Design and Characterization of Myristoylated and Non-Myristoylated Peptides Effective against Candida spp. Clinical Isolates.

The increasing resistance of fungi to antibiotics is a severe challenge in public health, and newly effective drugs are required. Promising potential medications are lipopeptides, linear antimicrobial peptides (AMPs) conjugated to a lipid tail, usually at the N-terminus. In this paper, we investigated the in vitro and in vivo antifungal activity of three short myristoylated and non-myristoylated peptides derived from a mutant of the AMP Chionodracine. We determined their interaction with anionic and zwitterionic membrane-mimicking vesicles and their structure during this interaction. We then investigated their cytotoxic and hemolytic activity against mammalian cells. Lipidated peptides showed a broad spectrum of activity against a relevant panel of pathogen fungi belonging to Candida spp., including the multidrug-resistant C. auris. The antifungal activity was also observed vs. biofilms of C. albicans, C. tropicalis, and C. auris. Finally, a pilot efficacy study was conducted on the in vivo model consisting of Galleria mellonella larvae. Treatment with the most-promising myristoylated peptide was effective in counteracting the infection from C. auris and C. albicans and the death of the larvae. Therefore, this myristoylated peptide is a potential candidate to develop antifungal agents against human fungal pathogens.

Structural Analysis and Design of Chionodracine-Derived Peptides Using Circular Dichroism and Molecular Dynamics Simulations.

Antimicrobial peptides have been identified as one of the alternatives to the extensive use of common antibiotics as they show a broad spectrum of activity against human pathogens. Among these is Chionodracine (Cnd), a host-defense peptide isolated from the Antarctic icefish Chionodraco hamatus, which belongs to the family of Piscidins. Previously, we demonstrated that Cnd and its analogs display high antimicrobial activity against ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species). Herein, we investigate the interactions with lipid membranes of Cnd and two analogs, Cnd-m3 and Cnd-m3a, showing enhanced potency. Using a combination of Circular Dichroism, fluorescence spectroscopy, and all-atom Molecular Dynamics (MD) simulations, we determined the structural basis for the different activity among these peptides. We show that all peptides are predominantly unstructured in water and fold, preferentially as α-helices, in the presence of lipid vesicles of various compositions. Through a series of MD simulations of 400 ns time scale, we show the effect of mutations on the structure and lipid interactions of Cnd and its analogs. By explaining the structural basis for the activity of these analogs, our findings provide structural templates to design minimalistic peptides for therapeutics.

Exploiting novel tailored immunotherapies of type 1 diabetes: Short interfering RNA delivered by cationic liposomes enables efficient down-regulation of variant PTPN22 gene in T lymphocytes.

In autoimmune diseases as Type 1 diabetes, the actual treatment that provides the missing hormones is not able, however, to interrupt the underlining immunological mechanism. Importantly, novel immunotherapies are exploited to protect and rescue the remaining hormone producing cells. Among probable targets of immunotherapy, the C1858T mutation in the PTPN22 gene, which encodes for the lymphoid tyrosine phosphatase (Lyp) variant R620W, reveals an autoimmunity related pathophysiological role. Our scope was to establish new C1858T PTPN22 siRNA duplexes delivered by liposomal carriers (lipoplexes) to patients' PBMC. Following lipoplexes treatment, CD3+ and CD3- immunotypes were efficiently transfected; cell integrity and viability were preserved. Specific target mRNA down-modulation was observed. After T cell receptor stimulation, in lipoplexes-treated PBMC Lyp function was restored by increased release of IL-2 in cultures. Results set-up the stage for ultimate trials in the treatment of autoimmunity based on the specific inhibitory targeting of C1858T PTPN22 by lipoplexes.

Effect of the Hydrophobic Tail of a Chiral Surfactant on the Chirality of Aggregates and on the Formation of Wormlike Micelles.

