SprA1 and SprA2 are small hydrophobic peptides that belong to the type I toxin-antitoxin systems expressed by Staphylococcus aureus. Both peptides induce S. aureus death when overexpressed. Although they share 71% of amino acids sequence similarity, SprA2 exhibits stronger hemolytic activity than SprA1. In this study, we investigated the mode of action of these toxins on both prokaryotic-like and eukaryotic-like membranes. We first confirmed that SprA2, like SprA1, is an alpha-helical peptide located at the S. aureus membrane. By overexpressing each toxin, we demonstrated that SprA1 forms stable pores in the S. aureus membrane, evidenced by concomitant membrane depolarization, permeabilization and ATP release leading to growth arrest, whereas SprA2 forms transient pores, causing concomitant membrane depolarization, ATP release, and growth arrest. We showed that the unique cysteine residue present in SprA1 and SprA2 is required for toxicity through disulfide bond formation. Next, we found that both synthetic peptides induce slight leakage in anionic DOPC-DOPG lipid vesicles mimicking prokaryotic membranes, concomitant with lipid vesicles aggregation and/or fusion. Moreover, we observed that SprA1 permeabilizes S. aureus protoplasts, via its ability to form stable pores, whereas SprA2 permeabilizes and lyses them. However, no permeabilization of intact bacteria was detected after the addition of SprA1 and SprA2 in the extracellular medium. Finally, we confirmed that SprA2 has strong activity on zwitterionic DOPC lipid vesicles mimicking eukaryotic membranes, without inducing aggregation. This work highlights the strong selectivity of SprA2 for eukaryotic membranes, suggesting that this toxin may play a role in S. aureus virulence.

Objectives Assessing the therapeutic potential of a novel antimicrobial pseudopeptide, Pep16, both in vitro and in vivo for the treatment of septic arthritis caused by Staphylococcus aureus.Methods Seven clinical isolates of S. aureus (two MRSA and five MSSA) were studied. MICs of Pep16 and comparators (vancomycin, teicoplanin, daptomycin and levofloxacin) were determined through the broth microdilution method. The intracellular activity of Pep16 and levofloxacin was assessed in two models of infection using non-professional (osteoblasts MG-63) or professional (macrophages THP-1) phagocytic cells. A mouse model of septic arthritis was used to evaluate the in vivo efficacy of Pep16 and vancomycin. A preliminary pharmacokinetic (PK) analysis was performed by measuring plasma concentrations using LC-MS/MS following a single subcutaneous injection of Pep16 (10 mg/kg).Results MICs of Pep16 were consistently at 8 mg/L for all clinical isolates of S. aureus (2- to 32-fold higher to those of comparators) while MBC/MIC ratios confirmed its bactericidal activity. Both Pep16 and levofloxacin (when used at 2 x MIC) significantly reduced the bacterial load of all tested isolates (two MSSA and two MRSA) within both osteoblasts and macrophages. In MSSA-infected mice, Pep16 demonstrated a significant (similar to 10-fold) reduction on bacterial loads in knee joints. PK analysis following a single subcutaneous administration of Pep16 revealed a gradual increase in plasma concentrations, reaching a peak of 5.6 mg/L at 12 h.Conclusions Pep16 is a promising option for the treatment of septic arthritis due to S. aureus, particularly owing to its robust intracellular activity.

Colistin is a drug of last resort to treat extreme drug-resistant Enterobacterales, but is limited by dose-dependent toxicity and the emergence of resistance. A recently developed antimicrobial pseudopeptide, Pep16, which acts on the cell membrane, may be synergistic with colistin and limit the emergence of resistance. We investigated Pep16 activity against Escherichia coli with varying susceptibility to colistin, in vitro and in a murine peritonitis model. Two isogenic derivatives of E. coli CFT073 (susceptible and resistant to colistin) and 2 clinical isolates (susceptible (B119) and resistant to colistin (Af31)) were used. Pep16 activity, alone and in combination with colistin, was determined in vitro (checkerboard experiments, time-kill curves, and flow cytometry to investigate membrane permeability). Toxicity and pharmacokinetic analyses of subcutaneous Pep16 were performed in mice, followed by the investigation of 10 mg/kg Pep16 + 10 mg/kg colistin (mimicking human concentrations) in a murine peritonitis model. Pep16 alone was inactive (MICs = 32-64 mg/L; no bactericidal effect). A concentration-dependent bactericidal synergy of Pep16 with colistin was evidenced on all strains, confirmed by flow cytometry. In vivo, Pep16 alone was ineffective. When Pep16 and colistin were combined, a significant decrease in bacterial counts in the spleen was evidenced, and the combination prevented the emergence of colistin-resistant mutants, compared to colistin alone. Pep16 synergizes with colistin in vitro, and the combination is more effective than colistin alone in a murine peritonitis by reducing bacterial counts and the emergence of resistance. Pep16 may optimize colistin use, by decreasing the doses needed, while limiting the emergence of colistin-resistant mutants.

