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Antibiotic resistance, particularly among Gram-negative bacteria, poses a significant healthcare challenge due to their ability to evade antibiotic action through various mechanisms. In this study, we explore the prediction of small molecule accumulation in Gram-negative bacteria by using machine learning techniques enhanced with statistical descriptors derived from molecular dynamics simulations. We begin by identifying a minimal set of molecular descriptors that maximize the model's predictive power while preserving human interpretability. We optimize model accuracy, precision, and the area under the receiver operating characteristic curve through an iterative process. We demonstrate that the inclusion of statistical descriptors significantly improves model performance across various prediction metrics. Particularly, the addition of statistical descriptors related to dipole moment and minimum projection radius enhances the model's predictive capabilities, shedding light on the physicochemical properties crucial for small molecule accumulation. Our findings highlight the importance of considering statistical moments beyond mean values in predictive modeling and suggest avenues for future research. Overall, our study provides insights into the complex dynamics of antibiotic accumulation in Escherichia coli bacterial cells, generalizable to other Gram-negative species, offering a promising approach for the discovery of effective antibacterial agents, identifying new hits, and improving them to define effective lead agents.

Drug resistance represents one of the great plagues of our time worldwide. This largely limits the treatment of common infections and requires the development of new antibiotics or other alternative approaches. Noteworthy, the indiscriminate use of antibiotics is mostly responsible for the selection of mutations that confer drug resistance to microbes. In this regard, recently, ozone has been raising interest for its unique biological properties when dissolved in natural oils. Ozonated oils have been reported to act in a non-specific way on microorganisms hindering the acquisition of advantageous mutations that result in resistance. Here, we focused on the antimicrobial effect of two commercial olive (OOO) and sunflower seeds (OSO) oils. Nuclear magnetic resonance spectroscopy and thermal analysis showed the change in the chemical composition of the oils after ozonation treatment. Different ozonated oil concentrations were then used to evaluate their antimicrobial profile against Candida albicans , Enterococcus faecalis , Staphylococcus aureus , Klebsiella pneumoniae , Pseudomonas aeruginosa , and Escherichia coli by agar diffusion and broth dilution methods. Cytotoxicity was also evaluated in keratinocytes and epithelial cells. Overall, our results revealed that both OOO and OSO showed a potent microbicidal effect, especially against C. albicans (IC50 = OOO: 0.3 mg/mL and OSO: 0.2 mg/mL) and E. faecalis (IC50 = OOO: 0.4 mg/mL and OSO: 2.8 mg/mL) albeit exerting a certain effect also against S. aureus and E. coli. Moreover, both OOO and OSO do not yield any relevant cytotoxic effect at the active concentrations in both cell lines. This indicates that the ozonated oils studied are not toxic for mammalian cells despite exerting a potent antimicrobial effect on specific microorganisms. Therefore, OOO and OSO may be considered to integrate standard therapies in the treatment of common infections, likely overcoming drug resistance issues.

Potentiometry is the primary pH measurement method, but alternatives are sought beyond glass electrodes operative limitations. In nuclear magnetic resonance (NMR) experiments, electrodeless pH sensing is important to track changes along titrations, during chemical reactions or inside compartmentalized environments inaccessible to electrodes, for instance. Although several interesting NMR pH indicators have been already presented, the potential of inorganic phosphate is overlooked, despite its common presence in NMR samples as the buffer main component. Its use for electrodeless pH determination can be expanded by exploiting all its three proton dissociations. This study was aimed at verifying the use of inorganic phosphate 31 P chemical shift to sense pH variations, and at exploring the complementary use of pyrophosphate ions to cover a wide pH range. A simple set of equations is presented to utilize both phosphate and pyrophosphate 31 P chemical shift in combination for accurate pH determination without a glass electrode over the 5-12 pH range, and without affecting the spectrum of other nuclei. The present study demonstrated an average deviation of 0.09 (maximum <0.2) pH unit from glass electrode measurements. The trimethylphosphate can be used as a suitable chemical shift reference for both 31 P and 1 H (also 13 C), with its hydrolysis being significant only at pH > 12. The method was also demonstrated by determining the pKa of three distinct molecules in a mixture and by comparing the results to those obtained when the glass electrode was used to measure the pH. The approach shown here can be easily tuned to different experimental conditions., (© 2024 John Wiley & Sons Ltd.)

Benzothiazole-based bacterial DNA gyrase and topoisomerase IV inhibitors are promising new antibacterial agents with potent activity against Gram-positive and Gram-negative bacterial strains. The aim of this study was to improve the uptake of these inhibitors into the cytoplasm of Gram-negative bacteria by conjugating them to the small siderophore mimics. The best conjugate 18b displayed potent Escherichia coli DNA gyrase and topoisomerase IV inhibition. The interaction analysis of molecular dynamics simulation trajectory showed the important contribution of the siderophore mimic moiety to binding affinity. By NMR spectroscopy, we demonstrated that the hydroxypyridinone moiety alone was responsible for the chelation of iron(iii). Moreover, 18b showed an enhancement of antibacterial activity against E. coli JW5503 in an iron-depleted medium, clearly indicating an increased uptake of 18b in this bacterial strain., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Herein we describe the binding abilities of Zn(II) complexes of [12]aneN 4 - (L1) and [9]aneN 3 -based receptors (L2, L3) towards the herbicides N -(phosphonomethyl)glycine (glyphosate, H 3 PMG) and 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid (glufosinate, H 2 GLU), and also aminomethylphosphonic acid (H 2 AMPA), the main metabolite of H 3 PMG, and phosphate. All ligands form stable Zn(II) complexes, whose coordination geometries allow a possible interaction of the metal center with exogenous anionic substrates. Potentiometric studies evidenced the marked coordination ability of the L2/Zn(II) system for the analytes considered, with a preferential binding affinity for H 3 PMG over the other substrates, in a wide range of pH values. 1 H and 31 P NMR experiments supported the effective coordination of such substrates by the Zn(II) complex of L2, while fluorescence titrations and a test strip experiment were performed to evaluate whether the H 3 PMG recognition processes could be detected by fluorescence signaling.

Impressive work has been completed in recent decades on the transmembrane anion transport capability of small synthetic transporters from many different structural classes. However, very few predicting models have been proposed for the fast screening of compound libraries before spending time and resources on the laboratory bench for their synthesis. In this work, a new approach is presented which aims at describing the transport process by taking all the steps into explicit consideration, and includes all possible experiment-derived parameters. The algorithm is able to simulate the macroscopic experiments performed with lipid vesicles to assess the ion-transport ability of the synthetic transporters following a non-electrogenic uniport mechanism. While keeping calculation time affordable, the final goal is the curve-fitting of real experimental data-so, to obtain both an analysis and a predictive tool. The role and the relative weight of the different parameters is discussed and the agreement with the literature is shown by using the simulations of a virtual benchmark case. The fitting of real experimental curves is also shown for two transporters of different structural type.

