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Physiological Reports Nov 2021We have previously demonstrated that inhibition of extracellularly oriented carbonic anhydrase (CA) isoforms protects the myocardium against ischemia-reperfusion injury....
We have previously demonstrated that inhibition of extracellularly oriented carbonic anhydrase (CA) isoforms protects the myocardium against ischemia-reperfusion injury. In this study, our aim was to assess the possible further contribution of CA intracellular isoforms examining the actions of the highly diffusible cell membrane permeant inhibitor of CA, ethoxzolamide (ETZ). Isolated rat hearts, after 20 min of stabilization, were assigned to the following groups: (1) Nonischemic control: 90 min of perfusion; (2) Ischemic control: 30 min of global ischemia and 60 min of reperfusion (R); and (3) ETZ: ETZ at a concentration of 100 μM was administered for 10 min before the onset of ischemia and then during the first 10 min of reperfusion. In additional groups, ETZ was administered in the presence of SB202190 (SB, a p38MAPK inhibitor) or chelerythrine (Chel, a protein kinase C [PKC] inhibitor). Infarct size, myocardial function, and the expression of phosphorylated forms of p38MAPK, PKCε, HSP27, and Drp1, and calcineurin Aβ content were assessed. In isolated mitochondria, the Ca response, Ca retention capacity, and membrane potential were measured. ETZ decreased infarct size by 60%, improved postischemic recovery of myocardial contractile and diastolic relaxation increased P-p38MAPK, P-PKCε, P-HSP27, and P-Drp1 expression, decreased calcineurin content, and normalized calcium and membrane potential parameters measured in isolated mitochondria. These effects were significantly attenuated when ETZ was administered in the presence of SB or Chel. These data show that ETZ protects the myocardium and mitochondria against ischemia-reperfusion injury through p38MAPK- and PKCε-dependent pathways and reinforces the role of CA as a possible target in the management of acute cardiac ischemic diseases.
Topics: Animals; Benzophenanthridines; Calcium; Carbonic Anhydrase Inhibitors; Enzyme Inhibitors; Ethoxzolamide; Heart; Imidazoles; Isolated Heart Preparation; Membrane Potential, Mitochondrial; Mitochondria, Heart; Mitochondrial Permeability Transition Pore; Myocardial Reperfusion Injury; Myocardium; Protein Kinase C; Pyridines; Rats; p38 Mitogen-Activated Protein Kinases
PubMed: 34806317
DOI: 10.14814/phy2.15093 -
Trends in Pharmacological Sciences Apr 2017The continued rise of antibiotic-resistant bacterial infections has motivated alternative strategies for target discovery and treatment of infections. Antivirulence... (Review)
Review
The continued rise of antibiotic-resistant bacterial infections has motivated alternative strategies for target discovery and treatment of infections. Antivirulence therapies function through inhibition of in vivo required virulence factors to disarm the pathogen instead of directly targeting viability or growth. This approach to treating bacteria-mediated diseases may have advantages over traditional antibiotics because it targets factors specific for pathogenesis, potentially reducing selection for resistance and limiting collateral damage to the resident microbiota. This review examines vulnerable molecular mechanisms used by bacteria to cause disease and the antivirulence compounds that sabotage these virulence pathways. By expanding the study of antimicrobial targets beyond those that are essential for growth, antivirulence strategies offer new and innovative opportunities to combat infectious diseases.
Topics: Anti-Bacterial Agents; Artemisinins; Bacteria; Bacterial Adhesion; Bacterial Proteins; Ethoxzolamide; Fimbriae, Bacterial; Histidine Kinase; Sulfonamides; Virulence
PubMed: 28209403
DOI: 10.1016/j.tips.2017.01.004 -
Journal of Enzyme Inhibition and... Dec 2019Ethoxzolamide (EZA), acetazolamide, and methazolamide are clinically used sulphonamide drugs designed to treat non-bacteria-related illnesses (e.g. glaucoma), but they...
