Linker-Free Synthesis of Antimicrobial Peptides Using a Novel Cleavage Reagent: Characterisation of the Molecular and Ionic Composition by nanoESI-HR MS.

The efficient preparation of novel bioactive peptide drugs requires the availability of reliable and accessible chemical methodologies together with suitable analytical techniques for the full characterisation of the synthesised compounds. Herein, we describe a novel acidolytic method with application to the synthesis of cyclic and linear peptides involving benzyl-type protection. The process consists of the in situ generation of anhydrous hydrogen bromide and a trialkylsilyl bromide that acts as protic and Lewis acid reagents. This method proved to be useful to effectively remove benzyl-type protecting groups and cleave Fmoc/tBu assembled peptides directly attached to 4-methylbenzhydrylamine (MBHA) resins with no need for using mild trifluoroacetic acid labile linkers. The novel methodology was successful in synthesising three antimicrobial peptides, including the cyclic compound polymyxin B3, dusquetide, and RR4 heptapeptide. Furthermore, electrospray mass spectrometry (ESI-MS) is successfully used for the full characterisation of both the molecular and ionic composition of the synthetic peptides.

Unveiling the Membrane and Cell Wall Action of Antimicrobial Cyclic Lipopeptides: Modulation of the Spectrum of Activity.

Antibiotic resistance is a major public health challenge, and Gram-negative multidrug-resistant bacteria are particularly dangerous. The threat of running out of active molecules is accelerated by the extensive use of antibiotics in the context of the COVID-19 pandemic, and new antibiotics are urgently needed. Colistin and polymyxin B are natural antibiotics considered as last resort drugs for multi-resistant infections, but their use is limited because of neuro- and nephrotoxicity. We previously reported a series of synthetic analogues inspired in natural polymyxins with a flexible scaffold that allows multiple modifications to improve activity and reduce toxicity. In this work, we focus on modifications in the hydrophobic domains, describing analogues that broaden or narrow the spectrum of activity including both Gram-positive and Gram-negative bacteria, with MICs in the low µM range and low hemolytic activity. Using biophysical methods, we explore the interaction of the new molecules with model membranes that mimic the bacterial inner and outer membranes, finding a selective effect on anionic membranes and a mechanism of action based on the alteration of membrane function. Transmission electron microscopy observation confirms that polymyxin analogues kill microbial cells primarily by damaging membrane integrity. Redistribution of the hydrophobicity within the polymyxin molecule seems a plausible approach for the design and development of safer and more selective antibiotics.

State-of-the-art polymeric nanoparticles as promising therapeutic tools against human bacterial infections.

Infectious diseases kill over 17 million people a year, among which bacterial infections stand out. From all the bacterial infections, tuberculosis, diarrhoea, meningitis, pneumonia, sexual transmission diseases and nosocomial infections are the most severe bacterial infections, which affect millions of people worldwide. Moreover, the indiscriminate use of antibiotic drugs in the last decades has triggered an increasing multiple resistance towards these drugs, which represent a serious global socioeconomic and public health risk. It is estimated that 33,000 and 35,000 people die yearly in Europe and the United States, respectively, as a direct result of antimicrobial resistance. For all these reasons, there is an emerging need to find novel alternatives to overcome these issues and reduced the morbidity and mortality associated to bacterial infectious diseases. In that sense, nanotechnological approaches, especially smart polymeric nanoparticles, has wrought a revolution in this field, providing an innovative therapeutic alternative able to improve the limitations encountered in available treatments and capable to be effective by theirselves. In this review, we examine the current status of most dangerous human infections, together with an in-depth discussion of the role of nanomedicine to overcome the current disadvantages, and specifically the most recent and innovative studies involving polymeric nanoparticles against most common bacterial infections of the human body.

Recent advances and perspectives in the design and development of polymyxins.

