Generation of Lasso Peptide-Based ClpP Binders

The Clp protease system fulfills a plethora of important functions in bacteria. It consists of a tetradecameric ClpP barrel holding the proteolytic centers and two hexameric Clp-ATPase rings, which recognize, unfold, and then feed substrate proteins into the ClpP barrel for proteolytic degradation. Flexible loops carrying conserved tripeptide motifs protrude from the Clp-ATPases and bind into hydrophobic pockets (H-pockets) on ClpP. Here, we set out to engineer microcin J25 (MccJ25), a ribosomally synthesized and post-translationally modified peptide (RiPP) of the lasso peptide subfamily, by introducing the conserved tripeptide motifs into the lasso peptide loop region to mimic the Clp-ATPase loops. We studied the capacity of the resulting lasso peptide variants to bind to ClpP and affect its activity. From the nine variants generated, one in particular (12IGF) was able to activate ClpP from Staphylococcus aureus and Bacillus subtilis. While 12IGF conferred stability to ClpP tetradecamers and stimulated peptide degradation, it did not trigger unregulated protein degradation, in contrast to the H-pocket-binding acyldepsipeptide antibiotics (ADEPs). Interestingly, synergistic interactions between 12IGF and ADEP were observed.     

Examination of a Structural Model of Peptidomimicry by Cyclic Acyldepsipeptide Antibiotics in Their Interaction with the ClpP Peptidase

The cyclic acyldepsipeptide (ADEP) antibiotics act by binding the ClpP peptidase and dysregulating its activity. Their exocyclic N‐acylphenylalanine is thought to structurally mimic the ClpP‐binding, (I/L)GF tripeptide loop of the peptidase's accessory ATPases. We found that ADEP analogues with exocyclic N‐acyl tripeptides or dipeptides resembling the (I/L)GF motif were weak ClpP activators and had no bioactivity. In contrast, ADEP analogues possessing difluorophenylalanine N‐capped with methyl‐branched acyl groups—like the side chains of residues in the (I/L)GF motifs—were superior to the parent ADEP with respect to both ClpP activation and bioactivity. We contend that the ADEP's N‐acylphenylalanine moiety is not simply a stand‐in for the ATPases' (I/L)GF motif; it likely has physicochemical properties that are better suited for ClpP binding. Further, our finding that the methyl‐branching on the acyl group of the ADEPs improves activity opens new avenues for optimization.     

Effect of Ester to Amide or N‐Methylamide Substitution on Bacterial Membrane Depolarization and Antibacterial Activity of Novel Cyclic Lipopeptides

Cyclic lipopeptides derived from the fusaricidin/LI‐F family of naturally occurring antibiotics represent particularly attractive candidates for the development of new antibacterial agents. In comparison with natural products, these derivatives may offer better stability under physiologically relevant conditions and lower nonspecific toxicity, while preserving their antibacterial activity. In this study we assessed the ability of cyclic lipodepsipeptide 1 and its analogues—amide 2 , N‐methylamide 3 , and linear peptide 4 —to interact with the cytoplasmic membranes of selected Gram‐positive bacteria. We also investigated their bacteriostatic/bactericidal modes of action and in vivo potency by using a Galleria mellonella model of MRSA infection. Cyclic lipopeptides 1 and 2 depolarize the cytoplasmic membranes of Gram‐positive bacteria in a concentration‐dependent manner. The degree of membrane depolarization was influenced by the structural and physical properties of 1 and 2 , with the more flexible and hydrophobic peptide 1 being most efficient. However, membrane depolarization does not correlate with bacterial cell lethality, suggesting that membrane‐targeting activity is not the main mode of action for this class of antibacterial peptides. Conversely, substitution of the depsipeptide bond in 1 with an N‐methylamide bond in 3 , or its hydrolysis to peptide 4 , lead to a complete loss of antibacterial activity and indicate that the conformation of cyclic lipopeptides plays a role in their antibacterial activities. Cyclic lipopeptides 1 and 2 are also capable of improving the survival of G. mellonella larvae infected with MRSA at varying efficiencies, reflecting their in vitro activities. Gaining more insight into the structure–activity relationship and mode of action of these cyclic lipopeptides may enable the development of new antibiotics of this class with improved antibacterial activity.     

