Hydrophobic substituents on isatin derivatives enhance their inhibition against bacterial peptidoglycan glycosyltransferase activity.

Moenomycin A, the well-known natural product inhibitor of peptidoglycan glycosyltransferase (PGT), is a large amphiphilic molecule of molecular mass of 1583 g/mol and its bioavailablity as a drug is relatively poor. In searching for small-molecule ligands with high inhibition ability targeting the enzyme, we found that the addition of hydrophobic groups to an isatin-based inhibitor of bacterial PGT significantly improves its inhibition against the enzyme, as well as its antibacterial activity. The improvement in enzymatic inhibition can be attributed to a better binding of the small molecule inhibitor to the hydrophobic region of the membrane-bound bacterial cell wall synthesis enzyme and the plasma membrane. In the present study, a total of 20 new amphiphilic compounds were systematically designed and the relationship between molecular hydrophobicity and the antibacterial activity by targeting at PGT was demonstrated. The in vitro lipid II transglycosylation inhibitory effects (IC50) against E. coli PBP1b and MICs of the compounds were investigated. Optimized results including MIC values of 6 µg/mL for MSSA, MRSA, B. subtilis and 12 µg/mL for E. coli were obtained with an isatin derivative 5m which has a molecular mass of 335 g/mol.

Effect of Lactamase Inhibitors on the Biosensor Penp during the Measurement of Lactam Antibiotics Concentration.

PenP is a fluorescent biosensor of lactam antibiotics (LA). It is structurally derived from the mutant lactamase TEM-1 comprising the substitution E166C, where fluorescein is covalently linked to cysteine. The presence of LA in the medium produces a change in the intrinsic fluorescence level of the biosensor, and the integral of the fluorescence level over time correlates directly with the LA concentration. Previously, we have successfully used PenP to determine the concentration of lactam antibiotics in clinical samples. The use of lactamase inhibitors (LI) is a common strategy to enhance the effect of LA due to the inhibition of an important resistance mechanism of pathogenic microorganisms. Structurally, LI and LA share the common element of recognition of lactamases (the lactam ring), but they differ in the reversibility of the mechanism of interaction with said enzyme. Because the biological recognition domain of PenP is derived from a lactamase, LI is expected to interfere with the PenP detection capabilities. Surprisingly, this work provides evidence that the effect of LI is marginal in the determination of LA concentration mediated by PenP.

Influences of disulfide connectivity on structure and antimicrobial activity of tachyplesin I.

Tachyplesin I is a potent antimicrobial peptide with broad spectrum of antimicrobial activity. It has 2 disulfide bonds and can form 3 disulfide bond isomers. In this study, the structure and antimicrobial activity of 3 tachyplesin I isomers (tachyplesin I, 3C12C, 3C7C) were investigated using molecular dynamic simulations, circular dichroism structural study, as well as antimicrobial activity and hemolysis assay. Our results suggest that in comparison to the native peptide, the 2 isomers (3C12C, 3C7C) have substantial structural and activity variations. The native peptide is in the ribbon conformation, while 3C12C and 3C7C possess remarkably different secondary structures, which are referred as "globular" and "beads" isomers, respectively. The substantially decreased hemolysis effects for these 2 isomers is accompanied by significantly decreased anti-gram-positive bacterial activity.

Evaluation of Meropenem Pharmacokinetics in an Experimental Acute Respiratory Distress Syndrome (ARDS) Model during Extracorporeal Membrane Oxygenation (ECMO) by Using a PenP ß-Lactamase Biosensor.

INTRODUCTION: The use of antibiotics is mandatory in patients during extracorporeal membrane oxygenation (ECMO) support. Clinical studies have shown high variability in the antibiotic concentrations, as well as sequestration of them by the ECMO circuit, suggesting that the doses and/or interval administration used during ECMO may not be adequate. Thus, a fast response sensor to estimate antibiotic concentrations in this setting would contribute to improve dose adjustments. The biosensor PenP has been shown to have a dynamic range, sensitivity and specificity useful for pharmacokinetic (PK) tests in healthy subjects. However, the use of this biosensor in the context of a complex critical condition, such as ECMO during acute respiratory distress syndrome (ARDS), has not been tested. OBJECTIVES: To describe, by using PenP Biosensor, the pharmacokinetic of meropenem in a 24-h animal ARDS/ECMO model. METHODS: The PK of meropenem was evaluated in a swine model before and during ECMO. RESULTS: The PK parameters such as maximum concentration (Cmax), elimination rate constant (Ke), and cleareance (Cl), were not significantly altered during ECMO support. CONCLUSIONS: (a) ECMO does not affect the PK of meropenem, at least during the first 24 h; and (b) PenP has the potential to become an effective tool for making medical decisions associated with the dose model of antibiotics in a critical patient context.

pZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis.