Ethoxzolamide (EZA), acetazolamide, and methazolamide are clinically used sulphonamide drugs designed to treat non-bacteria-related illnesses (e.g. glaucoma), but they also show antimicrobial activity against the gastric pathogen EZA showed the highest activity, and was effective against clinical isolates resistant to metronidazole, clarithromycin, and/or amoxicillin, suggesting that EZA kills mechanisms different from that of these antibiotics. The frequency of single-step spontaneous resistance acquisition by was less than 5 × 10, showing that resistance to EZA does not develop easily. Resistance was associated with mutations in three genes, including the one that encodes undecaprenyl pyrophosphate synthase, a known target of sulphonamides. The data indicate that EZA impacts multiple targets in killing . Our findings suggest that developing the approved anti-glaucoma drug EZA into a more effective anti- agent may offer a faster and cost-effective route towards new antimicrobials with a novel mechanism of action.
Topics: Anti-Bacterial Agents; Dose-Response Relationship, Drug; Ethoxzolamide; Helicobacter pylori; Microbial Sensitivity Tests; Molecular Structure; Structure-Activity Relationship
PubMed: 31530039
DOI: 10.1080/14756366.2019.1663416 -
Pathogens (Basel, Switzerland) Jun 2016Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes which catalyze the hydration of carbon dioxide to bicarbonate and protons. Many pathogenic bacteria encode such... (Review)
Review
Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes which catalyze the hydration of carbon dioxide to bicarbonate and protons. Many pathogenic bacteria encode such enzymes belonging to the α-, β-, and/or γ-CA families. In the last decade, enzymes from some of these pathogens, including Legionella pneumophila, have been cloned and characterized in detail. These enzymes were shown to be efficient catalysts for CO₂ hydration, with kcat values in the range of (3.4-8.3) × 10⁵ s(-1) and kcat/KM values of (4.7-8.5) × 10⁷ M(-1)·s(-1). In vitro inhibition studies with various classes of inhibitors, such as anions, sulfonamides and sulfamates, were also reported for the two β-CAs from this pathogen, LpCA1 and LpCA2. Inorganic anions were millimolar inhibitors, whereas diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid, and phenylarsonic acid were micromolar ones. The best LpCA1 inhibitors were aminobenzolamide and structurally similar sulfonylated aromatic sulfonamides, as well as acetazolamide and ethoxzolamide (KIs in the range of 40.3-90.5 nM). The best LpCA2 inhibitors belonged to the same class of sulfonylated sulfonamides, together with acetazolamide, methazolamide, and dichlorophenamide (KIs in the range of 25.2-88.5 nM). Considering such preliminary results, the two bacterial CAs from this pathogen represent promising yet underexplored targets for obtaining antibacterials devoid of the resistance problems common to most of the clinically used antibiotics, but further studies are needed to validate them in vivo as drug targets.
PubMed: 27322334
DOI: 10.3390/pathogens5020044 -
Antimicrobial Agents and Chemotherapy Aug 2015Mycobacterium tuberculosis must sense and adapt to host environmental cues to establish and maintain an infection. The two-component regulatory system PhoPR plays a...
Mycobacterium tuberculosis must sense and adapt to host environmental cues to establish and maintain an infection. The two-component regulatory system PhoPR plays a central role in sensing and responding to acidic pH within the macrophage and is required for M. tuberculosis intracellular replication and growth in vivo. Therefore, the isolation of compounds that inhibit PhoPR-dependent adaptation may identify new antivirulence therapies to treat tuberculosis. Here, we report that the carbonic anhydrase inhibitor ethoxzolamide inhibits the PhoPR regulon and reduces pathogen virulence. We show that treatment of M. tuberculosis with ethoxzolamide recapitulates phoPR mutant phenotypes, including downregulation of the core PhoPR regulon, altered accumulation of virulence-associated lipids, and inhibition of Esx-1 protein secretion. Quantitative single-cell imaging of a PhoPR-dependent fluorescent reporter strain demonstrates that ethoxzolamide inhibits PhoPR-regulated genes in infected macrophages and mouse lungs. Moreover, ethoxzolamide reduces M. tuberculosis growth in both macrophages and infected mice. Ethoxzolamide inhibits M. tuberculosis carbonic anhydrase activity, supporting a previously unrecognized link between carbonic anhydrase activity and PhoPR signaling. We propose that ethoxzolamide may be pursued as a new class of antivirulence therapy that functions by modulating expression of the PhoPR regulon and Esx-1-dependent virulence.