Covering: 1947-early 2017, particularly from 2005-early 2017The rise of bacterial pathogens with acquired resistance to almost all available antibiotics is becoming a serious public health issue. Polymyxins, antibiotics that were mostly abandoned a few decades ago because of toxicity concerns, are ultimately considered as a last-line therapy to treat infections caused by multi-drug resistant Gram-negative bacteria. This review surveys the progress in understanding polymyxin structure, and their chemistry, mechanisms of antibacterial activity and nephrotoxicity, biomarkers, synergy and combination with other antimicrobial agents and antibiofilm properties. An update of recent efforts in the design and development of a new generation of polymyxin drugs is also discussed. A novel approach considering the modification of the scaffold of polymyxins to integrate metabolism and detoxification issues into the drug design process is a promising new line to potentially prevent accumulation in the kidneys and reduce nephrotoxicity.

Synergistic Antipseudomonal Effects of Synthetic Peptide AMP38 and Carbapenems.

The aim was to explore the antimicrobial activity of a synthetic peptide (AMP38) and its synergy with imipenem against imipenem-resistant Pseudomonas aeruginosa. The main mechanism of imipenem resistance is the loss or alteration of protein OprD. Time-kill and minimal biofilm eradication concentration (MBEC) determinations were carried out by using clinical imipenem-resistant strains. AMP38 was markedly synergistic with imipenem when determined in imipenem-resistant P. aeruginosa. MBEC obtained for the combination of AMP38 and imipenem was of 62.5 µg/mL, whereas the MBEC of each antimicrobial separately was 500 µg/mL. AMP38 should be regarded as a promising antimicrobial to fight MDR P. aeruginosa infections. Moreover, killing effect and antibiofilm activity of AMP38 plus imipenem was much higher than that of colistin plus imipenem.

Cognitive mechanisms underlying Armoni: A computer-assisted cognitive training programme for individuals with intellectual disabilities / Mecanismos cognitivos que subyacen a Armoni: Un programa de entrenamiento cognitivo asistido por ordenador para personas con discapacidades intelectuales

Although a number of cognitive deficits have been described in individuals with intellectual disabilities (ID), few studies have examined the use of computer-assisted cognitive training programmes in this group of people. This study sought to determine the cognitive mechanisms underlying 16 activities included in Armoni, a computerized cognitive training programme for individuals with ID, in order to validate its use with this population. Fifty adults with ID from four residential care centres in Spain underwent neuropsychological testing tapping attention, verbal memory, visual memory, comprehension, visuoperception, visuoconstruction, naming ability, verbal fluency, verbal reasoning and motor function. In addition, they performed 16 activities included in the Armoni programme. The relationships between cognitive function and the computer-based activities were assessed using Spearman correlations. Stepwise multiple regression analyses were then used to explore how cognitive function predicted the performance of individuals with ID on the programme activities. Most programme activities correlated with visuoconstruction, comprehension and naming ability. Naming ability, visual memory, comprehension and visuoconstruction contributed the most to the predictive models regarding performance on the Armoni activities. Our findings support the validity of Armoni for cognitive training in individuals with ID

Aunque se han descrito varios déficits cognitivos en individuos con discapacidades intelectuales (DI), pocos estudios han examinado el uso de programas de entrenamiento cognitivo asistidos por ordenador en este grupo de personas. Este estudio trata de determinar los mecanismos cognitivos subyacentes a 16 actividades incluidas en Armoni, un programa de entrenamiento cognitivo computarizado para individuos con DI, con el fin de validar su uso en esta población. Cincuenta adultos con DI de cuatro centros de atención residencial en España fueron sometidos a pruebas neuropsicológicas de tapping atencional, memoria verbal, memoria visual, comprensión, visuopercepción, visuoconstructión, capacidad de nombrar, fluidez verbal, razonamiento verbal y función motora. Además, se llevaron a cabo las 16 actividades incluidas en el programa de Armoni. Las relaciones entre la función cognitiva y las actividades basadas en el ordenador se evaluaron utilizando correlaciones de Spearman. Después se utilizó análisis de regresión múltiple por pasos para explorar cómo la función cognitiva predice el rendimiento de las personas con DI en las actividades del programa. La mayoría de las actividades del programa correlacionaron con visuoconstructión, comprensión y capacidad de nombrar. La capacidad de nombrar, la memoria visual, la comprensión y la visuoconstructión fueron las que más contribuyeron en los modelos predictivos sobre el desempeño en las actividades de Armoni. Nuestros resultados apoyan la validez de Armoni para el entrenamiento cognitivo en las personas con DI