Synthesis and application of some novel antimicrobial monoazonaphthalimide dyes: synthesis and characterisation

A series of novel monoazo dyes based on N‐pyridine‐1,8‐naphthalimides were prepared using 4‐amino‐N‐2‐aminomethylpyridine‐1,8‐naphthalimide as the diazo component and N,N‐diethyl‐meta‐toluidine, 3‐(N,N‐diethylamino)acetanilide and N‐hydroxyethyl‐N‐ethylaniline as the coupling components. The synthesised dyes were purified by utilising column and preparative thin‐layer chromatography methods. The characterisations of the prepared dyes were carried out by differential scanning calorimetery, thin‐layer chromatography (R_f values), Fourier Transform infrared, and proton and carbon nuclear magnetic resonance techniques. The in vitro antibacterial activity of the novel synthesised compounds against Escherichia coli, Staphylococcus aureus, Bacillus subtilis and their antifungal activity against Candida albicans were evaluated by use of conventional agar dilution procedures and a minimum inhibitory concentration. Some of these synthesised dyes demonstrated antibacterial activity against Gram‐positive bacteria in addition to antifungal activities.     

Acyldepsipeptide Probes Facilitate Specific Detection of Caseinolytic Protease P Independent of Its Oligomeric and Activity State

Caseinolytic protease P (ClpP) is a tetradecameric peptidase that assembles with chaperones such as ClpX to gain proteolytic activity. Acyldepsipeptides (ADEPs) are small-molecule mimics of ClpX that bind into hydrophobic pockets on the apical site of the complex, thereby activating ClpP. Detection of ClpP has so far been facilitated with active-site-directed probes which depend on the activity and oligomeric state of the complex. To expand the scope of ClpP labeling, we took a stepwise synthetic approach toward customized ADEP photoprobes. Structure–activity relationship studies with small fragments and ADEP derivatives paired with modeling studies revealed the design principles for suitable probe molecules. The derivatives were tested for activation of ClpP and subsequently applied in labeling studies of the wild-type peptidase as well as enzymes bearing mutations at the active site and an oligomerization sensor. Satisfyingly, the ADEP photoprobes provided a labeling readout of ClpP independent of its activity and oligomeric state.     

Properties of different chitosan/low‐density polyethylene antibacterial plastics

A series of low‐density polyethylene (LDPE) antibacterial functional plastics were prepared by mechanical blending with commercial chitosan (CS), self‐made water‐soluble chitosan (W‐CS), and microchitosan as antibacterial agents. The effects of the antibacterial agent content on the elongation at break of the obtained plastics were tested, and the bacteriostatic effects against Escherichia coli, Bacillus subtilis, and Proteus species were investigated. The results indicate that the elongation at break of LDPE with antibacterial agent decreased and had a slower decline when the mass ratio of CS to LDPE was greater than 0.5 : 100. The LDPE‐based plastics showed different antibacterial activities against the three experimental strains, and plastics with W‐CS exhibited the best antibacterial activity against B. subtilis. However, the antibacterial content had little effect on the antibacterial ratio. Moreover, 6‐week soil burial tests indicated that the addition of CS caused a decrease in the resistance of LDPE to microbiological deterioration in a natural environment. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Synthesis and Characterization of Cationic Surfactants Based on N‐Hexamethylenetetramine as Active Microfouling Agents

Four cationic surfactants of quaternary hexammonium silane chloride based on hexamethylenetetramine and alkyl chloride were synthesized. The chemical structures of the prepared cationic surfactants were elucidated using Fourier transform infrared (FT‐IR) spectroscopy and mass spectrometry analysis. The surface and thermodynamic properties of the prepared surfactants were also studied. The performance of these cationic surfactants as microfouling agents against two strains of Gram‐negative bacteria, namely, Pseudomonas aeruginosa and Escherichia coli, and two strains of Gram‐positive bacteria, namely, Staphylococcus aureus and Bacillus subtilis, were evaluated as antimicrobial agents. The results showed that the maximum antimicrobial activity was detected for N‐hexamethylenetetramine‐N‐ethyl silane ammonium trichloride (Ah). The maximum and minimum antimicrobial activities were 73 and 60 % against S. aureus and E. coli, respectively, at a concentration of 5 mg/l, pH 7, and 37 °C.     

Synthesis and Evaluation of N‐Phenylpyrrolamides as DNA Gyrase B Inhibitors

ATP‐competitive inhibitors of DNA gyrase and topoisomerase IV are among the most interesting classes of antibacterial drugs that are unrepresented in the antibacterial pipeline. We developed 32 new N‐phenylpyrrolamides and evaluated them against DNA gyrase and topoisomerase IV from E. coli and Staphylococcus aureus. Antibacterial activities were studied against Gram‐positive and Gram‐negative bacterial strains. The most potent compound displayed an IC₅₀ of 47 nm against E. coli DNA gyrase, and a minimum inhibitory concentration (MIC) of 12.5 μm against the Gram‐positive Enterococcus faecalis. Some compounds displayed good antibacterial activities against an efflux‐pump‐deficient E. coli strain (MIC=6.25 μm ) and against wild‐type E. coli in the presence of efflux pump inhibitor PAβN (MIC=3.13 μm ). Here we describe new findings regarding the structure–activity relationships of N‐phenylpyrrolamide DNA gyrase B inhibitors and investigate the factors that are important for the antibacterial activity of this class of compounds.     