The micellization of chiral enantiopure surfactants, dodecyl- N, N-dimethyl- N-( S)-(1-phenyl)ethylammonium bromide and hexadecyl- N, N-dimethyl- N-( S)-(1-phenyl)ethylammonium bromide, was investigated by circular dichroism spectroscopy and isothermal titration calorimetry. The formation of wormlike micelles (WLMs) upon the addition of sodium salicylate to the aqueous solutions of the surfactant was observed only in the case of hexadecyl- N, N-dimethyl- N-( S)-(1-phenyl)ethylammonium bromide. The presence of WLMs was assessed by cryogenic transmission electron microscopy, rheology, and isothermal titration calorimetry experiments, and their supramolecular chirality was investigated by circular dichroism spectroscopy. Depending on the length of the hydrophobic tail, molecular chirality is transferred into a different chiral supramolecular trait. Our findings demonstrate that hydrophobic interactions by controlling the organization and functions of self-assemblies also control the transcription of the chiral information from molecules to complex supramolecular systems.

Proof of the Structure of the Stemodia chilensis Tetracyclic Diterpenoid (+)-19-Acetoxystemodan-12-ol by Synthesis from (+)-Podocarpic Acid: X-ray Structure Determination of a Key Intermediate.

The first synthesis of (+)-19-acetoxystemodan-12-ol (1), a stemodane diterpenoid isolated from Stemodia chilensis, is described. The structure was supported by an X-ray crystallographic analysis of intermediate (+)-9a, which confirmed the proposed structure and excluded the structure of (-)-19-hydroxystemod-12-ene as a possible candidate for the Chilean Calceolaria diterpenoid to which the (-)-19-hydroxystemar-13-ene structure (9b) had been erroneously assigned.

Achiral Dye/Surfactant Heteroaggregates for Chiral Sensing of Phosphocholines.

An investigation, based on absorption and circular dichroism spectroscopy, was carried out on assemblies formed in water upon the interaction of heteroaggregates, composed of dyes (Congo Red or Evans Blue) and cetyltrimethylammonium bromide (CTAB), with four enantiopure phopshocholines (DMPC, DPPC, DOPC, and POPC) characterized by the same polar head and different hydrophobic tails. The results show that the nature of the lipid as well as the concentration ratios influence sensitively the absorption and chiroptical properties of the supramolecular structure. Intriguingly, the transfer of chirality from the lipid to the assembly may be triggered or not, depending on the nature of the lipid hydrophobic chain. These findings confirm the fundamental role of hydrophobic interactions in the transcription of chirality from molecules to complex architectures.

Conformational isomerisms and nano-aggregation in substituted alkylammonium nitrates ionic liquids: an x-ray and computational study of 2-methoxyethylammonium nitrate.

In this study, we discuss, using molecular dynamics simulations and energy-dispersive x-ray diffraction data, how a conformational isomerism can dramatically alter the nanosegregation phenomena that take place in a prototypical ionic liquid. The diffraction patterns of liquid 2-methoxyethylammonium nitrate are compared with the results from molecular dynamics simulations. The simulations conditions and force field parameters have been varied producing different charge models and different populations of conformers of the cation. We show that, while the short range structure is relatively unchanged in the models, the long range aggregation phenomena deemed responsible for the appearance of low Q peaks in the X-ray patterns strongly depend on the choice of the charge model. In the title compound, the best agreement with the experiment, where no low Q peaks appear, occurs if the point charges are calculated using the gauche conformation of the cation, which is characterized by an intramolecular hydrogen bond between ammonium and ether groups.

Resveratrol and Resveratrol-Loaded Galactosylated Liposomes: Anti-Adherence and Cell Wall Damage Effects on Staphylococcus aureus and MRSA.

Antibiotic resistance due to bacterial biofilm formation is a major global health concern that makes the search for new therapeutic approaches an urgent need. In this context,, trans-resveratrol (RSV), a polyphenolic natural substance, seems to be a good candidate for preventing and eradicating biofilm-associated infections but its mechanism of action is poorly understood. In addition, RSV suffers from low bioavailability and chemical instability in the biological media that make its encapsulation in delivery systems necessary. In this work, the anti-biofilm activity of free RSV was investigated on Staphylococcus aureus and, to highlight the possible mechanism of action, we studied the anti-adherence activity and also the cell wall damage on a MRSA strain. Free RSV activity was compared to that of RSV loaded in liposomes, specifically neutral liposomes (L = DOPC/Cholesterol) and cationic liposomes (LG = DOPC/Chol/GLT1) characterized by a galactosylated amphiphile (GLT1) that promotes the interaction with bacteria. The results indicate that RSV loaded in LG has anti-adherence and anti-biofilm activity higher than free RSV. On the other side, free RSV has a higher bacterial-growth-inhibiting effect than encapsulated RSV and it can damage cell walls by creating pores; however, this effect can not prevent bacteria from growing again. This RSV ability may underlie its bacteriostatic activity.