Tryptophan-containing lipopeptide antibiotics derived from polymyxin B with activity against Gram positive and Gram negative bacteria.

Resistance to all known antibiotics is a growing concern worldwide, and has renewed the interest in antimicrobial peptides, a structurally diverse class of amphipathic molecules that essentially act on the bacterial membrane. Propelled by the antimicrobial potential of this compound class, we have designed three new lipopeptides derived from polymyxin B, sp-34, sp-96 and sp-100, with potent antimicrobial activity against both Gram positive and Gram negative bacteria. The three peptides bind with high affinity to lipopolysaccharide as demonstrated by monolayer penetration and dansyl-displacement. The interaction with the cytoplasmic membrane has been elucidated by biophysical experiments with model membranes of POPG or POPE/POPG (6:4), mimicking the Gram positive and Gram negative bacterial membrane. Trp-based fluorescence experiments including steady-state, quenching, anisotropy and FRET, reveal selectivity for anionic phospholipids and deep insertion into the membrane. All three lipopeptides induce membrane fusion and leakage from anionic vesicles, a process that is favored by the presence of POPE. The molecules bind to zwitterionic POPC vesicles, a model of the eukaryotic membrane, but in a different way, with lower affinity, less penetration into the bilayer and no fusion or permeabilization of the membrane. Results in model membranes are consistent with flow cytometry experiments in Escherichia coli and Staphylococcus aureus using a membrane potential sensitive dye (bis-oxonol) and a nucleic acid dye (propidium iodide), suggesting that the mechanism of action is based on membrane binding and collapse of membrane integrity by depolarization and permeabilization.

A bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity.

Bacterial resistance to almost all available antibiotics is an important public health issue. A major goal in antimicrobial drug discovery is the generation of new chemicals capable of killing pathogens with high selectivity, particularly multi-drug-resistant ones. Here we report the design, preparation and activity of new compounds based on a tunable, chemically accessible and upscalable lipopeptide scaffold amenable to suitable hit-to-lead development. Such compounds could become therapeutic candidates and future antibiotics available on the market. The compounds are cyclic, contain two D-amino acids for in vivo stability and their structures are reminiscent of other cyclic disulfide-containing peptides available on the market. The optimized compounds prove to be highly active against clinically relevant Gram-negative and Gram-positive bacteria. In vitro and in vivo tests show the low toxicity of the compounds. Their antimicrobial activity against resistant and multidrug-resistant bacteria is at the membrane level, although other targets may also be involved depending on the bacterial strain.

Gram-negative outer and inner membrane models: insertion of cyclic cationic lipopeptides.

