Authors: Joyanta K Modak, Alexandra Tikhomirova, Rebecca J Gorrell...

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

Review

Authors: Benjamin K Johnson, Robert B Abramovitch

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

Authors: Alejandro Ciocci Pardo, Luisa F González Arbeláez, Juliana C Fantinelli...

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

Review

Authors: Claudiu T Supuran

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

Authors: Nader S Abutaleb, Ahmed E M Elhassanny, Alessio Nocentini...

is a high-priority pathogen of concern due to the growing prevalence of resistance development against approved antibiotics. Herein, we report the anti-gonococcal activity of ethoxzolamide, the FDA-approved human carbonic anhydrase inhibitor. Ethoxzolamide displayed an MIC against a panel of isolates, of 0.125 µg/mL, 16-fold more potent than acetazolamide, although both molecules exhibited almost similar potency against the gonococcal carbonic anhydrase enzyme (NgCA) . Acetazolamide displayed an inhibition constant () versus NgCA of 74 nM, while Ethoxzolamides was estimated to 94 nM. Therefore, the increased anti-gonococcal potency of ethoxzolamide was attributed to its increased permeability in as compared to that of acetazolamide. Both drugs demonstrated bacteriostatic activity against , exhibited post-antibiotic effects up to 10 hours, and resistance was not observed against both. Taken together, these results indicate that acetazolamide and ethoxzolamide warrant further investigation for translation into effective anti- agents.

Topics: Acetazolamide; Anti-Bacterial Agents; Carbonic Anhydrase Inhibitors; Carbonic Anhydrases; Dose-Response Relationship, Drug; Ethoxzolamide; Microbial Sensitivity Tests; Molecular Structure; Neisseria gonorrhoeae; Structure-Activity Relationship; United States; United States Food and Drug Administration

PubMed: 34894972
DOI: 10.1080/14756366.2021.1991336

Authors: Asta Zubrienė, Jurgita Matulienė, Lina Baranauskienė...

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

Authors: Benjamin K Johnson, Christopher J Colvin, David B Needle...

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

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.

Authors: Song Gao, Jing Zhao, Taijun Yin...

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

Authors: Santiago Vilar, Elías Quezada, Eugenio Uriarte...

The development of computational methods to discover novel drug-target interactions on a large scale is of great interest. We propose a new method for virtual screening based on protein interaction profile similarity to discover new targets for molecules, including existing drugs. We calculated Target Interaction Profile Fingerprints (TIPFs) based on ChEMBL database to evaluate drug similarity and generated new putative compound-target candidates from the non-intersecting targets in each pair of compounds. A set of drugs was further studied in monoamine oxidase B (MAO-B) and cyclooxygenase-1 (COX-1) enzyme through molecular docking and experimental assays. The drug ethoxzolamide and the natural compound piperlongumine, present in Piper longum L, showed hMAO-B activity with IC values of 25 and 65 μM respectively. Five candidates, including lapatinib, SB-202190, RO-316233, GW786460X and indirubin-3'-monoxime were tested against human COX-1. Compounds SB-202190 and RO-316233 showed a IC in hCOX-1 of 24 and 25 μM respectively (similar range as potent inhibitors such as diclofenac and indomethacin in the same experimental conditions). Lapatinib and indirubin-3'-monoxime showed moderate hCOX-1 activity (19.5% and 28% of enzyme inhibition at 25 μM respectively). Our modeling constitutes a multi-target predictor for large scale virtual screening with potential in lead discovery, repositioning and drug safety.

Topics: Binding Sites; Computational Biology; Cyclooxygenase 1; Drug Evaluation, Preclinical; Humans; Imidazoles; Indoles; Lapatinib; Molecular Conformation; Molecular Docking Simulation; Monoamine Oxidase; Oximes; Protein Interaction Maps; Pyridines; Quinazolines

PubMed: 27845365
DOI: 10.1038/srep36969

Authors: Hideyuki Shimizu, Manabu Kodama, Masaki Matsumoto...

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