Regio- and diastereoselective synthesis and X-ray structure determination of (+)-2-deoxyoryzalexin S from (+)-podocarpic acid. Structural nonidentity with its nominal natural isolated enantiomer.

(+)-2-Deoxyoryzalexin S (1), the nominal enantiomer of a diterpenoid isolated in Chile from Calceolaria species, was regio- and diastereoselectively synthesized from (+)-podocarpic acid. (+)-2-Deoxyoryzalexin S (1) was characterized also as its acetyl derivative, (+)-2, whose structure was confirmed by X-ray crystallographic analysis. Surprisingly, comparison of the data recorded for (+)-1 and (+)-2 and those reported in the literature for the Calceolaria isolated diterpenoid 1 and its derivative (-)-2 showed some differences, suggesting that the latter do not possess the proposed structures.

Deracemization of bilirubin as the marker of the chirality of micellar aggregates.

The deracemization of bilirubin in micellar aggregates of structurally correlated chiral surfactants was studied by circular dichroism experiments and exploited as the marker of the expression of chirality of the aggregates. The obtained results suggest that the hydrophobic interactions control the transfer of chirality from the monomers to the aggregates, and that different regions of the same aggregate might feature opposite enantiorecognition capabilities.

Resveratrol loaded in cationic glucosylated liposomes to treat Staphylococcus epidermidis infections.

Glucosylated liposomes composed of the natural saturated phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol (Chol) and a cationic amphiphile featuring a glucosyl moiety (GL4), have been developed for delivering the antimicrobial trans-Resveratrol (RSV) to S. epidermidis, characterized by carbohydrate-specific adhesins able to recognize glucose. The cationic derivative of cholesterol, DC-Chol, was also included in liposome formulations, alone or in combination with GL4, in order to explore the role of both cationic charge and sugar moiety in the interaction of liposomes with bacterial cells. RSV was included inside glucosylated cationic liposomes by the thin film method, coupled with either extrusion or sonication; liposome mean diameter, polydispersity index, surface charge, RSV entrapment efficiency and concentration have been measured by DLS, electrophoretic mobility, and HPLC. The antimicrobial activity of RSV-loaded liposomes was evaluated by monitoring the bacterial growth curves of two cell lines of Staphylococcus epidermidis, a slime positive strain (i.e. a strain able to form a biofilm) and a slime negative one. Results point out that, when the glucosylamphiphile GL4 is included in the formulation, only the extrusion protocol allows obtaining monodisperse liposomes with high RSV entrapment efficiency. The mean diameters of empty and resveratrol-loaded liposomes are all around 120-140 nm and size distribution are narrow, except for samples including GL4 at 5 molar percentage. Here the higher polydispersity index may be the indication of the occurrence of a restructuring phenomenon. The microbiological tests put in evidence a different response of the two bacterial cell lines to liposome treatments, in fact, the slime negative bacterial cells, that are not able to produce the extracellular polymeric substances, are more susceptible to the cationic charge of the liposomes and to the detergent effect of GL4. The most interesting results concern DPPC/Chol/GL4 liposomes on the slime positive strain: this formulation, non-toxic in itself, displays an enhanced antibacterial efficacy with respect to free RSV, killing bacteria even at concentration tenfold under the MIC.

Improvement of Lipoplexes With a Sialic Acid Mimetic to Target the C1858T PTPN22 Variant for Immunotherapy in Endocrine Autoimmunity.