Most Gram-negative bacteria are susceptible to polymyxin B (PxB), and development of resistance to this cationic lipopeptide is very rare. PxB mechanism of action involves interaction with both the outer membrane (OM) and the inner membrane (IM) of bacteria. For the design of new antibiotics based on the structure of PxB and with improved therapeutic indexes, it is essential to establish the key features of PxB that are important for activity. We have used an approach based on mimicking the outer layers of the OM and the IM of Gram-negative bacteria using monolayers of lipopolysaccharide (LPS) or anionic 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG), respectively, and using a combination of penetration assay, analysis of pressure/area curves, and Brewster angle microscopy to monitor surface morphology changes. Synthetic analogue sp-B maintains the basic structural characteristics of the natural compound and interacts with the OM and the IM in a similar way. Analogue sp-C, with a mutation of the sequence [d-Phe6-Leu7] into [d-Phe6-Dab7], shows that this hydrophobic domain is involved in LPS binding. The significant role of the positive charges is demonstrated with sp-Dap analogue, where l-alpha,gamma-diaminobutyric acid residues Dab1 and Dab8 are replaced by l-alpha,gamma-diaminopropionic acid (Dap), resulting in lower degrees of insertion in both LPS and PG monolayers. The importance of the N-terminal acyl chain is demonstrated with polymyxin B nonapeptide (PxB-np). PxB-np shows lower affinity for LPS compared to PxB, sp-B, or sp-C, but it does not insert into PG monolayers, although it binds superficially to the anionic film. Since PxB microbial killing appears to be mediated by osmotic instability due to OM-IM phospholipid exchange, the ability of the different peptides to induce membrane-membrane lipid exchange has been studied by use of phospholipid unilamellar vesicles. Results indicate that cationic amphipathicity determines peptide activity.

Membrane association and contact formation by a synthetic analogue of polymyxin B and its fluorescent derivatives.

sP-B is a synthetic analogue of the natural lipopeptide antibiotic polymyxin B (PxB) that maintains the ability of the parent compound to form vesicle-vesicle contacts and induce lipid exchange. Exchange is selective, and only monoanionic phospholipids such as 1-palmitoyl-2-oleoyl-glycero-sn-3-phosphoglycerol (POPG) are transferred, whereas dianionic phospholipids such as 1-palmitoyl-2-oleoyl-glycero-sn-3-phosphate (POPA) are not, as shown by fluorescence experiments based on the excimer/monomer ratio of pyrene-labeled phospholipids. Synthetic fluorescent analogues of sP-B are used to investigate the peptide position and orientation in the intermembrane contacts: sP-Bw, an analogue that contains D-tryptophan (D-Trp) instead of the naturally occurring D-phenylalanine, and sP-Bpy, incorporating a pyrene group at the N-terminus. Tryptophan fluorescence, anisotropy, and quenching measurements performed with sP-Bw indicate that the peptide binds and inserts in anionic vesicles of POPG and POPA. However, significant differences are seen depending on the lipid composition, as also demonstrated by fluorescence resonance energy transfer (FRET) experiments from Trp to 7-nitro-2-1,3-benzoxadiazol (NBD) groups at the interface. Intermolecular FRET using sP-Bw as the donor and sP-Bpy as the acceptor indicates self-association of the peptide, possibly forming dimers, when bound to POPG vesicles at concentrations that induce the vesicle-vesicle contacts.

Synthesis and membrane action of polymyxin B analogues.

We have designed synthetic peptides that mimic the primary and secondary structure of the cationic lipopeptide antibiotic polymyxin B (PxB) in order to determine the structural requirements for membrane action and to assess possible therapeutic potential. Two analogues with related sequences to that of PxB, but including synthetic simplifications (disulphide bridge between two cysteines in positions 4 and 10, N-terminal nonanoic acid), have been synthesized. Peptide-lipid interactions have been studied by fluorescence resonance energy transfer between pyrene and 4,4-difluoro-5-methyl-4-bora-3alpha,4alpha-diaza-s-indacene-3-dodecanoyl (BODIPY)probes covalently linked to phospholipids, and the possibility of membrane disruption or permeabilization has been assessed by light scattering and fluorescence quenching assays. The synthetic peptide sP-B, which closely mimics the primary and secondary structures of PxB, binds to vesicles of anionic 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG) or of lipids extracted from Escherichia coli membranes, and induces apposition of the vesicles and selective lipid exchange without permeabilization of the membrane. We conclude that sP-B forms functional vesicle-vesicle contacts that are selective, as previously described for PxB. The second analogue, sP-C, has a permutation of two amino acids that breaks the hydrophobic patch formed by D-Phe and Leu residues on the cyclic part of the sequence. sP-C lipopeptide is more effective than sP-B in inducing lipid mixing, but shows no selectivity for the lipids that exchange through the vesicle-vesicle contacts, and at high concentrations has a membrane-permeabilizing effect. The deacylated and non-antibiotic derivative PxB-nonapeptide (PxB-NP) does not induce the formation of functional intervesicle contacts in the range of concentrations studied.