BACKGROUND: The ethanol-producing bacterium Zymomonas mobilis has attracted considerable scientific and commercial interest due to its exceptional physiological properties. Shuttle vectors derived from native plasmids have previously been successfully used for heterologous gene expression in this bacterium for a variety of purposes, most notably for metabolic engineering applications. RESULTS: A quantitative PCR (qPCR) approach was used to determine the copy numbers of two endogenous double stranded DNA plasmids: pZMO1A (1,647 bp) and pZMO7 (pZA1003; 4,551 bp) within the NCIMB 11163 strain of Z. mobilis. Data indicated pZMO1A and pZMO7 were present at ca. 3-5 and ca. 1-2 copies per cell, respectively. A ca. 1,900 bp fragment from plasmid pZMO7 was used to construct two Escherichia coli - Z. mobilis shuttle vectors (pZ7C and pZ7-184). The intracellular stabilities and copy numbers of pZ7C and pZ7-184 were characterized within the NCIMB 11163, ATCC 29191 and (ATCC 10988-derived) CU1 Rif2 strains of Z. mobilis. Both shuttle vectors could be stably maintained within the ATCC 29191 strain (ca. 20-40 copies per cell), and the CU1 Rif2 strain (ca. 2-3 copies per cell), for more than 50 generations in the absence of an antibiotic selectable marker. A selectable marker was required for shuttle vector maintenance in the parental NCIMB 11163 strain; most probably due to competition for replication with the endogenous pZMO7 plasmid molecules. N-terminal glutathione S-transferase (GST)-fusions of four endogenous proteins, namely the acyl-carrier protein (AcpP); 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA); DNA polymerase III chi subunit (HolC); and the RNA chaperone protein Hfq; were successfully expressed from pZ7C-derived shuttle vectors, and their protein-protein binding interactions were analyzed in Z. mobilis ATCC 29191. Using this approach, proteins that co-purified with AcpP and KdsA were identified. CONCLUSIONS: We show that a shuttle vector-based protein affinity 'pull-down' approach can be used to probe protein interaction networks in Z. mobilis cells. Our results demonstrate that protein expression plasmids derived from pZMO7 have significant potential for use in future biological or biotechnological applications within Z. mobilis.

Remineralising Dentine Caries Using Sodium Fluoride with Silver Nanoparticles: An In Vitro Study.

OBJECTIVE: To investigate the remineralizing and staining effects of sodium fluoride (NaF) solution with polyethylene glycol-coated silver nanoparticles (PEG-AgNPs) on artificial dentine caries. MATERIALS AND METHODS: Demineralized human dentine blocks were allocated to three groups. The blocks in group 1 underwent a topical application of a 12% silver diamine fluoride (SDF, 14,150 ppm fluoride) solution. The blocks in group 2 received a topical application of a 2.5% NaF (11,310 ppm fluoride) with PEG-AgNPs (400 ppm silver). The blocks in group 3 received deionized water. All blocks were subjected to pH cycling for 8 days. The surface morphology and cross-sectional features were investigated using scanning electron microscopy (SEM). The color parameters, crystal characteristics, lesion depth, and collagen degradation of the blocks were assessed using digital spectrophotometry, X-ray diffraction (XRD), micro-computed tomography, and spectrophotometry with a hydroxyproline assay, respectively. RESULTS: The SEM showed that dentine collagen was exposed in group 3 but not in groups 1 and 2. The mean lesion depths in groups 1 to 3 were 118±7 µm, 121±14 µm, and 339±20 µm, respectively (groups1,2<3; p<0.001). The data indicated that fluoridated PEG-AgNPs introduced no significant color effect on dentine, but SDF caused distinct discoloration. The XRD indicated that silver chloride was formed in group 1, and fluorapatite was detected in groups 1 and 2. The concentration of hydroxyproline liberated from collagen was significantly less in groups 1 and 2 than in group 3. CONCLUSION: The use of NaF solution with PEG-AgNPs can remineralize artificial dentine caries and inhibit collagen degradation without causing significant tooth staining.