Microtubules (MTs) are the principal target for drugs acting against mitosis. These compounds, called microtubule targeting agents (MTAs), cause a mitotic arrest during G2/M phase, subsequently inducing cell apoptosis. MTAs could be classified in two groups: microtubule stabilising agents (MSAs) and microtubule destabilising agents (MDAs). In this paper we present a new series of (E) (Z)-2-(5,6-difluoro-(1H)2H-benzo[d] [1,2,3]triazol-1(2)-yl)-3-(R)acrylonitrile (9a-j, 10e, 11a,b) and (E)-2-(1H-benzo[d] [1,2,3]triazol-1-yl)-3-(R)acrylonitrile derivatives (13d,j), which were recognised to act as MTAs agents. They were rationally designed, synthesised, characterised and subjected to different biological assessments. Computational docking was carried out in order to investigate the potential binding to the colchicine-binding site on tubulin. From this first prediction, the di-fluoro substitution seemed to be beneficial for the binding affinity with tubulin. The new fluorine derivatives, here presented, showed an improved antiproliferative activity when compared to the previously reported compounds. The biological evaluation included a preliminary antiproliferative screening on NCI60 cancer cells panel (1-10 μM). Compound 9a was selected as lead compound of the new series of derivatives. The in vitro XTT assay, flow cytometry analysis and immunostaining performed on HeLa cells treated with 9a showed a considerable antiproliferative effect, (IC50 = 3.2 μM), an increased number of cells in G2/M-phase, followed by an enhancement in cell division defects. Moreover, β-tubulin staining confirmed 9a as a MDA triggering tubulin disassembly, whereas colchicine-9a competition assay suggested that compound 9a compete with colchicine for the binding site on tubulin. Then, the co-administration of compound 9a and an extrusion pump inhibitor (EPI) was investigated: the association resulted beneficial for the antiproliferative activity and compound 9a showed to be client of extrusion pumps. Finally, structural superimposition of different colchicine binding site inhibitors (CBIs) in clinical trial and our MDA, provided an additional confirmation of the targeting to the predicted binding site. Physicochemical, pharmacokinetic and druglikeness predictions were also conducted and all the newly synthesised derivatives showed to be drug-like molecules., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

This study investigates the coordination chemistry of the tetradentate pyridine-containing 12-membered macrocycles L 1 - L 3 towards Platinum Group metal ions Pd II , Pt II , and Rh III . The reactions between the chloride salts of these metal ions and the three ligands in MeCN/H 2 O or MeOH/H 2 O (1:1 v / v ) are shown, and the isolated solid compounds are characterized, where possible, by mass spectroscopy and 1 H- and 13 C-NMR spectroscopic measurements. Structural characterization of the 1:1 metal-to-ligand complexes [Pd( L 1 )Cl] 2 [Pd 2 Cl 6 ], [Pt( L 1 )Cl](BF 4 ), [Rh( L 1 )Cl 2 ](PF 6 ), and [Rh( L 3 )Cl 2 ](BF 4 )·MeCN shows the coordinated macrocyclic ligands adopting a folded conformation, and occupying four coordination sites of a distorted square-based pyramidal and octahedral coordination environment for the Pd II /Pt II , and Rh III complexes, respectively. The remaining coordination site(s) are occupied by chlorido ligands. The reaction of L 3 with PtCl 2 in MeCN/H 2 O gave by serendipity the complex [Pt( L 3 )(m-1,3-MeCONH)PtCl(MeCN)](BF 4 ) 2 ·H 2 O, in which two metal centers are bridged by an amidate ligand at a Pt1-Pt2 distance of 2.5798(3) Å and feature one square-planar and one octahedral coordination environment. Density Functional Theory (DFT) calculations, which utilize the broken symmetry approach (DFT-BS), indicate a singlet d 8 - d 8 Pt II -Pt II ground-state nature for this compound, rather than the alleged d 9 - d 7 Pt I -Pt III mixed-valence character reported for related dinuclear Pt-complexes.

Skin secretions of the Mexican burrowing toad Rhinophrynus dorsalis (Rhinophrynidae) contain the proline-arginine-rich peptide, rhinophrynin-27 (RP-27; ELRLPEIARPVPEVLPARLPLPALPRN) with insulinotropic and immunomodulatory properties, together with a higher concentration of the biologically inactive form, rhinophrynin-33 (RP-33) that constitutes RP-27 extended from its C-terminus by the hexapeptide KMAKNQ. Determination of the conformation of RP-33 by NMR demonstrates that in both water and in a solvent that promotes protein folding (50% trifluoroethanol-water), the majority of the proline residues are found in a polyproline type II helical region. The peptide adopts a horseshoe (U-shaped) conformation with pronounced bends in the molecule of around 100°-120° at Glu 13 and Arg 18 . The hexapeptide extension adopts a α-helical conformation. When the hexapeptide is excised to generate RP-27, the molecule adopts an L-shaped conformation with a single bend at Glu 13 . A search of protein sequence databases indicated the P-X-P-XXX-P-XXX-P-X-P motif found in RP-27 and RP-33 occurs in a number of proteins although its functional implications are unclear. The data suggest that RP-33 represents a biosynthetic precursor of RP-27 that is activated by a protease cleaving at a single lysine residue of the type previously identified in Xenopus laevis skin secretions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Modern medicine relies upon antibiotics, but we have arrived to the point where our inability to come up with new effective molecules against resistant pathogens, together with the declining private investment, is resulting in the number of untreatable infections increasing worldwide at worrying pace. Among other pathogens, widely recognized institutions have indicated Gram-negative bacteria as particularly challenging, due to the presence of the outer membrane. The very first step in the action of every antibiotic or adjuvant is the permeation through this membrane, with small hydrophilic drugs usually crossing through protein channels. Thus, a detailed understanding of their properties at a molecular level is crucial. By making use of Molecular Dynamics simulations, we compared the two main porins of four members of the Enterobacteriaceae family, and, in this paper, we show their shared geometrical and electrostatic characteristics. Then, we used metadynamics simulations to reconstruct the free energy for permeation of selected diazobicyclooctans through OmpF. We demonstrate how porins features are coupled to those of the translocating species, modulating their passive permeation. In particular, we show that the minimal projection area of a molecule is a better descriptor than its molecular mass or the volume. Together with the magnitude and orientation of the electric dipole moment, these are the crucial parameters to gain an efficient compensation between the entropic and enthalpic contributions to the free energy barrier required for permeation. Our results confirm the possibility to predict the permeability of molecules through porins by using a few molecular parameters and bolster the general model according to which the free energy increase is mostly due to the decrease of conformational entropy, and this can be compensated by a favorable alignment of the electric dipole with respect to the channel intrinsic electric field.

A family of acyclic squaramide receptors (L1-L5) have been synthesised with the aim to bind anions in a competitive solvent mixture and to evaluate how the presence of additional H-bond donor groups on the squaramide scaffold could affect the affinity towards anions and the transmembrane transport ability.

The synthesis and coordination properties of two fluorescent chemosensors, featuring [9]aneN 3 (1,4,7-triazacyclononane; L1) and [12]aneNS 3 (1-aza-4,7,10-trithiacyclododecane; L2) as receptor units, and a quinoline pendant arm with an amide group as a functional group spacer are described. The optical responses of L1 and L2 in the presence of several metal ions were analysed in MeCN/H 2 O (1 : 4 v/v) solutions. A selective chelation enhancement of fluorescence (CHEF) effect was observed in the presence of Zn 2+ in the case of L1, and in the presence of Cd 2+ in the case of L2, following the formation of a 1 : 1 and a 1 : 2 metal/ligand complex, respectively, which was also confirmed by potentiometric measurements. 1 H and 13 C NMR measurements in CD 3 CN/CDCl 3 in combination with molecular mechanics calculations show that for both complexes of L1 and L2 with Zn 2+ and Cd 2+ , respectively, the coordination of the carbonyl group from the pendant arm could be the origin of the observed optical selectivity., (© 2020 Wiley-VCH GmbH.)

