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In this paper, the efficiency of the carbonic anhydrase (CA) enzyme in accelerating the hydration of CO2 is evaluated using a measurement system which consists of a vessel in which a gaseous flow of mixtures of nitrogen and CO2 is bubbled into water or water solutions containing a known quantity of CA enzyme. The pH value of the solution and the CO2 concentration at the measurement system gas exhaust are continuously monitored. The measured CO2 level allows for assessing the quantity of CO2, which, subtracted from the gaseous phase, is dissolved into the liquid phase and/or hydrated to bicarbonate. The measurement procedure consists of inducing a transient and observing and modelling the different kinetics involved in the steady-state recovery with and without CA. The main contribution of this work is exploiting dynamical system theory and chemical kinetics modelling for interpreting measurement results for characterising the activity of CA enzymes. The data for model fitting are obtained from a standard bioreactor, in principle equal to standard two-phase bioreactors described in the literature, in which two different techniques can be used to move the process itself away from the steady-state, inducing transients.

Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes involved in biosynthetic processes, transport, supply, and balance of CO2/HCO(3)(-)into the cell. In Bacteria, CAs avoid the depletion of the dissolved CO2/HCO(3)(-)from the cell, providing them to the central metabolism that is compromised without the CA activity. The involvement of CAs in the survival, pathogenicity, and virulence of several bacterial pathogenic species is recent. Here, we report the kinetic properties of the recombinant gamma-CA (EcoCA gamma) encoded in the genome ofEscherichia coli. EcoCA gamma is an excellent catalyst for the physiological CO(2)hydration reaction to bicarbonate and protons, with a k(cat)of 5.7 x 10(5) s(-1)and k(cat)/K(M)of 6.9 x 10(6) M(-1)s(-1). The EcoCA gamma inhibition profile with a broad series of known CA inhibitors, the substituted benzene-sulphonamides, and clinically licenced drugs was explored. Benzolamide showed a K(I)lower than 100 nM. Our study reinforces the hypothesis that the synthesis of new drugs capable of interfering selectively with the bacterial CA activity, avoiding the inhibition of the human alpha-CAs, is achievable and may lead to novel antibacterials.

Coumarins are widely diffused secondary metabolites possessing a plethora of biological activities. It has been established that coumarins represent a peculiar class of human carbonic anhydrase (hCA) inhibitors having a distinct mechanism of action involving a non-classical binding with amino acid residues paving the entrance of hCA catalytic site. Herein, we report the synthesis of a small series of new coumarin derivatives 7-11, 15, 17 prepared via classical Pechmann condensation starting from resorcinol derivatives and suitable β-ketoesters. The evaluation of inhibitory activity revealed that these compounds possessed nanomolar affinity and high selectivity towards tumour-associated hCA IX and XII over cytosolic hCA I and hCA II isoforms. To investigate the binding mode of these new coumarin-inspired inhibitors, the most active compounds 10 and 17 were docked within hCA XII catalytic cleft.

The inhibition of δ- and η-class carbonic anhydrases (CAs; EC 4.2.1.1) was poorly investigated so far. Only one δ-CA, TweCA from the diatom Thalassiosira weissflogii, and one η-CA, PfCA, from Plasmodium falciparum, have been cloned and characterised to date. To enrich δ- and η-CAs inhibition profiles, a panel of 22 phenols was investigated for TweCA and PfCA inhibition. Some derivatives showed effective, sub-micromolar inhibition of TweCA (KIs 0.81–65.4 µM) and PfCA (KIs 0.62–78.7 µM). A subset of compounds demonstrated a significant selectivity for the target CAs over the human physiologically relevant ones. This study promotes the identification of new potent and selective inhibitors of TweCA and PfCA, which could be considered as leads for finding molecular probes in the study of carbon fixation processes (in which TweCA and orthologue enzymes are involved) or drug candidates in the treatment of malaria.