Structure‐Based Design of Microsomal Prostaglandin E2 Synthase‐1 (mPGES‐1) Inhibitors using a Virtual Fragment Growing Optimization Scheme

A small library of 2,3‐dihydroxybenzamide‐ and N‐(2,3‐dihydroxyphenyl)‐4‐sulfonamide‐based microsomal prostaglandin E₂ synthase‐1 (mPGES‐1) inhibitors was identified following a step‐by‐step optimization of small aromatic fragments selected to interact in focused regions in the active site of mPGES‐1. During the virtual optimization process, the 2,3‐dihydroxybenzamide moiety was first selected as a backbone of the proposed new chemical entities; the identified compounds were then synthesized and biologically evaluated, identifying derivatives with very promising inhibitory activities in the micromolar range. Subsequent structure‐guided replacement of the 2,3‐dihydroxybenzamide by the N‐(2,3‐dihydroxyphenyl)sulfonamide moiety led to the identification of N‐(2,3‐dihydroxyphenyl)‐4‐biphenylsulfonamide ( 6 ), the most potent small molecule of the series (IC₅₀=0.53±0.04 μm ). The simple synthetic procedure and the possibility of enhancing the potency of this class of inhibitors through additional structural modifications pave the way for further development of new molecules with mPGES‐1‐inhibitory activity, with potential application as anti‐inflammatory and anticancer agents.     

Cationic starch iodophores

Cross‐linked cationic starches N‐(2‐hydroxyl)propyl‐3‐trimethyl ammonium starch chloride (CQS chloride), N‐(2‐hydroxyl)propyl‐3‐trimethyl ammonium starch iodide (CQS iodide), and N‐(2‐hydroxyl)propyl‐3‐trimethyl ammonium starch iodide–iodine (CQS triiodide) with the degree of substitution (DS) according to cationic groups from 0.04 to 0.62, as well as cross‐linked starch–iodine complexes were synthesized and tested as potential antibacterial agents. Cationic starch iodine derivatives were obtained during ion exchange reaction between CQS chloride and iodide or iodide–iodine anions in aqueous solutions. CQS_DS≤0.3 chloride can form several types of iodine complexes, such as the blue amylose–iodine inclusion complex and ionic CQS⁺I^?·(I₂)_m complex (m ≥ 1). The antibacterial activity of modified starches–iodine samples against different pathogenic bacterial cultures and contaminated water microorganisms was evaluated. CQS chloride and CQS iodide were found to be bacteriostatic. A strong antibacterial activity was characteristic of CQS triiodides in which molecular iodine is present in both ionic and inclusion complexes. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Synthesis and water absorbency of polyampholytic hydrogels with antibacterial activity

Amphoteric terpolymers of acrylic acid (AA), acrylamide (AM), and N,N′‐dimethyl‐N‐ethylmeth‐acryloxylethylammoniumbromide (DMAEA‐EB) with varied compositions P[AA‐AM‐(DMAEA‐EB)] were synthesized by inverse suspension polymerization. The components of P[AA‐AM‐(DMAEA‐EB)] were verified by FTIR spectroscopy. The water absorption ability and antibacterial activity of the copolymer against Escherichia coli(E. coli) and Staphylococcus hyicus(S. hyicus) suspended in sterilized physiological saline were investigated. The introduction of  N⁺R₄ may increase the water absorbency of P[AA‐AM‐(DMAEA‐EB)] in some degree because of the excellent hydrophilicity of  N⁺R₄. The AA‐AM‐(DMAEA‐EB) hydrogels exhibited high antibacterial activity against bacteria tested. The process of adsorption between live bacteria cells and resins was at least partially reversible. A peak of antibacterial efficiency existed with increasing contact time. The resin killed 96.6% E. coli organisms and 90.3% S. hyicus organisms, respectively, within 30 min of contact at dosage of 0.1g. The concentration of DMAEA‐EB has a special effect on the antibacterial activity of the polyampholytic hydrogels, which is different from polycation. It was observed that the antibacterial activity of the resin with 2 mol % of DMAEA‐EB is superior to the copolymers tested with other compositions. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Synthesis and Properties of Di‐Chain Esterquat Surfactants