Topics: Animals; Antigens, Bacterial; Bacterial Proteins; Carbonic Anhydrase Inhibitors; Carbonic Anhydrases; Cells, Cultured; Down-Regulation; Ethoxzolamide; Gene Expression Regulation, Bacterial; Macrophages; Mice; Mice, Inbred C57BL; Mutation; Mycobacterium tuberculosis; Regulon; Tuberculosis; Virulence
PubMed: 25987613
DOI: 10.1128/AAC.00719-15 -
International Journal of Molecular... Jun 2009The analysis of tight protein-ligand binding reactions by isothermal titration calorimetry (ITC) and thermal shift assay (TSA) is presented. The binding of radicicol to...
The analysis of tight protein-ligand binding reactions by isothermal titration calorimetry (ITC) and thermal shift assay (TSA) is presented. The binding of radicicol to the N-terminal domain of human heat shock protein 90 (Hsp90alphaN) and the binding of ethoxzolamide to human carbonic anhydrase (hCAII) were too strong to be measured accurately by direct ITC titration and therefore were measured by displacement ITC and by observing the temperature-denaturation transitions of ligand-free and ligand-bound protein. Stabilization of both proteins by their ligands was profound, increasing the melting temperature by more than 10 masculineC, depending on ligand concentration. Analysis of the melting temperature dependence on the protein and ligand concentrations yielded dissociation constants equal to 1 nM and 2 nM for Hsp90alphaN-radicicol and hCAII-ethoxzolamide, respectively. The ligand-free and ligand-bound protein fractions melt separately, and two melting transitions are observed. This phenomenon is especially pronounced when the ligand concentration is equal to about half the protein concentration. The analysis compares ITC and TSA data, accounts for two transitions and yields the ligand binding constant and the parameters of protein stability, including the Gibbs free energy and the enthalpy of unfolding.
Topics: Calorimetry; Carbonic Anhydrase II; Ethoxzolamide; HSP90 Heat-Shock Proteins; Humans; Kinetics; Ligands; Macrolides; Models, Theoretical; Protein Binding; Thermodynamics
PubMed: 19582223
DOI: 10.3390/ijms10062662 -
Microbiology Spectrum Jun 2023The ADC (AmpC) β-lactamase is universally present in the Acinetobacter baumannii chromosome, suggesting it may have a yet-to-be-identified cellular function. Using...
The ADC (AmpC) β-lactamase is universally present in the Acinetobacter baumannii chromosome, suggesting it may have a yet-to-be-identified cellular function. Using peptidoglycan composition analysis, we show that overexpressing the ADC-7 β-lactamase in A. baumannii drives changes consistent with altered l,d-transpeptidase activity. Based on this, we tested whether cells overexpressing ADC-7 would exhibit new vulnerabilities. As proof of principle, a screen of transposon insertions revealed that an insertion in the distal 3' end of , encoding carbonic anhydrase, resulted in a significant loss of viability when the gene was overexpressed. A deletion mutant exhibited a more pronounced loss of viability than the transposon insertion, and this became amplified when cells overexpressed ADC-7. Interestingly, overexpression of the OXA-23 or TEM-1 β-lactamases also led to a pronounced loss of viability in cells with reduced carbonic anhydrase activity. In addition, we demonstrate that reduced CanB activity led to increased sensitivity to peptidoglycan synthesis inhibitors and to the carbonic anhydrase inhibitor ethoxzolamide. Furthermore, this strain exhibited a synergistic interaction with the peptidoglycan inhibitor fosfomycin and ethoxzolamide. Our results highlight the impact of ADC-7 overexpression on cell physiology and reveal that the essential carbonic anhydrase CanB may represent a novel target for antimicrobial agents that would exhibit increased potency against β-lactamase-overexpressing A. baumannii. Acinetobacter baumannii has become resistant to all classes of antibiotics, with β-lactam resistance responsible for the majority of treatment failures. New classes of antimicrobials are needed to treat this high-priority pathogen. This study had uncovered a new genetic vulnerability in β-lactamase-expressing A. baumannii, where reduced carbonic anhydrase activity becomes lethal. Inhibitors of carbonic anhydrase could represent a new method for treating A. baumannii infections.
Topics: Humans; Acinetobacter baumannii; Ethoxzolamide; Peptidoglycan; Acinetobacter Infections; beta-Lactamases; Anti-Bacterial Agents; Cell Physiological Phenomena; Microbial Sensitivity Tests
PubMed: 37074187
DOI: 10.1128/spectrum.04646-22 -
Gut Pathogens 2020With the rise of bacterial resistance to conventional antibiotics, re-purposing of Food and Drug Administration (FDA) approved drugs currently used to treat non-bacteria...
