Kinetic study of the effect of histidines 240 and 164 on TEM-149 enzyme probed by ß-lactam inhibitors.

In the present study, we performed a detailed kinetic analysis of the enzymes TEM-149, TEM-149(H240), and TEM-149(H164-H240) versus a large panel of inhibitors/inactivators, including penicillins, penems, carbapenems, monobactams, cephamycin, and carbacephem. These compounds behaved as poor substrates versus TEM-149, TEM-149(H240), and TEM-149(H164-H240) ß-lactamases, and the Ki (inhibition constant), K (dissociation constant of the Henri-Michaelis complex), k+2 and k+3 (first-order acylation and deacylation constants, respectively), and k+2/K values were calculated.

Inhibition of the transcriptional repressor LexA: Withstanding drug resistance by inhibiting the bacterial mechanisms of adaptation to antimicrobials.

AIMS: LexA protein is a transcriptional repressor which regulates the expression of more than 60 genes belonging to the SOS global regulatory network activated by damages to bacterial DNA. Considering its role in bacteria, LexA represents a key target to counteract bacterial resistance: the possibility to modulate SOS response through the inhibition of LexA autoproteolysis may lead to reduced drug susceptibility and acquisition of resistance in bacteria. In our study we investigated boron-containing compounds as potential inhibitors of LexA self-cleavage. MAIN METHODS: The inhibition of LexA self-cleavage was evaluated by following the variation of the first-order rate constant by LC-MS at several concentrations of inhibitors. In silico analysis was applied to predict the binding orientations assumed by the inhibitors in the protein active site, upon covalent binding to the catalytic Ser-119. Bacterial filamentation assay was used to confirm the ability of (3-aminophenyl)boronic acid to interfere with SOS induced activation. KEY FINDINGS: Boron-containing compounds act as inhibitors of LexA self-cleavage, as also confirmed by molecular modelling where the compounds interact with the catalytic Ser-119, via the formation of an acyl-enzyme intermediate. A new equation for the description of the inhibition potency in an autoproteolytic enzyme is also disclosed. Bacterial filamentation assays strongly support the interference of our compounds with the SOS response activation through inhibition of septum formation. SIGNIFICANCE: The obtained results demonstrated that phenylboronic compounds could be exploited in a hit-to-lead optimization process toward effective LexA self-cleavage inhibitors. They would sustain the rehabilitation in therapy of several dismissed antibiotics.

Cerium oxide nanoparticles as potential antibiotic adjuvant. Effects of CeO2 nanoparticles on bacterial outer membrane permeability.

BACKGROUND: Therapeutic options against Multi Drug Resistant (MDR) pathogens are limited and the overall strategy would be the development of adjuvants able to enhance the activity of therapeutically available antibiotics. Non-specific outer membrane permeabilizer, like metal-oxide nanoparticles, can be used to increase the activity of antibiotics in drug-resistant pathogens. The study aims to investigate the effect of cerium oxide nanoparticles (CeO2 NPs) on bacterial outer membrane permeability and their application in increasing the antibacterial activity of antibiotics against MDR pathogens. METHODS: The ability of CeO2 NPs to permeabilize Gram-negative bacterial outer membrane was investigated by calcein-loaded liposomes. The extent of the damage was evaluated using lipid vesicles loaded with FITC-dextran probes. The effect on bacterial outer membrane was evaluated by measuring the coefficient of permeability at increasing concentrations of CeO2 NPs. The interaction between CeO2 NPs and beta-lactams was evaluated by chequerboard assay against a Klebsiella pneumoniae clinical isolate expressing high levels of resistance against those antibiotics. RESULTS: Calcein leakage increases as NPs concentrations increase while no leakage was observed in FITC-dextran loaded liposomes. In Escherichia coli the outer membrane permeability coefficient increases in presence of CeO2 NPs. The antibacterial activity of beta-lactam antibiotics against K. pneumoniae was enhanced when combined with NPs. CONCLUSIONS: CeO2 NPs increases the effectiveness of antimicrobials which activity is compromised by drug resistance mechanisms. The synergistic effect is the result of the interaction of NPs with the bacterial outer membrane. The low toxicity of CeO2 NPs makes them attractive as antibiotic adjuvants against MDR pathogens.

SOS response in bacteria: Inhibitory activity of lichen secondary metabolites against Escherichia coli RecA protein.

BACKGROUND: RecA is a bacterial multifunctional protein essential to genetic recombination, error-prone replicative bypass of DNA damages and regulation of SOS response. The activation of bacterial SOS response is directly related to the development of intrinsic and/or acquired resistance to antimicrobials. Although recent studies directed towards RecA inactivation via ATP binding inhibition described a variety of micromolar affinity ligands, inhibitors of the DNA binding site are still unknown. PURPOSE: Twenty-seven secondary metabolites classified as anthraquinones, depsides, depsidones, dibenzofurans, diphenyl-butenolides, paraconic acids, pseudo-depsidones, triterpenes and xanthones, were investigated for their ability to inhibit RecA from Escherichia coli. They were isolated in various Chilean regions from 14 families and 19 genera of lichens. METHODS: The ATP hydrolytic activity of RecA was quantified detecting the generation of free phosphate in solution. The percentage of inhibition was calculated fixing at 100µM the concentration of the compounds. Deeper investigations were reserved to those compounds showing an inhibition higher than 80%. To clarify the mechanism of inhibition, the semi-log plot of the percentage of inhibition vs. ATP and vs. ssDNA, was evaluated. RESULTS: Only nine compounds showed a percentage of RecA inhibition higher than 80% (divaricatic, perlatolic, alpha-collatolic, lobaric, lichesterinic, protolichesterinic, epiphorellic acids, sphaerophorin and tumidulin). The half-inhibitory concentrations (IC50) calculated for these compounds were ranging from 14.2µM for protolichesterinic acid to 42.6µM for sphaerophorin. Investigations on the mechanism of inhibition showed that all compounds behaved as uncompetitive inhibitors for ATP binding site, with the exception of epiphorellic acid which clearly acted as non-competitive inhibitor of the ATP site. Further investigations demonstrated that epiphorellic acid competitively binds the ssDNA binding site. Kinetic data were confirmed by molecular modelling binding predictions which shows that epiphorellic acid is expected to bind the ssDNA site into the L2 loop of RecA protein. CONCLUSION: In this paper the first RecA ssDNA binding site ligand is described. Our study sets epiphorellic acid as a promising hit for the development of more effective RecA inhibitors. In our drug discovery approach, natural products in general and lichen in particular, represent a successful source of active ligands and structural diversity.

Interaction between lichen secondary metabolites and antibiotics against clinical isolates methicillin-resistant Staphylococcus aureus strains.

The in vitro antimicrobial activities of five compounds isolated from lichens, collected in several Southern regions of Chile (including the Chilean Antarctic Territory), were evaluated alone and in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. MIC90, MIC50, as well as MBC90 and MBC50, for the lichen compounds were evaluated. The MIC90 was ranging from 32 µg/ml for perlatolic acid to 128 µg/ml for α-collatolic acid. MBC90 was ranging from onefold up to twofold the MIC90 for each compound. A synergistic action was observed in combination with gentamicin, whilst antagonism was observed for some lichen compounds in combination with levofloxacin. All combinations with erythromycin were indifferent, whilst variability was observed for clindamycin and oxacillin combinations. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index and the response surface approach using the ΔE model. Discrepancies were found between both methods for some combinations. These could mainly be explained by the failure of FIC approach, being too much subjective and sensitive to experimental errors. These findings suggest, however, that the natural compounds from lichens are good candidates for the individuation of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.