The study investigates conformational analysis and the in vitro cytokine-mediated immunomodulatory and insulin-releasing activities of rhinophrynin-27 (ELRLPEIARPVPEVLPARLPLPALPRN; RP-27), a proline-arginine-rich peptide first isolated from skin secretions of the Mexican burrowing toad Rhinophrynus dorsalis (Rhinophrynidae). In both water and 50% trifluoroethanol-water, the peptide adopts a polyproline type II helical conformation with a high degree of deviation from the canonical collagen-like folding and a pronounced bend in the molecule at the Glu 13 residue. Incubation of mouse peritoneal cells with RP-27 significantly (P < 0.05) inhibited production of the pro-inflammatory cytokines TNF-α and IL-1β and stimulated production of the anti-inflammatory cytokine IL-10. The peptide significantly (P < 0.01) stimulated release of insulin from BRIN-BD11 rat clonal β-cells at concentrations ≥ 1 nM while maintaining the integrity of the plasma membrane and also stimulated insulin release from isolated mouse islets at a concentration of 10 -6  M. Increasing the cationicity of RP-27 by substituting glutamic acid residues in the peptide by arginine and increasing hydrophobicity by substituting alanine residues by tryptophan did not result in analogues with increased activity with respect to cytokine production and insulin release. The combination of immunosuppressive and insulinotropic activities together with very low cytotoxicity suggests that RP-27 may represent a template for the development of an agent for use in anti-inflammatory and Type 2 diabetes therapies., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

We describe here the synthesis and coordination properties of three new derivatives of [9]aneN3 containing phenyl/quinoline pendant arm derivatives (L1, L2 and L3, respectively) also featuring urea (L1-L2) or amide (L3) functions as "non-innocent" spacers. At first, L1, L2 and L3 were studied considering the interaction with a series of anions (AcO-, BzO-, H2PO4-, F-, and Cl-) by means of 1H NMR measurements. Subsequently, the optical responses of L2 and L3 in the presence of several metal ions Cd2+, Co2+, Cu2+, Fe3+, Hg2+, K+, Mg2+, Mn2+, Ni2+, Zn2+ and Pb2 were analysed in MeCN/H2O (4 : 1 v/v). As observed by spectrophotometric and spectrofluorimetric titrations, there were significant changes in the absorbance and fluorescent emission of L2 upon addition of increasing amounts of Cd2+, Zn2+, Pb2+ and Cu2+ in MeCN/H2O (4 : 1 v/v). In particular, titrations of L2 with Cd2+, Zn2+ or Pb2+ showed an almost comparable CHEF effect up to an M2+/L2 molar ratio of 1. Overall, no significant optical selectivity was observed in the case of L2. Conversely, L3 revealed an OFF-ON selective response only in the presence of the Zn2+ ion in MeCN/H2O (4 : 1 v/v), which can be attributed to the formation of both 1 : 1 and 1 : 2 metal-to-ligand complexes, as also confirmed by potentiometric measurements. Finally, crystals of [ZnL1(Ac)](Ac) (1), [CuL1(Cl)](Cl)·H2O (2) and [CuL3](NO3) (3) were grown and analysed by X-ray diffraction. 1 and 3 feature the metal center in a pseudo-octahedral coordination geometry coordinated also by the carbonyl group from one pendant arm, while in the case of 2, one of the six coordination sites in the final distorted octahedral geometry is occupied by the nitrogen donor from the urea group of one pendant arm.

Nuclear magnetic resonance and infrared spectroscopy have been used to investigate the formation of complexes of BAL30072 with Fe 3+ and Ga 3+ in solution and to collect geometrical parameters supporting reliable 3D structure models. Structural models for the ligand-metal complexes with different stoichiometries have been characterized using density functional theory calculations. Blind ensemble docking to the PiuA receptor from P. aeruginosa was performed for the different complexes to compare binding affinities and statistics of the residues most frequently contacted. When compared to analogues, BAL30072 was found to have an intrinsic propensity to form complexes with low ligand-to-metal stoichiometry. By using one of the sulfate oxygen atoms as a third donor in addition to the bidentate pyridinone moiety, BAL30072 can form a L 2 M complex, which was predicted to be the one with the best binding affinity to PiuA. The example of BAL30072 strongly suggests that a lower stoichiometry might be the one recognized by the receptor, so that to focus only on the highest stoichiometry might be misleading for siderophores with less than six donors.

New strategies are urgently required to develop antibiotics. The siderophore uptake system has attracted considerable attention, but rational design of siderophore antibiotic conjugates requires knowledge of recognition by the cognate outer-membrane transporter. Acinetobacter baumannii is a serious pathogen, which utilizes (pre)acinetobactin to scavenge iron from the host. We report the structure of the (pre)acinetobactin transporter BauA bound to the siderophore, identifying the structural determinants of recognition. Detailed biophysical analysis confirms that BauA recognises preacinetobactin. We show that acinetobactin is not recognised by the protein, thus preacinetobactin is essential for iron uptake. The structure shows and NMR confirms that under physiological conditions, a molecule of acinetobactin will bind to two free coordination sites on the iron preacinetobactin complex. The ability to recognise a heterotrimeric iron-preacinetobactin-acinetobactin complex may rationalize contradictory reports in the literature. These results open new avenues for the design of novel antibiotic conjugates (trojan horse) antibiotics., Competing Interests: LM, IS, MS, EO, MP, MC, JN No competing interests declared, (© 2018, Moynié et al.)

Voltage-dependent anion-selective channels (VDACs) are primarily located in the mitochondrial outer membrane (MOM). They are essential for the regulation of ion and metabolite exchanges. In particular, their role in energy-related nucleotide exchange has many implications in apoptosis, cancer, and neurodegenerative diseases. It has been proposed that VDACs' functions are regulated by mobility of the N-terminal helical domain, which is bound to the inner wall of the main β-barrel domain but exists in equilibrium between the bound-folded and the unbound-unfolded state. When the N-terminal domain detaches from the channel's wall and eventually leaves the lumen, it can either stay exposed to the cytosolic environment or interact with the outer leaflet of the MOM; then, it may also interact with other protein partners. In humans, three different VDAC isoforms are expressed at different tissue-specific levels with evidence of distinct roles. Although the N-terminal domains share high sequence similarity, important differences do exist, with the functionality of the entire protein mostly attributed to them. In this work, the three-dimensional structure and membrane affinity of the three isolated hVDAC N-terminal peptides have been compared through Fourier-transform infrared and NMR spectroscopy in combination with molecular dynamics simulations, and measurement of the surface pressure of lipid monolayers. Although peptides were studied as isolated from the β-barrel domain, the observed differences are relevant for those whole protein's functions in which a protein-protein interaction is mediated by the N-terminal domain., Competing Interests: The authors declare no competing financial interest.

Antimicrobial peptides attracted increasing interest in last decades due to the rising concern of multi-drug resistant pathogens. Dendrimeric peptides are branched molecules with multiple copies of one peptide functional unit bound to the central core. Compared to linear analogues, they usually show improved activity and lower susceptibility to proteases. Knowledge of structure-function relationship is fundamental to tailor their properties. This work is focused on SB056, the smallest example of dendrimeric peptide, whose amino acid sequence is WKKIRVRLSA. Two copies are bound to the α- and ε- nitrogen of one lysine core. An 8-aminooctanamide was added at the C-terminus to improve membrane affinity. Its propensity for β-type structures is also interesting, since helical peptides were already thoroughly studied. Moreover, SB056 maintains activity at physiological osmolarity, a typical limitation of natural peptides. An optimized analogue with improved performance was designed, β-SB056, which differs only in the relative position of the first two residues (KWKIRVRLSA). This produced remarkable differences. Structure order and aggregation behavior were characterized by using complementary techniques and membrane models with different negative charge. Infrared spectroscopy showed different propensity for ordered β-sheets. Lipid monolayers' surface pressure was measured to estimate the area/peptide and the ability to perturb lipid packing. Fluorescence spectroscopy was applied to compare peptide insertion into the lipid bilayer. Such small change in primary structure produced fundamental differences in their aggregation behavior. A regular amphipathic peptide's primary structure was responsible for ordered β-sheets in a charge independent fashion, in contrast to unordered aggregates formed by the former analogue., (Copyright © 2018 Elsevier Inc. All rights reserved.)