Synthesis and Characterization of Fluoridated Silver Nanoparticles and Their Potential as a Non-Staining Anti-Caries Agent.

OBJECTIVES: The first objective of this study was to prepare sodium fluoride (NaF) solution with various concentrations of polyethylene glycol-coated silver nanoparticles (PEG-AgNPs). The second objective was to study the antibacterial activity against Streptococcus mutans and the tooth-staining effect of the solution. METHODS: PEG-AgNPs were prepared via the one-step chemical reduction of silver acetate with thiolated polyethylene glycol. The PEG-AgNPs were characterized with ultraviolet-visible spectrometry and transmission electron microscopy. The half maximal inhibitory concentration (IC50) for the PEG-AgNPs against Streptococcus mutans and human gingival fibroblasts (HGF-1) were determined. The staining effect on dentin and enamel for the 2.5% NaF solutions with PEG-AgNPs at 12,800, 6400, 1600, and 400 ppm was investigated using digital spectrophotometry. The IC50 of the fluoridated silver nanoparticles against Streptococcus mutans were measured. RESULTS: The PEG-AgNPs have an average diameter of 2.56±0.43 nm and showed excellent stability at high ionic strength (2.5% NaF) for 18 months. The IC50 of PEG-AgNPs against Streptococcus mutans was found to be 21.16±1.08 ppm silver, which was half of IC50 against HGF-1 cells (42.36±1.12 ppm), providing a working range to kill bacteria with no harm to human cells. The formulations with different concentrations of PEG-AgNPs showed no significant staining of teeth. Combining PEG-AgNPs with NaF significantly expanded the therapeutic window against Streptococcus mutans by reducing its IC50. CONCLUSION: A biocompatible solution of NaF with PEG-AgNPs was developed. Because it has antibacterial activity against Streptococcus mutans and no tooth-staining effect, it can be used as an anti-caries agent.

Enhanced Activity against Multidrug-Resistant Bacteria through Coapplication of an Analogue of Tachyplesin I and an Inhibitor of the QseC/B Signaling Pathway.

Tachyplesin I (TPI) is a cationic ß-hairpin antimicrobial peptide with broad-spectrum, potent antimicrobial activity. In this study, the all d-amino acid analogue of TPI (TPAD) was synthesized, and its structure and activity were determined. TPAD has comparable antibacterial activity to TPI on 14 bacterial strains, including four drug-resistant bacteria. Importantly, TPAD has significantly improved stability against enzymatic degradation and decreased hemolytic activity compared to TPI, indicating that it has better therapeutic potential. The induction of bacterial resistance using low concentrations of TPAD resulted in the activation of the QseC/B two-component system. Deletion of this system resulted in at least five-fold improvement of TPAD activity, and the combined use of TPAD with LED209, a QseC/B inhibitor, significantly enhanced the bactericidal effect against three classes of multidrug-resistant bacteria.

Plasma and Renal Cortex Meropenem Concentrations in Patients Undergoing Percutaneous Renal Biopsy.

BACKGROUND: Urinary tract infection (UTI) is the most common bacterial infection in the world. Some cases can have serious complication as death by septic shock. With the increasing spread of multidrug-resistant bacteria, the therapeutic possibilities against the complicated UTI are exhausted, forcing the use of broad-spectrum antibiotics such as meropenem. OBJECTIVES: To evaluate the penetrating ability of meropenem to renal tissue using an enzymatic biosensor in samples of renal cortex and its correlation with plasma levels. METHOD: We conducted a descriptive study in humans with indication of kidney biopsy. Meropenem was administered 1 hour before performing the biopsy, and the concentrations of meropenem in a series of samples of plasma and renal biopsy were determined. RESULTS: Renal biopsy and plasma samples of 14 patients, 64% women with body mass index of 26.3 kg/m2 (SD ± 2.9) and estimated glomerular filtration rate of 57.5 mL/min/1.73 m2 (SD ± 44.1), were examined. Renal biopsy was done at 68.9 minutes (SD ± 20.3), and the second plasma sample was obtained at 82.1 minutes (SD ± 21.2) and the third at 149.6 minutes (SD ± 31.5). The mean kidney meropenem concentration was 3.1 µg/mL (SD ± 1.9). For each patient, a decay curve of plasma meropenem concentration was constructed. The proportion of meropenem concentrations in renal tissue and plasma at biopsy moment was 14% (SD ± 10) with an interquartile range of 5.5-20.3%. With normal renal function, meropenem can achieve a bactericidal effect towards bacteria with MIC-90 < 0.76 µg/mL in the renal parenchyma. CONCLUSIONS: Meropenem is effective to treat the most frequent uropathogens with the bactericidal effect. Nevertheless, for resistant bacteria, it is necessary to adjust the dose to achieve adequate parenchymal concentration.