A series of cationic surfactants, di‐chain esterquat surfactants (EQs), were synthesized from N‐methyl diethanolamine, hydrochloric acid, ethylene oxide, and caprylic acid, decylic acid, or lauric acid by a three‐step process. The synthesized surfactants were characterized by IR, MS, and ¹HNMR. Surface properties, antibacterial activity, and biodegradability of the synthesized surfactants were investigated. The results showed that synthesized EQs exhibit high surface activities and good biodegradability. Further, the synthesized surfactant, N,N‐di‐[O‐decanoyl‐2‐hydroxyethyl]‐N‐hydroxyethyl‐N‐methylammonium chloride, exhibits excellent antibacterial activity against E. coli and S. aureus.     

Homoserine Lactones,Methyl Oligohydroxybutyrates,and Other Extracellular Metabolites of Macroalgae‐Associated Bacteria of the Roseobacter Clade: Identification and Functions

Twenty‐four strains of marine Roseobacter clade bacteria were isolated from macroalgae and investigated for the production of quorum‐sensing autoinducers, N‐acylhomoserine lactones (AHLs). GC/MS analysis of the extracellular metabolites allowed us to evaluate the release of other small molecules as well. Nineteen strains produced AHLs, ranging from 3‐OH‐C10:0‐HSL (homoserine lactone) to (2E,11Z)‐C18:2‐HSL, but no specific phylogenetic or ecological pattern of individual AHL occurrence was observed when cluster analysis was performed. Other identified compounds included indole, tropone, methyl esters of oligomers of 3‐hydroxybutyric acid, and various amides, such as N‐9‐hexadecenoylalanine methyl ester (9‐C16:1‐NAME), a structural analogue of AHLs. Several compounds were tested for their antibacterial and antialgal activity on marine isolates likely to occur in the habitat of the macroalgae. Both AHLs and 9‐C16:1‐NAME showed high antialgal activity against Skeletonema costatum, whereas their antibacterial activity was low.     

Functional Characterisation of ClpP Mutations Conferring Resistance to Acyldepsipeptide Antibiotics in Firmicutes

Acyldepsipeptide (ADEP) is an exploratory antibiotic with a novel mechanism of action. ClpP, the proteolytic core of the caseinolytic protease, is deregulated towards unrestrained proteolysis. Here, we report on the mechanism of ADEP resistance in Firmicutes. This bacterial phylum contains important pathogens that are relevant for potential ADEP therapy. For Staphylococcus aureus, Bacillus subtilis, enterococci and streptococci, spontaneous ADEP-resistant mutants were selected in vitro at a rate of 10⁻⁶. All isolates carried mutations in clpP. All mutated S. aureus ClpP proteins characterised in this study were functionally impaired; this increased our understanding of the mode of operation of ClpP. For molecular insights, crystal structures of S. aureus ClpP bound to ADEP4 were determined. Well-resolved N-terminal domains in the apo structure allow the pore-gating mechanism to be followed. The compilation of mutations presented here indicates residues relevant for ClpP function and suggests that ADEP resistance will occur at a lower rate during the infection process.     

Antibacterial materials: structure–bioactivity relationship of epoxy–amine resins containing quaternary ammonium compounds covalently attached

Bifunctional aminoalkyldimethylpropylammonium salts (N‐(3‐aminopropyl)‐N,N‐dimethylpentylammonium chloride, N‐(3‐aminopropyl)‐N,N‐dimethyloctylammonium chloride, N‐(3‐aminopropyl)‐N,N‐dimethyldecylammonium chloride, N‐(3‐aminopropyl)‐N,N‐dimethyldodecylammonium chloride) are synthesized and their structure‐dependent antibacterial effect against Gram‐negative Escherichia coli and Gram‐positive Lactococcus lactis is investigated. To this end, resins prepared from bisphenol A diglycidyl ether (2,2‐bis[4‐(glycidyloxy)phenyl]propane) and diethylenetriamine (2,2′‐diaminodiethylamine) as matrix and the bifunctional aminoalkyldimethylpropylammonium salts in a ratio of 6 mol% compared to epoxy components are used. A dependence of antibacterial effect on alkyl chain length of the quaternary ammonium compounds is observed for both species. Furthermore, resins with N‐(3‐aminopropyl)‐N,N‐dimethyldecylammonium chloride in varying concentrations up to 16 mol% for both organisms show a concentration‐dependent antibacterial effect of the quaternary ammonium salt. The antibacterial materials are characterized by differential scanning calorimetry, infrared spectroscopy and rheological studies. © 2013 Society of Chemical Industry     