NiP alloys are very often used in industry, due to their outstanding performance in corrosion and wear. Alloys with high phosphorus content (≥16 atom % P) are amorphous and show high corrosion resistance in both neutral and acidic solutions irrespective of the presence of chloride ions. The reason for this behavior is attributed to the formation of a "P-enriched surface layer" whose exact nature is still under debate. In this work, an iterative algorithm based on the application of maximum entropy method on nondestructive angle-resolved X-ray photoelectron spectroscopy data has been applied to the investigation of the surface layer grown on Ni18P alloys following mechanical polishing and anodic polarization in sulfate solutions. The results show that the outermost region of the examined alloy has a complex layered structure: (1) an uppermost hydrocarbon contamination layer about 1 nm thick, which includes also adsorbed water; (2) a nickel (poly)phosphate layer of about 1 nm; (3) a highly phosphorus-enriched interface being about 2 nm thick with a marked phosphorus concentration gradient, from 70 to 20 atom %; and (4) bulk alloy with the stoichiometric composition. These findings, together with the chemical state of the different phosphorus compounds, allow us to conclude that the high corrosion and wear resistance of NiP alloys might be ascribed to the presence of a thin, self-repairing nickel (poly)phosphate layer grown on a strongly P-enriched interface. Because the Auger parameter of P at the interface is similar to that of elemental P, it might be also concluded that the interface is enriched in elemental phosphorus., Competing Interests: The authors declare no competing financial interest.

Pseudomonas aeruginosa is a major cause of chronic lung infections in cystic fibrosis (CF) patients. The ability of the bacterium to form biofilms and the presence of a thick and stagnant mucus in the airways of CF patients largely contribute to antibiotic therapy failure and demand for new antimicrobial agents able to act in the CF environment. The present study investigated the anti- P. aeruginosa activity of lin-SB056-1, a recently described semi-synthetic antimicrobial peptide, used alone and in combination with the cation chelator ethylenediaminetetraacetic acid (EDTA). Bactericidal assays were carried out in standard culture conditions and in an artificial sputum medium (ASM) closely resembling the CF environment. Peptide's structure and interaction with large unilamellar vesicles in media with different ionic strengths were also investigated through infrared spectroscopy. Lin-SB056-1 demonstrated fast and strong bactericidal activity against both mucoid and non-mucoid strains of P. aeruginosa in planktonic form and, in combination with EDTA, caused significant reduction of the biomass of P. aeruginosa mature biofilms. In ASM, the peptide/EDTA combination exerted a strong bactericidal effect and inhibited the formation of biofilm-like structures of P. aeruginosa . Overall, the results obtained highlight the potential of the lin-SB056-1/EDTA combination for the treatment of P. aeruginosa lung infections in CF patients., Competing Interests: The authors declare no conflict of interest.

Transport of molecules through biological membranes is a fundamental process in biology, facilitated by selective channels and general pores. The architecture of some outer membrane pores in Gram-negative bacteria, common to other eukaryotic pores, suggests them as prototypes of electrostatically regulated nanosieve devices. In this study, we sensed the internal electrostatics of the two most abundant outer membrane channels of Escherichia coli, using norfloxacin as a dipolar probe in single molecule electrophysiology. The voltage dependence of the association rate constant of norfloxacin interacting with these nanochannels follows an exponential trend, unexpected for neutral molecules. We combined electrophysiology, channel mutagenesis, and enhanced sampling molecular dynamics simulations to explain this molecular mechanism. Voltage and temperature dependent ion current measurements allowed us to quantify the transversal electric field inside the channel as well as the distance where the applied potential drops. Finally, we proposed a general model for transport of polar molecules through these electrostatic nanosieves. Our model helps to further understand the basis for permeability in Gram-negative pathogens, contributing to fill in the innovation gap that has limited the discovery of effective antibiotics in the last 20 years.

The increasing level of antibiotic resistance in Gram-negative bacteria, together with the lack of new potential drug scaffolds in the pipeline, make the problem of infectious diseases a global challenge for modern medicine. The main reason that Gram-negative bacteria are particularly challenging is the presence of an outer cell-protecting membrane, which is not present in Gram-positive species. Such an asymmetric bilayer is a highly effective barrier for polar molecules. Several protein systems are expressed in the outer membrane to control the internal concentration of both nutrients and noxious species, in particular: (i) water-filled channels that modulate the permeation of polar molecules and ions according to concentration gradients, and (ii) efflux pumps to actively expel toxic compounds. Thus, besides expressing specific enzymes for drugs degradation, Gram-negative bacteria can also resist by modulating the influx and efflux of antibiotics, keeping the internal concentration low. However, there are no direct and robust experimental methods capable of measuring the permeability of small molecules, thus severely limiting our knowledge of the molecular mechanisms that ultimately control the permeation of antibiotics through the outer membrane. This is the innovation gap to be filled for Gram-negative bacteria. This review is focused on the permeation of small molecules through porins, considered the main path for the entry of polar antibiotics into Gram-negative bacteria. A fundamental understanding of how these proteins are able to filter small molecules is a prerequisite to design/optimize antibacterials with improved permeation. The level of sophistication of modern molecular modeling algorithms and the advances in new computer hardware has made the simulation of such complex processes possible at the molecular level. In this work we aim to share our experience and perspectives in the context of a multidisciplinary extended collaboration within the IMI-Translocation consortium. The synergistic combination of structural data, in vitro assays and computer simulations has proven to give new insights towards the identification and description of physico-chemical properties modulating permeation. Once similar general rules are identified, we believe that the use of virtual screening techniques will be very helpful in searching for new molecular scaffolds with enhanced permeation, and that molecular modeling will be of fundamental assistance to the optimization stage.

A major challenge in the discovery of the new antibiotics against Gram-negative bacteria is to achieve sufficiently fast permeation in order to avoid high doses causing toxic side effects. So far, suitable assays for quantifying the uptake of charged antibiotics into bacteria are lacking. We apply an electrophysiological zero-current assay using concentration gradients of β-lactamase inhibitors combined with single-channel conductance to quantify their flux rates through OmpF. Molecular dynamic simulations provide in addition details on the interactions between the nanopore wall and the charged solutes. In particular, the interaction barrier for three β-lactamase inhibitors is surprisingly as low as 3-5 kcal/mol and only slightly above the diffusion barrier of ions such as chloride. Within our macroscopic constant field model, we determine that at a zero-membrane potential a concentration gradient of 10 μM of avibactam, sulbactam, or tazobactam can create flux rates of roughly 620 molecules/s per OmpF trimer.

Microbial resistance to conventional antibiotics is one of the most outstanding medical and scientific challenges of our times. Despite the recognised need for new anti-infective agents, however, very few new drugs have been brought to the market and to the clinic in the last three decades. This review highlights the properties of a new class of antibiotics, namely dendrimeric peptides. These intriguing novel compounds, generally made of multiple peptidic sequences linked to an inner branched core, display an array of antibacterial, antiviral and antifungal activities, usually coupled to low haemolytic activity. In addition, several peptides synthesized in oligobranched form proved to be promising tools for the selective treatment of cancer cells.