With the rise of bacterial resistance to conventional antibiotics, re-purposing of Food and Drug Administration (FDA) approved drugs currently used to treat non-bacteria related diseases as new leads for antibacterial drug discovery has become an attractive alternative. Ethoxzolamide (EZA), an FDA-approved diuretic acting as a human carbonic anhydrase inhibitor, is known to kill the gastric pathogenic bacterium in vitro via an, as yet, unknown mechanism. To date, EZA activity and resistance have been investigated for only one strain, P12. We have now performed a susceptibility and resistance study with strains SS1 and 26695. Mutants resistant to EZA were isolated, characterized and their genomes sequenced. Resistance-conferring mutations were confirmed by backcrossing the mutations into the parent strain. As with P12, resistance to EZA in strains SS1 and 26695 does not develop easily, since the rate of spontaneous resistance acquisition was less than 10. Acquisition of resistance was associated with mutations in 3 genes in strain SS1, and in 6 different genes in strain 26695, indicating that EZA targets multiple systems. All resistant isolates had mutations affecting cell wall synthesis and control of gene expression. EZA's potential for treating duodenal ulcers has already been demonstrated. Our findings suggest that EZA may be developed into a novel anti- drug.
PubMed: 32318117
DOI: 10.1186/s13099-020-00358-5 -
Journal of Chromatography. B,... Apr 2015The purpose of this study is to develop and validate an UPLC-MS/MS method to quantify ethoxzolamide in plasma (EZ) and apply the method to absorption, brain...
Development and validation of an UPLC-MS/MS method for the quantification of ethoxzolamide in blood, brain tissue, and bioequivalent buffers: applications to absorption, brain distribution, and pharmacokinetic studies.
The purpose of this study is to develop and validate an UPLC-MS/MS method to quantify ethoxzolamide in plasma (EZ) and apply the method to absorption, brain distribution, as well as pharmacokinetic studies. A C₁₈ column was used with 0.1% of formic acid in acetonitrile and 0.1% of formic acid in water as the mobile phases to resolve EZ. The mass analysis was performed in a triple quadrupole mass spectrometer using multiple reaction monitoring (MRM) with positive scan mode. The results show that the linear range of EZ is 4.88-10,000.00 nM. The intra-day variance is less than 12.43% and the accuracy is between 88.88 and 108.00%. The inter-day variance is less than 12.87% and accuracy is between 89.27 and 115.89%. Protein precipitation was performed using methanol to extract EZ from plasma and brain tissues. Only 40 μL of plasma is needed for analysis due to the high sensitivity of this method, which could be completed in less than three minutes. This method was used to study the pharmacokinetics of EZ in SD rats, and the transport of EZ in Caco-2 and MDCK-MDR1 overexpressing cell culture models. Our data show that EZ is not a substrate for p-glycoprotein (P-gp) and its entry into the brain may not limited by the blood-brain barrier.
Topics: Administration, Intravenous; Animals; Brain Chemistry; Caco-2 Cells; Chromatography, High Pressure Liquid; Ethoxzolamide; Humans; Linear Models; Male; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Sensitivity and Specificity; Tandem Mass Spectrometry
PubMed: 25706567
DOI: 10.1016/j.jchromb.2015.01.034 -
IScience Nov 2022One of the bottlenecks in the application of basic research findings to patients is the enormous cost, time, and effort required for high-throughput screening of...
One of the bottlenecks in the application of basic research findings to patients is the enormous cost, time, and effort required for high-throughput screening of potential drugs for given therapeutic targets. Here we have developed LIGHTHOUSE, a graph-based deep learning approach for discovery of the hidden principles underlying the association of small-molecule compounds with target proteins. Without any 3D structural information for proteins or chemicals, LIGHTHOUSE estimates protein-compound scores that incorporate known evolutionary relations and available experimental data. It identified therapeutics for cancer, lifestyle related disease, and bacterial infection. Moreover, LIGHTHOUSE predicted ethoxzolamide as a therapeutic for coronavirus disease 2019 (COVID-19), and this agent was indeed effective against alpha, beta, gamma, and delta variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that are rampant worldwide. We envision that LIGHTHOUSE will help accelerate drug discovery and fill the gap between bench side and bedside.
PubMed: 36246574
DOI: 10.1016/j.isci.2022.105314