The C1858T variant of the protein tyrosine phosphatase N22 (PTPN22) gene is associated with pathophysiological phenotypes in several autoimmune conditions, namely, Type 1 diabetes and autoimmune thyroiditis. The R620W variant protein, encoded by C1858T, leads to a gain of function mutation with paradoxical reduced T cell activation. We previously exploited a novel personalized immunotherapeutic approach based on siRNA delivered by liposomes (lipoplexes, LiposiRNA) that selectively inhibit variant allele expression. In this manuscript, we functionalize lipoplexes carrying siRNA for variant C1858T with a high affinity ligand of Siglec-10 (Sig10L) coupled to lipids resulting in lipoplexes (LiposiRNA-Sig10L) that enhance delivery to Siglec-10 expressing immunocytes. LiposiRNA-Sig10L lipoplexes more efficiently downregulated variant C1858T PTPN22 mRNA in PBMC of heterozygous patients than LiposiRNA without Sig10L. Following TCR engagement, LiposiRNA-Sig10L more significantly restored IL-2 secretion, known to be paradoxically reduced than in wild type patients, than unfunctionalized LiposiRNA in PBMC of heterozygous T1D patients.

Transport of cationic liposomes in a human blood brain barrier model: Role of the stereochemistry of the gemini amphiphile on liposome biological features.

HYPOTHESIS: The positive charge on liposome surface is known to promote the crossing of the Blood brain barrier (BBB). However, when diastereomeric cationic gemini amphiphiles are among lipid membrane components, also the stereochemistry may affect the permeability of the vesicle across the BBB. EXPERIMENTS: Liposomes featuring cationic diasteromeric gemini amphiphiles were formulated, characterized, and their interaction with cell culture models of BBB investigated. FINDINGS: Liposomes featuring the gemini amphiphiles were internalized in a monolayer of brain microvascular endothelial cells derived from human induced pluripotent stem cells (hiPSC) through an energy dependent transport, internalization involving both clathrin- and caveolae-mediated endocytosis. On the same formulations, the permeability was also evaluated across a human derived in vitro BBB transport model. The permeability of liposomes featuring the gemini amphiphiles was significantly higher compared to that of neutral liposomes (DPPC/Cholesterol), that were not able to cross BBB. Most importantly, the permeability was influenced by the stereochemistry of the gemini and pegylation of these formulations did not result in a drastic reduction of the crossing ability. The in vitro iPSC-derived BBB models used in this work represent an important advancement in the drug discovery research of novel brain delivery strategies and therapeutics for central nervous system diseases.

Diastereoselective total synthesis of (+)-13-stemarene by fourth generation methods: a formal total synthesis of (+)-18-deoxystemarin.

The problem of constructing diastereoselectively the C/D ring system of stemarane diterpenes from a bicyclo[2.2.2]octane intermediate was solved resulting in very simple synthesis of (+)-13-stemarene 1. The obtaining of the latter represents also a formal synthesis of (+)-18-deoxystemarin 2. In the key step, the epimeric mixture 10, dissolved in toluene, was converted by the action of TsOH into (+)-stemar-13-en-15-one 28.

Structural characterization of zinc(II) chloride in aqueous solution and in the protic ionic liquid ethyl ammonium nitrate by x-ray absorption spectroscopy.

Extended x-ray absorption fine structure (EXAFS) spectroscopy has been used to investigate the species and structures existing in a series of ZnCl(2)-H(2)O-NaCl solutions with different chloride/zinc ratios and in a solution of ZnCl(2) in the protic ionic liquid ethyl ammonium nitrate (EAN). The average coordination numbers and distances of zinc species were determined from the analysis of the EXAFS data. In aqueous solution the number of chloride ions tightly bounded to Zn(2+) is significantly related to the chloride/zinc ratio, and no inner complex formation between Zn(2+) and Cl(-) ions has been detected for low ZnCl(2) concentration (0.1 and 0.2 M). Conversely, in the same concentration range (0.13 M) the ZnCl(2) species do not dissociate in EAN and the Zn(2+) first coordination shell has two chloride ions and is completed by two oxygen atoms of the nitrate anion. The results of this investigation show that notwithstanding the existence of similar characteristics between EAN and water, the solvation properties of the two solvents are markedly different.

How stereochemistry of lipid components can affect lipid organization and the route of liposome internalization into cells.

Though liposome-based drugs are in clinical use, the mechanism of cell internalization of liposomes is yet an object of controversy. The present experimental investigation, carried out on human glioblastoma cells, indicated different internalization routes for two diastereomeric liposomes. Molecular dynamics simulations of the lipid bilayers of the two formulations indicated that the different stereochemistry of a lipid component controls some parameters such as area per lipid molecule and fluidity of lipid membranes, surface potential and water organization at the lipid/water interface, all of which affect the interaction with biomolecules and cell components.