Dendrimeric peptides make a versatile group of bioactive peptidomimetics and a potential new class of antimicrobial agents to tackle the pressing threat of multi-drug resistant pathogens. These are branched supramolecular assemblies where multiple copies of the bioactive unit are linked to a central core. Beyond their antimicrobial activity, dendrimeric peptides could also be designed to functionalize the surface of nanoparticles or materials for other medical uses. Despite these properties, however, little is known about the structure-function relationship of such compounds, which is key to unveil the fundamental physico-chemical parameters and design analogues with desired attributes. To close this gap, we focused on a semi-synthetic, two-branched peptide, SB056, endowed with remarkable activity against both Gram-positive and Gram-negative bacteria and limited cytotoxicity. SB056 can be considered the smallest prototypical dendrimeric peptide, with the core restricted to a single lysine residue and only two copies of the same highly cationic 10-mer polypeptide; an octanamide tail is present at the C-terminus. Combining NMR and Molecular Dynamics simulations, we have determined the 3D structure of two analogues. Fluorescence spectroscopy was applied to investigate the water-bilayer partition in the presence of vesicles of variable charge. Vesicle leakage assays were also performed and the experimental data were analyzed by applying an iterative Monte Carlo scheme to estimate the minimum number of bound peptides needed to achieve the release. We unveiled a singular beta hairpin-type structure determined by the peptide chains only, with the octanamide tail available for further functionalization to add new potential properties without affecting the structure.

Four host-defense peptides belonging to the tigerinin family (tigerinin-1O: RICTPIPFPMCY; tigerinin-2O: RTCIPIPLVMC; tigerinin-3O: RICTAIPLPMCL; and tigerinin-4O: RTCIPIPPVCF) were isolated from skin secretions of the African crowned bullfrog Hoplobatrachus occipitalis. In aqueous solution at pH 4.8, the cyclic domain of tigerinin-2O adopts a rigid amphipathic conformation that incorporates a flexible N-terminal tail. The tigerinins lacked antimicrobial (MIC > 100 μM) and hemolytic (LC50 > 500 μM) activities but, at a concentration of 20 μg/mL, significantly (P < 0.05) inhibited production of interferon-γ (IFN-γ) by peritoneal cells from C57BL/6 mice without affecting production of IL-10 and IL-17. Tigerinin-2O and -4O inhibited IFN-γ production at concentrations as low as 1 μg/mL. The tigerinins significantly (P ≤ 0.05) stimulated the rate of insulin release from BRIN-BD11 clonal β-cells without compromising the integrity of the plasma membrane. Tigerinin-1O was the most potent (threshold concentration 1 nM) and the most effective (395% increase over basal rate at a concentration of 1 μM). Tigerinin-4O was the most potent and effective peptide in stimulating the rate of glucagon-like peptide-1 release from GLUTag enteroendocrine cells (threshold concentration 10 nM; 289% increase over basal rate at 1 μM). Tigerinin peptides have potential for development into agents for the treatment of patients with type 2 diabetes.

Background: In Gram-negative bacteria, the outer-membrane represents an additional barrier for antibiotics to permeate inside pathogens. Our inability to come up with novel effective antibiotics mostly relies upon insufficient understanding of the molecular basis behind outer-membrane penetration., Results: Polar antibiotics can permeate through water-filled porins, such as OmpF and OmpC from Escherichia coli. Through molecular modeling, permeation of imipenem and meropenem was found to be strongly dependent upon capability of drugs to properly align their electric dipole to the internal electric field in the restricted region of the pore. Electrostatics differences between OmpF and OmpC, and modifications along a series of OmpC mutants from E. coli-resistant clinical strains identify a 'preorientation' region, which dramatically affects antibiotic pathway., Conclusion: A novel perspective is presented, suggesting new molecular properties to be included in drug design.

Multi-drug resistance bacteria are a challenging problem of contemporary medicine. This is particularly critical for Gram-negative bacteria, where antibiotics are hindered by the outer membrane to reach internal targets. Here more polar antibiotics make use of nanometric water-filled channels to permeate inside. We present in this work a computational all-atom approach, using water as a probe, for the calculation of the macroscopic electric field inside water-filled channels. The method allows one to compare not only different systems but also the same system under different conditions, such as pH and ion concentration. This provides a detailed picture of electrostatics in biological nanopores shedding more light on how the charged residues of proteins determine the electric field inside, and also how medium can tune it. These details are central to unveil the filtering mechanism behind the permeation of small polar molecules through nanometric water-filled channels.

The human VDAC channel exists in three isoforms characterized by high sequence homology and structural similarity. Yet the function and mode of action of hVDAC3 are still elusive. The presence of six surface cysteines exposed to the oxidizing environment of the mitochondrial inter-membrane space suggests the possible establishment of intramolecular disulfide bonds. Two natural candidates for disulfide bridge formation are Cys2 and Cys8 that, located on the flexible N-terminal domain, can easily come in contact. A third potentially important residue is Cys122 that is close to Cys2 in the homology model of VDAC3. Here we analyzed the impact of SS bonds through molecular dynamics simulations of derivatives of hVDAC3 (dubbed SS-2-8, SS-2-122, SS-8-122) including a single disulfide bond. Simulations showed that in SS-8-122, the fragment 1-7 crosses the top part of the barrel partially occluding the pore and causing a 20% drop of conductance. In order to identify other potential channel-occluding disulfide bonds, we used a set of neural networks and structural bioinformatics algorithms, after filtering with the steric constraints imposed by the 3D-structure. We identified other three species, namely SS-8-65, SS-2-36 and SS-8-36. While the conductance of SS-8-65 and SS-2-36 is about 30% lower than that of the species without disulfide bonds, the conductance of SS-8-36 was 40-50% lower. The results show how VDAC3 is able to modulate its pore size and current by exploiting the mobility of the N-terminal and forming, upon external stimuli, disulfide bridges with cysteine residues located on the barrel and exposed to the inter-membrane space., (Copyright © 2016 Elsevier B.V. All rights reserved)

Peptide-based antibiotics might help containing the rising tide of antimicrobial resistance. We developed SB056, a semi-synthetic peptide with a dimeric dendrimer scaffold, active against both Gram-negative and Gram-positive bacteria. Being the mechanism of SB056 attributed to disruption of bacterial membranes, we enhanced the amphiphilic profile of the original, empirically derived sequence [WKKIRVRLSA-NH2] by interchanging the first two residues [KWKIRVRLSA-NH2], and explored the effects of this modification on the interaction of peptide, both in linear and dimeric forms, with model membranes and on antimicrobial activity. Results obtained against Escherichia coli and Staphylococcus aureus planktonic strains, with or without salts at physiological concentrations, confirmed the added value of dendrimeric structure over the linear one, especially at physiological ionic strength, and the impact of the higher amphipathicity obtained through sequence modification on enhancing peptide performances. SB056 peptides also displayed intriguing antibiofilm properties. Staphylococcus epidermidis was the most susceptible strain in sessile form, notably to optimized linear analog lin-SB056-1 and the wild-type dendrimer den-SB056. Membrane affinity of all peptides increased with the percentage of negatively charged lipids and was less influenced by the presence of salt in the case of dendrimeric peptides. The analog lin-SB056-1 displayed the highest overall affinity, even for zwitterionic PC bilayers. Thus, in addition to electrostatics, distribution of charged/polar and hydrophobic residues along the sequence might have a significant role in driving peptide-lipid interaction. Supporting this view, dendrimeric analog den-SB056-1 retained greater membrane affinity in the presence of salt than den-SB056, despite the fact that they bear exactly the same net positive charge.