The good halogen bond donor N-bromosaccharin (NBrSac) has been cocrystallized with four para-substituted pyridines (PyX) as good halogen bond acceptors of different strengths (PyCN, PyCF3, PyCO2Me, and PyMe). Their crystal structures have been determined and permitted to observe the formation of adducts with a significantly weakened N-SacBr bonding interaction with respect to the NBrSac donor and the formation of a strong BrN-Py interaction. The topological properties at the N-SacBrN-Py bond critical points (BCPs) of the adducts frozen at experimental geometries indicate the increasing strength of the studied acceptors along the series PyCN < PyCF3 < PyCO2Me < PyMe, correlating with the structural distances and the deeper negative molecular electrostatic potential values in the individual molecules. The polarizability calculated in individual molecules clearly points out the atomic dipole moments of the N-Sac, Br, and N-Py atoms, increasing in magnitude within the adducts and demonstrating their sensitivity to external electric fields mimicking molecular environments. In order to explore the effect of polarizing environments on the N-SacBrN-Py halogen bonding motif, an external electric field has been applied (-40 x 10(-4) < epsilon < 100 x 10(-4) a.u.) to adducts extracted from experimental geometries with all atoms frozen except Br, which has been optimized as a function of epsilon. It has been shown that an effective electric field ranging from 1.28 to 2.96 GV m(-1) is necessary to recover the experimental position of the Br-atom in the adducts (from that calculated in gas-phase optimization), providing an evaluation of the effect of the crystalline environment. At any investigated geometry (i.e., within the full range of applied fields), the adducts exhibit a significant covalence degree at both donor and acceptor sides (measured by 1 < |V|/G < 2), in addition to a nonnegligible delocalization index (DI(N-Sac|N-Py)) in the range of 0.085-0.099. These features indicate that the adducts should be considered as a unique molecular entity rather than two while pointing to an assembly with a small but nonnegligible contribution of a three-center four-electrons interaction. The formal border of the halogen atom transfer between donor and acceptor moieties is established by the topological magnitudes of rho and |V|/G at both BCPs, as well as that of DI, which all balance almost simultaneously in donor and acceptor regions. The position of the halogen atom within the adduct is straightforwardly driven by the external polarization induced by epsilon, permitting control of the significant variation of the dipole moment of the adducts.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Antibiotics are a medical wonder, but an increasing frequency of resistance among most human pathogens is rendering them ineffective. If this trend continues, the consequences for public health and for the general community could be catastrophic. The current clinical pipeline, however, is very limited and is dominated by derivatives of established classes, the me too compounds. Here, we have exploited our recent identification of a bacterial toxin to transform it into antibiotics active on multidrug-resistant (MDR) gram-positive and -negative bacterial pathogens. We generated a new family of peptidomimetics-cyclic heptapseudopeptides-inspired from a natural bacterial peptide. Out of the 4 peptides studied, 2 are effective against methicillin-resistant Staphylococcus aureus (MRSA) in mild and severe sepsis mouse models without exhibiting toxicity on human erythrocytes and kidney cells, zebrafish embryos, and mice. These new compounds are safe at their active doses and above, without nephrotoxicity. Efficacy was also demonstrated against Pseudomonas aeruginosa and MRSA in a mouse skin infection model. Importantly, these compounds did not result in resistance after serial passages for 2 weeks and 4 or 6 days' exposure in mice. Activity of heptapseudopeptides was explained by the ability of unnatural amino acids to strengthen dynamic association with bacterial lipid bilayers and to induce membrane permeability, leading to bacterial death. Based on structure determination, we showed that cationic domains surrounded by an extended hydrophobic core could improve bactericidal activity. Because 2 peptide analogs, Pep 16 and Pep19, are effective against both MRSA and P. aeruginosa in severe sepsis and skin infection models, respectively, we believe that these peptidomimetics are promising lead candidates for drug development. We have identified potential therapeutic agents that can provide alternative treatments against antimicrobial resistance. Because the compounds are potential leads for therapeutic development, the next step is to start phase I clinical trials.