Study of Benzofuroquinolinium Derivatives as a New Class of Potent Antibacterial Agent and the Mode of Inhibition Targeting FtsZ.

New generation of antibacterial agents are urgently needed in order to fight the emergence of multidrug-resistant bacteria. FtsZ is currently identified as a promising target for new types of antimicrobial compounds development because of its conservative characteristics and its essential role played in bacterial cell division. In the present study, the antibacterial activity of a series of benzofuroquinolinium derivatives was investigated. The results show that the compounds possess potent antibacterial activity against drug resistant pathogens including MRSA, VREF and NDM-1 Escherichia coli. Biological studies reveal that the compound is an effective inhibitor that is able to suppress FtsZ polymerization and GTPase activity and thus stopping the cell division and causing cell death. More importantly, this series of compounds shows low cytotoxicity on mammalian cells and therefore they could be new chemotypes for the development of new antibacterial agents targeting the cell-division protein FtsZ.

New application of tiplaxtinin as an effective FtsZ-targeting chemotype for an antimicrobial study.

The filamenting temperature-sensitive mutant Z (FtsZ) protein is generally recognized as a promising antimicrobial drug target. In the present study, a small organic molecule (tiplaxtinin) was identified for the first time as an excellent cell division inhibitor by using a cell-based screening approach from a library with 250 compounds. Tiplaxtinin possesses potent antibacterial activity against Gram-positive pathogens. Both in vitro and in vivo results reveal that the compound is able to disrupt dynamic assembly of FtsZ and Z-ring formation effectively through the mechanism of stimulating FtsZ polymerization and impairing GTPase activity.

A Thiazole Orange Derivative Targeting the Bacterial Protein FtsZ Shows Potent Antibacterial Activity.

The prevalence of multidrug resistance among clinically significant bacteria calls for the urgent development of new antibiotics with novel mechanisms of action. In this study, a new small molecule exhibiting excellent inhibition of bacterial cell division with potent antibacterial activity was discovered through cell-based screening. The compound exhibits a broad spectrum of bactericidal activity, including the methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and NDM-1 Escherichia coli. The in vitro and in vivo results suggested that this compound disrupts the dynamic assembly of FtsZ protein and Z-ring formation through stimulating FtsZ polymerization. Moreover, this compound exhibits no activity on mammalian tubulin polymerization and shows low cytotoxicity on mammalian cells. Taken together, these findings could provide a new chemotype for development of antibacterials with FtsZ as the target.

'Light up' protein-protein interaction through bioorthogonal incorporation of a turn-on fluorescent probe into ß-lactamase.

Fluorescent labeling of biomacromolecules to 'light up' biological events through non-invasive methods is of great importance, but is still challenging in terms of fluorophore properties and the labeling methods used. Herein, we designed and synthesized a biocompatible and conformation sensitive tetraphenylethene derivative EPB with aggregation induced emission (AIE) properties. By introducing EPB into TEM-1 ß-lactamase (TEM-1 Bla) through a two-step approach, a conformation-dependent fluorescent sensor EPB104-Bla was genetically engineered, which was applied to monitor the protein-protein interaction (PPI) with ß-lactamase inhibitor protein (BLIP). The fluorescence signal of EPB104-Bla increases by an approximately 5-fold upon binding to BLIP, indicating that EPB-104 Bla is capable of lighting up the PPI. The dissociation constant (Kd) between EPB104-Bla and BLIP was estimated to be 0.6 µM, which is consistent with that derived from the kinetic inhibition assay. This study demonstrates that genetic modification of proteins with AIE probes might open up new opportunities to develop biosensors in PPI analysis.