Three fluorescent asymmetric bis-urea receptors (L1-L3) have been synthesised. The binding properties of L1-L3 towards different anions (fluoride, acetate, hydrogencarbonate, dihydrogen phosphate, and hydrogen pyrophosphate HPpi(3-)) have been studied by means of (1)H-NMR, UV-Vis and fluorescence spectroscopy, single crystal X-ray diffraction, and theoretical calculations. In particular, a remarkable affinity for HPpi(3-) has been observed in the case L1 (DMSO-d6/0.5% H2O) which also acts as a fluorimetric chemosensor for this anion. Interestingly, when L1 is included in cetyltrimethylammonium (CTAB) micelles, hydrogen pyrophosphate recognition can also be achieved in pure water.

Integral membrane proteins known as porins are the major pathway by which hydrophilic antibiotics cross the outer membrane of Gram-negative bacteria. Single point mutations in porins can decrease the permeability of an antibiotic, either by reduction of channel size or modification of electrostatics in the channel, and thereby confer clinical resistance. Here, we investigate four mutant OmpC proteins from four different clinical isolates of Escherichia coli obtained sequentially from a single patient during a course of antimicrobial chemotherapy. OmpC porin from the first isolate (OmpC20) undergoes three consecutive and additive substitutions giving rise to OmpC26, OmpC28, and finally OmpC33. The permeability of two zwitterionic carbapenems, imipenem and meropenem, measured using liposome permeation assays and single channel electrophysiology differs significantly between OmpC20 and OmpC33. Molecular dynamic simulations show that the antibiotics must pass through the constriction zone of porins with a specific orientation, where the antibiotic dipole is aligned along the electric field inside the porin. We identify that changes in the vector of the electric field in the mutated porin, OmpC33, create an additional barrier by "trapping" the antibiotic in an unfavorable orientation in the constriction zone that suffers steric hindrance for the reorientation needed for its onward translocation. Identification and understanding the underlying molecular details of such a barrier to translocation will aid in the design of new antibiotics with improved permeation properties in Gram-negative bacteria., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes.

Pseudhymenochirin-1Pb (Ps-1Pb; IKIPSFFRNILKKVGKEAVSLIAGALKQS) and pseudhymenochirin-2Pa (Ps-2Pa; GIFPIFAKLLGKVIKVASSLISKGRTE) are amphibian peptides with broad spectrum antimicrobial activities and cytotoxicity against mammalian cells. In the membrane-mimetic solvent 50% (v/v) trifluoroethanol-H2O, both peptides adopt a well-defined α-helical conformation that extends over almost all the sequence and incorporates a flexible bend. Both peptides significantly (p < 0.05) stimulate the rate of release of insulin from BRIN-BD11 clonal β-cells at concentrations ≥ 0.1 nM but produce loss of integrity of the plasma membrane at concentrations ≥ 1 μM. Increasing cationicity by the substitution Glu(17) → l-Lys in Ps-1Pb and Glu(27) → l-Lys in Ps-2Pa generates analogues with increased cytotoxicity and reduced insulin-releasing potency. In contrast, the analogues [R8r]Ps-1Pb and [K8k,K19k]Ps-2Pa, incorporating d-amino acid residues to destabilize the α-helical domains, retain potent insulin-releasing activity but are nontoxic to BRIN-BD11 cells at concentrations of 3 μM. [R8r]Ps-1Pb produces a significant increase in insulin release rate at 0.3 nM and [K8k,K19k]Ps-2Pa at 0.01 nM. Both analogues show low hemolytic activity (IC50 > 100 μM) but retain broad-spectrum antimicrobial activity and remain cytotoxic to a range of human tumor cell lines, albeit with lower potency than the naturally occurring peptides. These analogues show potential for development into agents for type 2 diabetes therapy.

As a contribution to understand the interactions between mineral surfaces and organic molecules, this study reports an accurate characterization of the bis-(2-ethylhexyl) phthalate (DEHP)-Hydrozincite (DEHP-HY), that has been conduced combining the following techniques: FTIR, NMR, XAS spectroscopies and XRD. XRD patterns indicate that the HY is made of nanocrystals whose size is not influenced by the presence of DEHP. The (1)H NMR analysis of DEHP-HY samples points out the presence of interactions of DEHP with HY. CPMAS NMR analysis suggests that the interaction is operated by ester carbonyl groups while the aliphatic chain, as expected, is not involved. MAS and CPMAS NMR measurements, performed on (13)C ester carbonyl enriched DEHP, allow to demonstrate that there are two ester carbonyl linkage sites interacting at the HY surface: an acid site with a strong link and a second one with weak chemical interactions. Zn K-edge XAS spectroscopy demonstrates that the local atomic structure around Zn in DEHP-HY sample remains essentially unchanged with respect to that of HY. Such a weak structural effect suggests that HY interaction with DEHP is limited to the nanoparticle surface., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The objective of this study is to identify the structural features governing the transport of molecules through nanometric channel proteins at a molecular level. Our focus is to come up with a precise understanding of the structure and dynamics of the outer membrane porin OprD of the Gram-negative bacterium Pseudomonas aeruginosa by studying the translocation of natural amino acid residues/substrates through it. We used in silico electrophysiology and metadynamics simulation techniques as they can provide precise information on the molecule/channel interactions at the atomic scale that allows testing quantitative structure-function relationships. We performed our simulations on the whole OprD protein, with all loops modelled and without any constraints to keep the channel open. Dynamics of both internal and external loops and the polar nature of the eyelet region play important roles in modulating the translocation of molecules through OprD by creating two alternative paths for translocation. All positive residues take the main path upon binding in the negative pocket just above the constriction region. The same factor is unfavourable for negative substrates and hence they have a relatively high barrier for translocation. Differently, neutral substrates do not show any specificity and they can follow the two alternative paths.

The voltage-dependent anion channel (VDAC) is the main mitochondrial porin allowing the exchange of ions and metabolites between the cytosol and the mitochondrion. In addition, VDAC was found to actively interact with proteins playing a fundamental role in the regulation of apoptosis and being of central interest in cancer research. VDAC is a large transmembrane β-barrel channel, whose N-terminal helical fragment adheres to the channel interior, partially closing the pore. This fragment is considered to play a key role in protein stability and function as well as in the interaction with apoptosis-related proteins. Three VDAC isoforms are differently expressed in higher eukaryotes, for which distinct and complementary roles are proposed. In this work, the folding propensity of their N-terminal fragments has been compared. By using multiple spectroscopic techniques, and complementing the experimental results with theoretical computer-assisted approaches, we have characterized their conformational equilibrium. Significant differences were found in the intrinsic helical propensity of the three peptides, decreasing in the following order: hVDAC2 > hVDAC3 > hVDAC1. In light of the models proposed in the literature to explain voltage gating, selectivity, and permeability, as well as interactions with functionally related proteins, our results suggest that the different chemicophysical properties of the N-terminal domain are possibly correlated to different functions for the three isoforms. The overall emerging picture is that a similar transmembrane water accessible conduit has been equipped with not identical domains, whose differences can modulate the functional roles of the three VDAC isoforms.

Although the role of general bacterial porins is well established as main pathway for polar antibiotics, the molecular details of their mode-of-action are still under debate. Using molecular dynamics simulations and water as a probe, we demonstrated the strong ordering of water molecules, differently tuned along the axis of diffusion in the transversal direction. Preserved features and important differences were characterized for different channels, allowing to put forward a general model for molecular filtering. The intrinsic electric field, responsible for water ordering, (i) filters those dipolar molecules that can compensate the entropy decrease by dipole alignment in the restricted region and (ii) might create an additional barrier by changing direction when escaping from the restricted region. We tested this model using two antibiotics, cefepime and cefotaxime, through metadynamics free energy calculations. A rational drug design should take this into account for screening molecules with improved permeation properties.