Bacterial type I toxin-antitoxin (TA) systems are widespread, and consist of a stable toxic peptide whose expression is monitored by a labile RNA antitoxin. We characterized Staphylococcus aureus SprA2/SprA2(AS) module, which shares nucleotide similarities with the SprA1/SprA1(AS) TA system. We demonstrated that SprA2/SprA2(AS) encodes a functional type I TA system, with the cis-encoded SprA2(AS) antitoxin acting in trans to prevent ribosomal loading onto SprA2 RNA. We proved that both TA systems are distinct, with no cross-regulation between the antitoxins in vitro or in vivo. SprA2 expresses PepA2, a toxic peptide which internally triggers bacterial death. Conversely, although PepA2 does not affect bacteria when it is present in the extracellular medium, it is highly toxic to other host cells such as polymorphonuclear neutrophils and erythrocytes. Finally, we showed that SprA2(AS) expression is lowered during osmotic shock and stringent response, which indicates that the system responds to specific triggers. Therefore, the SprA2/SprA2(AS) module is not redundant with SprA1/SprA1(AS), and its PepA2 peptide exhibits an original dual mode of action against bacteria and host cells. This suggests an altruistic behavior for S.aureus in which clones producing PepA2 in vivo shall die as they induce cytotoxicity, thereby promoting the success of the community.

Dendritic oligopeptides have been reported as useful building blocks for many interactions. Starting from hydrazine, we described an approach to create new dendritic pseudopeptides linked with biological systems, such as cell membrane, as chelate metal, Ni 2+ -nitrilotriacetic acid moieties which could target histidine rich peptides or proteins. Depending on the nature of these new chemical recognition units, they could be integrated into a peptide by coupling in C or N -termini. Graphical abstract: Dendrimer formation

We compare here the halogen bond characteristics of bimolecular adducts involving either N-bromo- or N-iodosaccharin as strong halogen bond donors, with 4-picoline as a common XB acceptor. In the NBSac·Pic system, the bromine atom of NBSac is displaced toward the picoline, almost at a median position between the two nitrogen atoms, NSac and N'Pic, with NSacBr and BrN'Pic distances at 2.073(6) and 2.098(6) Å respectively. This extreme situation contrasts with the analogous iodine derivative, NISac·Pic, where the NSac-I and IN'Pic distances amount to 2.223(4) and 2.301(4) Å respectively. Periodic DFT calculations, and molecular calculations of adducts (PBEPBE-D2 aug-cc-pVTZ) either at the experimental frozen geometry or with optimization of the halogen position, indicate a more important degree of covalency (i.e. shared-shell character) in the adduct formed with the bromine atom. A stronger charge transfer to the picoline is also found for the bromine (+0.27 |e|) than for the iodine (+0.18 |e|) system. This inversion of halogen bond strength between I and Br finds its origin in the strong covalent character of the interaction in these adducts, in line with the strength of covalent N-Br and N-I bonds. Detailed characterization of the critical points (CPs) of the L(r) = -∇2ρ(r) function along bonding directions has permitted the adducts to be distinguished and they can be respectively described as "neutral" NISac/Pic and "intermediate" NSac/Br/Pic, the latter with Br being close to formal equivalent NSacBr and BrN'Pic interactions but still more associated to the XB donor than to the picoline, as indicated by the topological and energetic properties of the ρ(r) function at the bond critical points (BCPs).

The distinction between cocrystals and salts is usually investigated in hydrogen-bonded systems as A−H···B⇆ [A]−···[H −B]+, where the position of the hydrogen atom actually defines the ionicity of the complex. The same distinction, but in halogen-bonded systems, is addressed here, in complexes formed out of N-iodoimide derivatives as halogen bond donors, and pyridines as halogen-bond acceptors, anticipating that the position of the iodine atom in these A−I···B ⇆ [A]−···[I−B]+ systems will also define their degree of ionicity. We show that the crystalline halogen-bonded complexes of Niodosuccinimide (NIS) with pyridine, 4-methylpyridine, and 4-dimethylaminopyridine can be described as “close-to-neutral” cocrystals while the crystalline halogen-bonded complex of N-iodosaccharin (NISac) with 4-dimethylaminopyridine (DMAP) adopts a “close-to-ionic” structure [1].

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

N,N'-Dibromohydantoins, known as electrophilic bromination reagents, are successfully used here as halogen bond (XB) donors, as demonstrated in their crystalline adducts with para-substituted pyridines acting as halogen bond acceptors. 1 : 1 adducts of the achiral 5,5-dimethyl- N, N'-dibromohydantoin (DBH) are crystallized with methylisonicotinate, 4- trifluoromethylpyridine and 4- cyanopyridine, while both nitrogen atoms of pyrazine are engaged in halogen bonding in the 2 : 1 adduct (DBH)(2).(pyrazine). A strengthening of the XB interaction between the imidic N-Br group of DBH and the pyridinic nitrogen atom is observed with the more electron rich pyridines in the order Py-CO2Me > Py-CF3 > Py-CN > pyrazine. Chiral hydantoins and their N, N'-dibromo derivatives are obtained in good yields from different amino acids (phenylglycine, phenylalanine, valine and leucine). The ability of such enantiopure N-iodoimide derivatives to act as halogen-bond donors is demonstrated in the 1 : 1 methylisonicotinate adduct with (S)-5-isobutyl-N, N'-dibromohydantoin.

