptides based on physicochemical properties (amphipathic helical structure, cationicity) and use of the D‐descriptor model to predict the therapeutic index (relation between the minimum inhibitory concentration and the concentration giving 50% haemolysis). This analysis also suggested mutations to probe the role of the primary structure in determining potency and selectivity. Selected sequences were chemically synthesized and the antimicrobial activity of the peptides was confirmed. In particular, a short (21‐residue) sequence, likely of sticklefish origin, showed potent activity and it was possible to tune the spectrum of action and/or selectivity by combining three directed mutations. Membrane permeabilization studies on both bacterial and host cells indicate that the mode of action was prevalently membranolytic. This method opens up the possibility for more effective searching of the vast and continuously growing expressed sequence tag databases for novel antimicrobial peptides, which are likely abundant, and the efficient identification of the most promising candidates among them.

Gold nanoparticles with patterned surface monolayers for nanomedicine: current perspectives

Molecular self-assembly is a topic attracting intense scientific interest. Various strategies have been developed for construction of molecular aggregates with rationally designed properties, geometries, and dimensions that promise to provide solutions to both theoretical and practical problems in areas such as drug delivery, medical diagnostics, and biosensors, to name but a few. In this respect, gold nanoparticles covered with self-assembled monolayers presenting nanoscale surface patterns—typically patched, striped or Janus-like domains—represent an emerging field. These systems are particularly intriguing for use in bio-nanotechnology applications, as presence of such monolayers with three-dimensional (3D) morphology provides nanoparticles with surface-dependent properties that, in turn, affect their biological behavior. Comprehensive understanding of the physicochemical interactions occurring at the interface between these versatile nanomaterials and biological systems is therefore crucial to fully exploit their potential. This review aims to explore the current state of development of such patterned, self-assembled monolayer-protected gold nanoparticles, through step-by-step analysis of their conceptual design, synthetic procedures, predicted and determined surface characteristics, interactions with and performance in biological environments, and experimental and computational methods currently employed for their investigation.

Designing High-Affinity Peptides for Organic Molecules by Explicit Solvent Molecular Dynamics.

Short peptides offer a cheap alternative to antibodies for developing sensing units in devices for concentration measurement. We here describe a computational procedure that allows designing peptides capable of binding with high affinity a target organic molecule in aqueous or nonstandard solvent environments. The algorithm is based on a stochastic search in the space of the possible sequences of the peptide, and exploits finite temperature molecular dynamics simulations in explicit solvent to check if a proposed mutation improves the binding affinity or not. The procedure automatically produces peptides which form thermally stable complexes with the target. The estimated binding free energy reaches the 13 kcal/mol for Irinotecan anticancer drug, the target considered in this work. These peptides are by construction solvent specific; namely, they recognize the target only in the solvent in which they have been designed. This feature of the algorithm calls for applications in devices in which the peptide-based sensor is required to work in denaturants or under extreme conditions of pressure and temperature.

Peptide biosensors for anticancer drugs: Design in silico to work in denaturizing environment

One of the main targets in current clinical oncology is the development of a cheap device capable of monitoring in real-time the concentration of a drug in the blood of a patient. This would allow fine-tuning the dosage according to the patients metabolism, a key condition to reduce side effects. By using surface plasmon resonance and fluorescence spectroscopy we here show that short peptides designed in silico by a recently developed algorithm are capable of binding the anticancer drug irinotecan (CPT-11) with micromolar affinity. Importantly, the recognition takes place in the denaturating solution used in standard therapeutic drug monitoring to detach the drug from the proteins that are present in human plasma, and some of the peptides are capable of distinguishing CPT-11 from its metabolite SN-38. These results suggest that the in silico design of small artificial peptides is now a viable route for designing sensing units, opening a wide range of applications in diagnostic and clinical areas.

Effect of targeted minimal sequence variations on the structure and biological activities of the human cathelicidin LL-37

LL‐37 is an innate immune peptide derived from the human cathelicidin, which exerts pleiotropic roles in host defense and healing. These activities in part depend on its capacity to adopt an amphipathic helical structure in physiological solutions and then oligomerizing. Orthologues from other primates, such as rhesus RL‐37, remain monomeric and disordered under the same conditions. Intramolecular salt‐bridges, arising from appropriately spaced anionic and cationic residues in its sequence, may play a relevant role in determining the particular structure adopted by LL‐37. To probe this, we have effected minimal, targeted residue variations such as replacement of a single residue (K15→G), or inversion of one or both sets of two residues (E10 K11→ K10 E11 or E16 K18→ K16 E18). This could alter the pattern of intramolecular salt bridging without affecting other functionally relevant parameters such as overall hydrophobicity, helix amphipathicity or charge. The structural and functional effects were analyzed using CD spectroscopy, surface plasmon resonance, antimicrobial activity assays, and bacterial membrane permeabilization to fluorescent probes of increasing sizes, using flow cytometry. Analogs were functionally different from both LL‐37 and RL‐37, so it was not possible to switch from the function of one to that of the other simply by altering the salt‐bridging pattern in this manner. This indicates that the particular structure/function characteristics of LL‐37 likely depend quite subtly, and in a precise and complex manner, on a complex pattern of intramolecular interactions.