SB056 is a novel semi-synthetic antimicrobial peptide with a dimeric dendrimer scaffold. Active against both Gram-negative and -positive bacteria, its mechanism has been attributed to a disruption of bacterial membranes. The branched peptide was shown to assume a β-stranded conformation in a lipidic environment. Here, we report on a rational modification of the original, empirically derived linear peptide sequence [WKKIRVRLSA-NH2, SB056-lin]. We interchanged the first two residues [KWKIRVRLSA-NH2, β-SB056-lin] to enhance the amphipathic profile, in the hope that a more regular β-strand would lead to a better antimicrobial performance. MIC values confirmed that an enhanced amphiphilic profile indeed significantly increases activity against both Gram-positive and -negative strains. The membrane binding affinity of both peptides, measured by tryptophan fluorescence, increased with an increasing ratio of negatively charged/zwitterionic lipids. Remarkably, β-SB056-lin showed considerable binding even to purely zwitterionic membranes, unlike the original sequence, indicating that besides electrostatic attraction also the amphipathicity of the peptide structure plays a fundamental role in binding, by stabilizing the bound state. Synchrotron radiation circular dichroism and solid-state 19F-NMR were used to characterize and compare the conformation and mobility of the membrane bound peptides. Both SB056-lin and β-SB056-lin adopt a β-stranded conformation upon binding POPC vesicles, but the former maintains an intrinsic structural disorder that also affects its aggregation tendency. Upon introducing some anionic POPG into the POPC matrix, the sequence-optimized β-SB056-lin forms well-ordered β-strands once electro-neutrality is approached, and it aggregates into more extended β-sheets as the concentration of anionic lipids in the bilayer is raised. The enhanced antimicrobial activity of the analogue correlates with the formation of these extended β-sheets, which also leads to a dramatic alteration of membrane integrity as shown by 31P-NMR. These findings are generally relevant for the design and optimization of other membrane-active antimicrobial peptides that can fold into amphipathic β-strands.

The fluorescent ligand (L) based on the N2S2 pyridinophane macrocycle and the 7-nitrobenzo[1,2,5]oxadiazole (NBD) fluorogenic fragment has been synthesized to coordinate Pd(II) ions. Loaded on dye-doped silica nanoparticles, L can be used as a ratiometric fluorescent chemosensor for Pd(II) in water with high selectivity toward other metal ions including the platinum group ones.

Antimicrobial peptides (AMPs) are effectors of the innate immunity of most organisms. Their role in the defense against pathogen attack and their high selectivity for bacterial cells make them attractive for the development of a new class of antimicrobial drugs. The N-terminal fragment of the frog-skin peptide esculentin-1b (Esc(1-18)) has shown broad-spectrum antimicrobial activity. Similarly to most cationic AMPs, it is supposed to act by binding to and damaging the negatively charged plasma membrane of bacteria. Differently from many other AMPs, Esc(1-18) activity is preserved in biological fluids such as serum. In this work, a structural investigation was performed through NMR spectroscopy. The 3D structure was obtained in the presence of either zwitterionic or negatively charged micelles as membrane models for eukaryotic and prokaryotic membranes, respectively. Esc(1-18) showed a higher affinity for and deeper insertion into the latter and adopted an amphipathic helical structure characterized by a kink at the residue G8. These findings were confirmed by measuring penetration into lipid monolayers. The presence of negatively charged lipids in the bilayer appears to be necessary for Esc(1-18) to bind, to fold in the right three-dimensional structure, and, ultimately, to exert its biological role as an AMP.

Hymenochirin-1Pa (LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2) is a host-defense peptide first isolated from skin secretions of the frog Pseudhymenochirus merlini (Pipidae). A nuclear magnetic resonance structural investigation demonstrates that the peptide has a random coil conformation in water but, in the membrane-mimetic solvent 50% (v/v) trifluoroethanol-water adopts a well-defined conformation characterized by two α-helical domains from residues K6 to G17 and from G21 to M28, with the N-terminal region unfolded. The presence of a GXXXG domain, the most common structural motif found at the interface between interacting trans-membrane helices, between residues 17 and 21, introduces a kink corresponding to a deviation from linearity of 93 ± 31°. Hymenochirin-1Pa shows broad spectrum anti-bacterial activity, including high potency against multidrug-resistant clinical isolates of Staphylococcus aureus, Acinetobacter baumannii, and Stenotrophomonas maltophilia. The peptide also shows high cytotoxic potency against human non-small lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells but its therapeutic potential as an anti-cancer agent is limited by moderate hemolytic activity against human erythrocytes and lack of selectivity for tumor cells. Increasing cationicity of the peptide by substituting the Asp(9) residue by either L-Lys (K) or D-Lys (k) has relatively minor effects on antimicrobial and anti-tumor potencies but the [D9k] analog is non-hemolytic LC50 > 400 μM. Thus, [D9k]hymenochirin-1Pa may serve as a template for the design of non-toxic antimicrobial agents for use against multidrug-resistant pathogenic bacteria., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Voltage Dependent Anion-selective Channels (VDACs) are pore-forming proteins located in the outer mitochondrial membrane. They are responsible for the access of ions and energetic metabolites into the inner membrane transport systems. Three VDAC isoforms exist in mammalian, but their specific role is unknown. In this work we have performed extensive (overall ∼5 µs) Molecular Dynamics (MD) simulations of the human VDAC isoforms to detect structural and conformational variations among them, possibly related to specific functional roles of these proteins. Secondary structure analysis of the N-terminal domain shows a high similarity among the three human isoforms of VDAC but with a different plasticity. In particular, the N-terminal domain of the hVDAC1 is characterized by a higher plasticity, with a ∼20% occurrence for the 'unstructured' conformation throughout the folded segment, while hVDAC2, containing a peculiar extension of 11 amino acids at the N-terminal end, presents an additional 310-helical folded portion comprising residues 10' to 3, adhering to the barrel wall. The N-terminal sequences of hVDAC isoforms are predicted to have a low flexibility, with possible consequences in the dynamics of the human VDACs. Clear differences were found between hVDAC1 and hVDAC3 against hVDAC2: a significantly modified dynamics with possible important consequence on the voltage-gating mechanism. Charge distribution inside and at the mouth of the pore is responsible for a different preferential localization of ions with opposite charge and provide a valuable rationale for hVDAC1 and hVDAC3 having a Cl-/K+ selectivity ratio of 1.8, whereas hVDAC2 of 1.4. Our conclusion is that hVDAC isoforms, despite sharing a similar scaffold, have modified working features and a biological work is now requested to give evidence to the described dissimilarities.

Plasticin-L1 (GLVNGLLSSVLGGGQGGGGLLGGIL) is a conformationally flexible glycine/leucine-rich peptide originally isolated from norepinephrine-stimulated skin secretions of the South-American Santa Fe frog Leptodactylus laticeps (Leptodactylidae). A nuclear magnetic resonance/molecular dynamics characterization of plasticin-L1 in the presence of dodecylphosphocholine (DPC) and DPC/sodium dodecylsulphate micelles as membrane-mimetic models showed that the peptide has affinity for both neutral and anionic membranes. The peptide adopts a stable helical conformation at the N-terminal region and a more disordered helix at the C-terminal region, separated by an unstructured loop wherein the highest number of glycines is localized. In both micelle environments, plasticin-L1 slowly inserts between the detergent head groups but always remains localized at the micelle/water interface. Plasticin-L1 lacks direct antimicrobial activity but stimulates cytokine production by macrophages. Incubation with plasticin-L1 (20 μg/mL) significantly (P < 0.05) increased the production of the proinflammatory cytokines IL-1β, IL-12, IL-23, and TNF-α from unstimulated peritoneal macrophages from both C57BL/6 and BALB/C mice. The peptide also increased IL-6 production by unstimulated (P < 0.01) and lipopolysaccharide-stimulated (P < 0.01) macrophages, whereas the effects on production of the anti-inflammatory cytokine IL-10 were not significant. These findings suggest that plasticin-L1 may play an immunomodulatory role in vivo by stimulating cytokine production from frog skin macrophages in response to microbial pathogens. This peptide may represent a template for the design of peptides with therapeutic applications as immunostimulatory agents.