The halogen bonding ability of ditopic halogen bond donors can be assessed from the maximum value of the molecular surface electrostatic potential, called c-hole, at the two halogen atoms. We show here that in N,N'-diodo-dimethylhydantoin (DIH), the halogen bonding (XB) ability of the two nitrogen-bound iodine atoms does not parallel the calculated c-hole amplitude. The cocrystallization of DIH with a series of para-substituted pyridines, noted Py-R (R = pyrrolidinyl, NMe2, Me, H, CO2Me, CF3, CN), affords bis-adducts DIH(Py-R)(2) with the more electron-rich pyridines, while mono-adducts DIH (Py-R) are favored with the more electron-poor pyridines (R = CO2Me, CF3, CN). Analysis of the structural characteristics of these mono- and bis-adducts, combined with theoretical calculations, demonstrates that the formation of a first N-I N'(py-R) XB deeply modifies the XB ability (and associated sigma-hole) of the second uncoordinated iodine atom. Under these conditions, the latter might associate through I center dot center dot center dot O XB to the carbonyl oxygen atom of a neighboring mono-adduct in the crystal rather than to a second pyridine. These studies show that when working with polytopic XB donors, one should always consider the deactivation of the remaining halogen atoms following sequential XB formation.

The metalloporphyrin-catalyzed oxidation of SSR180575, a ligand of the peripheral benzodiazepine receptor, that contains both N-methylindole and N,N-dimethylamide functions, produces a high yield of a stable carbinolamide derivative. (C) 2013 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.

The effect of the streaming current flowing through the porous structure of composite membranes during tangential electrokinetic measurements was investigated. It was shown that neglecting this additional path for streaming current may have dramatic implications in the interpretation of the experimental data and on the determination of the membrane zeta potential. Experimental measurements of both streaming current and electrical conductance were performed with two different composite polymer membranes. By following the procedure proposed by Yaroshchuk and Luxbacher, Langmuir 26 (2010) 10882–10889, in the present work it was possible to determine separately the zeta potential of the membrane surfaces and that of their underlying porous structures. This experimental procedure was shown to provide useful information on the functionalization of an ultrafiltration polyethersulfone membrane by positively charged 4-benzyltriphenylphosphonium groups. Notably we found that the chemical modification leads to a charge reversal (from negative to positive) of the porous substructure of the membrane while the overall charge of the external surface remains negative, although with diminished magnitude.

Ultrafiltration polyethersulfone membranes were modified covalently by chemical reduction of aryl diazonium salts. Functionalizations were performed with four aryl diazonium salts bearing different functional groups (4-benzyltriphenylphosphonium diazonium, 4-nitrophenyl diazonium, 4-benzonitrile diazonium and 4-phenylacetic acid diazonium) so as to demonstrate the versatility of the method. The efficiency of the different functionalizations was checked with various characterization techniques. Attenuated total reflectance–Fourier transform infra red spectroscopy revealed the presence of 4-nitrophenyl, 4-benzonitrile and 4-phenylacetic acid groups at the surface of the different modified membranes but no characteristic vibration band was detected on the surface of the membrane modified with 4-benzyltriphenylphosphonium diazonium. The presence of 4-benzyltriphenylphosphonium, however, could be demonstrated by both Energy Dispersive X-ray spectroscopy (detection of the Kα ray of phosphorous at 2.015keV) and streaming current measurements (shift of the membrane isoelectric point). Finally, dead-end filtration of an antibiotic (tylosin) was carried out with the unmodified membrane and the membrane modified by 4-benzyltriphenyl-phosphonium diazonium. Experiments revealed that the transport properties of the grafted membrane were significantly modified, with a significant increase in rejection mainly due to electrostatic repulsions between the surface of the modified membrane and tylosin.

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Different substrates have been modified through electroreduction of tetraarylporphyrin diazonium salts, H2TPP-N2+ or H2TPP-(N2+)4, generated in situ in acidic aqueous media from monoamino-TPP (H2TPP-NH2) or tetraamino-TPP (H2TPP-(NH2)4) in low concentration (5.10−4M). The modification of the electrodes with H2TPP-(NH2)4 leads to a purple deposit (PolyH2TPP-1) strongly linked to the electrode surface whereas those obtained from H2TPP-NH2 were not covalently linked to the electrode surface, soluble in organic solvents and identified as tetraphenylporphyrin (H2TPP). The deposits have been characterized by electrochemistry, atomic force microscopy (AFM) and UV–Visible absorption spectroscopy. Cyclic voltammetry of PolyH2TPP-1 revealed an electroactivity akin to that of polytetraarylporphyrin obtained by anodic electropolymerization of H2TPP (~2.10−3M) in strictly anhydrous electrolytic medium (PolyH2TPP-2). In light of their UV–vis spectra, the two related polymers present different intermolecular interactions and macromolecular organization.