Synthesis and Characterization of Mitochondria-Targeted Triphenylphosphonium Bolaamphiphiles.

In this chapter we describe: (1) the procedure for the synthesis of four single chain bolaamphiphiles, displaying chains of 12, 16, 20 and 30 methylene units and triphenylphosphonium moieties as headgroups (TPP1-TPP4); (2) the methods used to characterize TPP1-TPP4 spontaneous aggregation in aqueous solution. We illustrate the determination of Krafft point and cac by conductivity measurements and the procedures used to investigate dimensions, morphology, and stability by dynamic and dielectrophoretic laser light scattering, dialysis, transmission electron microscopy, and Raman spectroscopy measurements.

Trematocine, a Novel Antimicrobial Peptide from the Antarctic Fish Trematomus bernacchii: Identification and Biological Activity.

Antimicrobial peptides (AMPs) are short peptides active against a wide range of pathogens and, therefore, they are considered a useful alternative to conventional antibiotics. We have identified a new AMP in a transcriptome derived from the Antarctic fish Trematomus bernacchii. This peptide, named Trematocine, has been investigated for its expression both at the basal level and after in vivo immunization with an endemic Antarctic bacterium (Psychrobacter sp. TAD1). Results agree with the expected behavior of a fish innate immune component, therefore we decided to synthesize the putative mature sequence of Trematocine to determine the structure, the interaction with biological membranes, and the biological activity. We showed that Trematocine folds into a α-helical structure in the presence of both zwitterionic and anionic charged vesicles. We demonstrated that Trematocine has a highly specific interaction with anionic charged vesicles and that it can kill Gram-negative bacteria, possibly via a carpet like mechanism. Moreover, Trematocine showed minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against selected Gram-positive and Gram-negative bacteria similar to other AMPs isolated from Antarctic fishes. The peptide is a possible candidate for a new drug as it does not show any haemolytic or cytotoxic activity against mammalian cells at the concentration needed to kill the tested bacteria.

Enantiodiscrimination of bilirubin-IXalpha enantiomers in biomembrane models: has chirality a role in bilirubin toxicity?

Simple biomembrane models, namely micellar aggregates formed by enantiopure sodium N-acylprolinates, are able to convert the racemic mixture of bilirubin-IXalpha into an enantiomerically enriched mixture, thus suggesting a possible role of chirality in bilirubin toxicity due to the perturbation of neuron membrane dynamics. The length of alkyl chain does not influence the extent of equilibrium displacement, however, it affects the conformation of bilirubin, thus confirming the role of lipid structure in the membrane/bilirubin interaction, and suggesting a non-superficial main site of association.

Aggregation behaviour of triphenylphosphonium bolaamphiphiles.

HYPOTHESIS: Bolaamphiphiles are characterized by wide polymorphism of their aggregates, due to the connection of the headgroups that renders their investigation very intriguing in several technological applications. Some bolaamphiphiles displaying the triphenylphosphonium motif (TPP-bolaamphiphiles) were previously explored for their ability in crossing the mitochondrial membranes but their colloidal features, which are crucial for the potential development of an effective drug delivery system, were never investigated. EXPERIMENTS: Single chain TPP-bolaamphiphiles, featuring chains of 12, 16, 20 and 30 methylene units, were synthesized and their aggregation features (Krafft point, cac, dimensions, morphology, stability) were investigated by conductivity, dialysis, transmission electron microscopy, Raman spectroscopy, dynamic and dielectrophoretic laser light scattering measurements. FINDINGS: All the TPP-bolaamphiphiles spontaneously self-assemble into vesicles, independently of the chain length. The bolaamphipile with the longest chain forms monodispersed vesicles whereas for the other bolaamphiphiles two distinct populations of vesicles are observed. All vesicles are not equilibrium systems, in particular vesicles formed by the bolaamphiphiles featuring 20 and 30 methylene units result notably stable to dilution thanks to both the tightening of molecular packing at increasing chain length and the progressive reduction of the monomer percentage in U-shaped conformation. These features make these TPP-bolaamphiphiles very attractive as minor components for the development of novel mitochondriotropic liposomes.