This work presents an in-depth investigation of the molecular interactions in the incorporation mechanism of colloidal hydrophobic-capped nanoparticles into the hydrophilic core of reverse microemulsions. (1)H Nuclear Magnetic Resonance (NMR) was employed to obtain molecular level details of the interaction between the nanoparticles capping amphiphiles and the microemulsion surfactants. The model system of choice involved oleic acid (OAC) and oleylamine (OAM) as capping molecules, while igepal-CO520 was the surfactant. The former were studied both in their "free" state and "ligated" one, i.e., bound to nanoparticles. The latter was investigated either in cyclohexane (micellar solution) or in water/cyclohexane microemulsions. The approach was extremely useful to gain a deeper understanding of the equilibria involved in this complex system (oleic acid capped-Bi2S3 in igepal/water/cyclohexane microemulsions). In difference to previously proposed mechanisms, the experimental data showed that the high affinity of the capping ligands for the reverse micelle interior was the drivingforce for the incorporation of the nanoparticles. A simple ligand-exchange mechanism could be ruled out. The collected information about the nanoparticle incorporation mechanism is extremely useful to develop new synthetic routes with an improved/tuned coating efficiency, in order to tailor the core-shell structure preparation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The human hepcidin 25 (hep-25) and its isoform hepcidin 20 (hep-20) are histidine-containing, cystein rich, β-sheet structured peptides endowed with antimicrobial activity. We previously reported that, similar to other histidine-containing peptides, the microbicidal effects of hep-25 and hep-20 are highly enhanced at acidic pH. In the present study, we investigated whether pH influences the mode of action of hep-25 and hep-20 on Escherichia coli American Type Culture Collection 25922 and model membranes. A striking release of β-galactosidase by hepcidin-treated E. coli was observed at pH 5.0, whereas no inner membrane permeabilization capacity was seen at pH 7.4, even at bactericidal concentrations. Similar results were obtained by flow cytometry when assessing the internalization of propidium iodide by hepcidin-treated E. coli. Scanning electron microscope imaging revealed that both peptides induced the formation of numerous blebs on the surface of bacterial cells at acidic pH but not at neutral pH. Moreover, a phospholipid/polydiacetylene colourimetric vesicle assay revealed a more evident membrane damaging effect at pH 5.0 than at pH 7.4. The leakage of entrapped dextrans of increasing molecular size from liposomes was also assessed at pH 7.4. Consistent with the lack of β-galactosidase release from whole E. coli observed at such a pH value, evident leakage of only the smallest 4-kDa dextran (and not of dextrans of 20 or 70 kDa) was observed, indicating a poor ability of hepcidin peptides to permeabilize liposome vesicles at pH 7.4. Altogether, the data obtained in the present study using different approaches strongly suggest that the ability of hepcidins to perturb bacterial membranes is markedly pH-dependent., (© 2013 FEBS.)

This work is focused on the two more expressed human myoglobin isoforms. In the literature, their different overexpression in high-altitude natives was proposed to be related to alternative/complementary functions in hypoxia. Interestingly, they differ only at residue-54, lysine or glutamate, which is external and far from the main binding site. In order to ascertain whether these two almost identical myoglobins might exert different functions and to contribute to a deeper understanding about myoglobin's oxygen-level dependent functioning, they have been compared with respect to dynamics, heme electronic structure, and NO reactivity at different O2 levels. Electron paramagnetic resonance (EPR) spectroscopy was employed to investigate the electronic structure of the nitrosyl-form, obtaining fundamental clues about a different bond interaction between the heme-iron and the proximal histidine and highlighting striking differences in NO reactivity, especially at a very low pO2. The experimental results well matched with the information provided by molecular dynamics simulations, which showed a significantly different dynamics for the two proteins only in the absence of O2. The single mutation differentiating the two myoglobins resulted in strongly affecting the plasticity of the CD-region (C-helix-loop-D-helix), whose fluctuations, being coupled to the solvent, were found to be correlated with the dynamics of the distal binding site. In the absence of O2, on the one hand a significantly different probability for the histidine-gate opening has been shown by MD simulations, and on the other a different yield of myoglobin-NO formation was experimentally observed through EPR.

The complexity of biological membranes leads to the use of extremely simplified models in biophysical investigations of membrane-bound proteins and peptides. Liposomes are probably the most widely used membrane models due, especially, to their versatility in terms of electric charge and size. However, liquid-state NMR suffers the lack of such a model, because even the smallest liposomes slowly tumble in solution, resulting in a dramatic signals broadening. Micelles are typically used as good substitutes, with sodium dodecylsulphate (SDS) and dodecylphosphocholine (DPC) being the most widely employed surfactants. However, they are always used separately to mimic prokaryotic and eukaryotic membranes, respectively, and accurate investigations as a function of surface charge cannot be performed. In this work, the critical micelle concentration (CMC) of binary mixtures with different SDS/DPC ratios has been determined by following the chemical shift variation of selected (1)H and (31)P NMR signals as a function of total surfactant concentration. The regular solution theory and the Motomura's formalism have been applied to characterize the micellization both in water and in phosphate buffer saline, and results were compared with those obtained directly from the experimental NMR chemical shift. The ζ-potential and size distribution of the mixed micelles have been estimated with dynamic light scattering measurements. Results showed that SDS and DPC are synergic and can be used together to prepare mixed micelles with different negative/zwitterionic surfactants molar ratio., (Copyright © 2013 John Wiley & Sons, Ltd.)

Antimicrobial peptides (AMPs) are found in various classes of organisms as part of the innate immune system. Despite high sequence variability, they share common features such as net positive charge and an amphipathic fold when interacting with biologic membranes. Esculentin-1b is a 46-mer frog-skin peptide, which shows an outstanding antimicrobial activity. Experimental studies revealed that the N-terminal fragment encompassing the first 18 residues, Esc(1-18), is responsible for the antimicrobial activity of the whole peptide, with a negligible toxicity toward eukaryotic cells, thus representing an excellent candidate for future pharmaceutical applications. Similarly to most of the known AMPs, Esc(1-18) is expected to act by destroying/permeating the bacterial plasma-membrane but, to date, its 3D structure and the detailed mode of action remains unexplored. Before an in-depth investigation on peptide/membranes interactions could be undertaken, it is necessary to characterize peptide's folding propensity in solution, to understand what is intrinsically due to the peptide sequence, and what is actually driven by the membrane interaction. Circular dichroism and nuclear magnetic resonance spectroscopy were used to determine the structure adopted by the peptide, moving from water to increasing amounts of trifluoroethanol. The results showed that Esc(1-18) has a clear tendency to fold in a helical conformation as hydrophobicity of the environment increases, revealing an intriguing amphipathic structure. The helical folding is adopted only by the N-terminal portion of the peptide, while the rest is unstructured. The presence of a hydrophobic cluster of residues in the C-terminal portion suggests its possible membrane-anchoring role., (Copyright © 2012 Wiley Periodicals, Inc.)