Antibacterial finishing of polyester/cotton blend fabrics using neem (Azadirachta indica): A natural bioactive agent

The study focused on the development of biofunctional polyester/cotton blend fabric using a natural product. An antimicrobial agent extracted from the seeds of Neem tree (Azadirachta indica) was used for imparting antibacterial property to the blend fabric. Resin and catalyst concentrations were optimized to get the maximum crosslinking in the fabric blends using glyoxal/glycol as a crosslinking agent. The optimized concentrations were used to treat the fabric with the antimicrobial agent along with the crosslinking agent. Quantitative analysis was carried out to measure the antimicrobial activity against Gram‐positive and Gram‐negative bacteria. The results showed that the treated fabrics inhibited the growth of Gram‐positive bacteria (Bacillus subtilis) by more than 90% as compared to the control sample. Antimicrobial activity against Gram‐ positive bacteria was retained up to five machine washes and decreased thereafter. The antibacterial activity was higher against Gram‐positive bacteria as compared to Gram‐ negative bacteria (Proteus vulgaris). The treated fabrics also showed improved crease recovery property although the tensile property showed a marginal decrease. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007     

Modification of narrow‐spectrum peptidomimetic polyurethanes with fatty acid chains confers broad‐spectrum antibacterial activity

Each year, thousands of patients die from antimicrobial‐resistant bacterial infections that fail to respond to conventional antibiotic treatment. Antimicrobial polymers are a promising new method of combating antibiotic‐resistant bacterial infections. We have previously reported the synthesis of a series of narrow‐spectrum peptidomimetic antimicrobial polyurethanes that are effective against Gram‐negative bacteria, such as Escherichia coli; however, these polymers are not effective against Gram‐positive bacteria, such as Staphylococcus aureus. With the aim of understanding the correlation between chemical structure and antibacterial activity, we have subsequently developed three structural variants of these antimicrobial polyurethanes using post‐polymerization modification with decanoic acid and oleic acid. Our results show that such modifications converted the narrow‐spectrum antibacterial activity of these polymers into broad‐spectrum activity against Gram‐positive species such as S. aureus, however, also increasing their toxicity to mammalian cells. Mechanistic studies of bacterial membrane disruption illustrate the differences in antibacterial action between the various polymers. The results demonstrate the challenge of balancing antimicrobial activity and mammalian cell compatibility in the design of antimicrobial polymer compositions. © 2019 Society of Chemical Industry     

N-acyl-L-leucines of biologically active uncommon fatty acids: Synthesis and antibacterial activity

The effect of structural variation in the fatty acid chain on the antibacterial activity ofN-acyl-L-leucines was investigated. PureN-acyl leucines of some structurally different and biologically active uncommon fatty acids were synthesized for the first time and tested for antimicrobial activity.N-Stearoyl-,N-oleoyl- andN-ricinoleoyl leucines were also evaluated for comparison. TheN-acyl leucines exhibited greater activity in acid form than the methyl ester form and against gram positive bacteria than gram-negative bacteria. The presence of a cyclopropane or a hydroxy group or unsaturation in the acyl chain increased the antibacterial activity. Shifting the hydroxyl group toward the amide linkage resulted in a diminished effect on the antibacterial activity.     

Antibacterial activity and cytotoxicity of hydrogel–nanosilver composites based on copolymers from 2‐acrylamido‐2‐methylpropanesulfonate sodium

In this research, we contributed to the search for potential hydrogel–silver dressings by generating hydrogel–silver nanoparticles (AgNPs) composites prepared by the dipping of the crosslinked hydrogel poly(N‐vinylpyrrolidone‐co‐2‐acrylamido‐2‐methylpropanesulfonate sodium) (1:1) and poly(acrylamide‐co‐2‐acrylamido‐2‐methylpropanesulfonate sodium) (1:1) into an aqueous suspension of citrate‐stabilized AgNPs. The composites obtained were evaluated by an antibacterial activity assay on Staphylococcus aureus and Escherichia coli and subjected to an in vitro cytotoxicity assay for human fibroblasts. The composite formed from the hydrogel poly(N‐vinylpyrrolidone‐co‐2‐acrylamido‐2‐methylpropanesulfonate sodium) with 3 mol % N,N‐methylene bisacrylamide showed the highest antibacterial activity and the least cytotoxicity among the composites tested; this makes it an excellent alternative as a potential dressing for the treatment of deep and exudative wounds. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 39644.