Polymyxin B-lipid interactions in Langmuir-Blodgett monolayers of Escherichia coli lipids: a thermodynamic and atomic force microscopy study.

The dramatically increased frequency of antibiotic resistance has led to intensive efforts towards developing new families of antibiotics. Membrane-active antibiotic peptides such as polymyxin B (PxB) hold promise as the next generation of antibiotics, since they rarely spur the evolution of resistance. At low concentrations in the membrane, PxB forms vesicle-vesicle contacts and induces lipid exchange without leakage or fusion, a phenomenon that can explain its specificity towards gram-negative bacteria by contact formation between the two phospholipids interfaces in the periplasmatic space. In this work, the interaction of PxB and the nonantibiotic derivative polymyxin B nonapeptide (PxB-NP) with monolayers of Escherichia coli membrane lipids (ECL) has been studied by thermodynamic and structural methods. PxB inserts itself into ECL monolayers as a conformation that forms intermembrane contacts with vesicles injected underneath, and induces lipid exchange when the monolayer surface pressure is set at 32 mN/m (membrane equivalence pressure) or net transfer vesicle-to-monolayer at lower surface pressures. Thermodynamic analysis of the compression isotherms of mixed monolayers indicates that PxB inserts into the monolayer with an expansion of the mean molecular area, implying that peptide and lipids form nonideal mixtures. At low concentrations, corresponding to the membrane-membrane contact form of PxB, the mixed monolayers present positive excess energy values (deltaGm(Ex)), and atomic force microscopy (AFM) imaging reveals structures of approximately 120-nm diameter that protrude from the lipid surface approximately 0.7 nm. At concentrations of PxB above 4 mol %, thermodynamic analysis gives a very high deltaGm(Ex), corresponding to nonfavorable interactions, and AFM images show round structures of 20-30 nm diameter. PxB-NP behaves in a totally different way, in agreement with its inability to form vesicle-vesicle contacts and its lack of antibiotic effect. These results are discussed in the light of the mechanism of action of PxB on the membrane of gram-negative bacteria.

Origins of delays in monolayer kinetics: phospholipase A2 paradigm.

The interfacial kinetic paradigm is adopted to model the kinetic behavior of pig pancreatic phospholipase A(2) (PLA2) at the monolayer interface. A short delay of about a minute to the onset of the steady state is observed under all monolayer reaction progress conditions, including the PLA2-catalyzed hydrolysis of didecanoylphosphatidyl-choline (PC10) and -glycerol (PG10) monolayers as analyzed in this paper. This delay is independent of enzyme concentration and surface pressure and is attributed to the equilibration time by stationary diffusion of the enzyme added to the stirred subphase to the monolayer through the intervening unstirred aqueous layer. The longer delays of up to several hours, seen with the PC10 monolayers at >15 mN/m, are influenced by surface pressure as well as enzyme concentration. Virtually all features of the monolayer reaction progress are consistent with the assumption that the product accumulates in the substrate monolayer, although the products alone do not spread as a compressible monolayer. These results rule out models that invoke slow "activation" of PLA2 on the monolayer. The observed steady-state rate on monolayers after the delays is <1% of the rate observed with micellar or vesicles substrates of comparable substrate. Together these results suggest that the monolayer steady-state rate includes contributions from steps other than those of the interfacial turnover cycle. Additional considerations that provide understanding of the pre-steady-state behaviors and other nonideal effects at the surface are also discussed.