A primary strategy to combat antimicrobial resistance is the identification of novel therapeutic targets and anti-infectives with alternative mechanisms of action. The inhibition of the metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1) from pathogens (bacteria, fungi, and protozoa) was shown to produce an impairment of the microorganism growth and virulence. As phosphonamidates have been recently validated as human α-CA inhibitors (CAIs) and no phosphorus-based zinc-binding group have been assessed to date against β-class CAs, herein we report an inhibition study with this class of compounds against β-CAs from pathogenic bacteria, fungi, and protozoa. Our data suggest that phosphonamidates are among the CAIs with the best selectivity for β-class over human isozymes, making them interesting leads for the development of new anti-infectives.

Proteins are relevant antimicrobial drug targets, and among them, enzymes represent a significant group, since most of them catalyze reactions essential for supporting the central metabolism, or are necessary for the pathogen vitality. Genomic exploration of pathogenic and non-pathogenic microorganisms has revealed genes encoding for a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1). CAs catalyze the physiologically crucial reversible reaction of the carbon dioxide hydration to bicarbonate and protons. Herein, we investigated the sulfonamide inhibition profile of the recombinant ?-CA (CynT2) identified in the genome of the Gram-negative bacterium Escherichia coli. This biocatalyst is indispensable for the growth of the microbe at atmospheric pCO2. Surprisingly, this enzyme has not been investigated for its inhibition with any class of CA inhibitors. Here, we show that CynT2 was strongly inhibited by some substituted benzene-sulfonamides and the clinically used inhibitor sulpiride (KIs in the range of 82&ndash, 97 nM). This study may be relevant for identifying novel CA inhibitors, as well as for another essential part of the drug discovery pipeline, such as the structure&ndash, activity relationship for this class of enzyme inhibitors.

The interconversion of CO2 and HCO3&minus, is catalyzed by a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1), which maintain the equilibrium between dissolved inorganic CO2 and HCO3&minus, In the genome of Escherichia coli, a Gram-negative bacterium typically colonizing the lower intestine of warm-blooded organisms, the cyn operon gene includes the CynT gene, encoding for a &beta, CA, and CynS gene, encoding for the cyanase. CynT (&beta, CA) prevents the depletion of the cellular bicarbonate, which is further used in the reaction catalyzed by cyanase. A second &beta, CA (CynT2 or Can or yadF), as well as a &gamma, and &iota, CAs were also identified in the E. coli genome. CynT2 is essential for bacterial growth at atmospheric CO2 concentration. Here, we characterized the kinetic properties and the anion inhibition profiles of recombinant CynT2. The enzyme showed a good activity for the physiological CO2 hydratase reaction with the following parameters: kcat = 5.3 &times, 105 s&minus, 1 and kcat/KM = of 4.1 &times, 107 M&minus, 1 s&minus, 1. Sulfamide, sulfamate, phenylboronic acid, phenylarsonic acid, and diethyldithiocarbamate were the most effective CynT2 inhibitors (KI = 2.5 to 84 &micro, M). The anions allowed for a detailed understanding of the interaction of inhibitors with the amino acid residues surrounding the catalytic pocket of the enzyme and may be used as leads for the design of more efficient and specific inhibitors.

In this paper the efficiency of carbonic anhydrase (CA) enzyme in accelerating the hydration of CO 2 is evaluated using a measurement system which consists of a vessel in which a gaseous flow of mixture of air and a fixed concentration of CO 2 (20%) is bubbled into a water solution containing a known quantity of CA enzyme. The pH value of the solution and the CO 2 concentration at the measurement system gas exhaust are continuously monitored. The measured CO 2 concentration allows for assessing the quantity of CO 2 , which is subtracted from the gaseous phase, dissolved into the liquid phase and/or hydrated. The measurement procedure consists of inducing a transient, and observing and modeling the different kinetics involved in the recovery of the steady state with and without CA.

Carbonic anhydrases (CAs) are ubiquitous metalloenzymes, which started to be investigated in detail in pathogenic, as well as non-pathogenic species since their pivotal role is to accelerate the physiological CO2 hydration/dehydration reaction significantly. Here, we propose the marine unicellular diatom Phaeodactylum tricornutum as a model organism for testing the membrane penetrability of CA inhibitors (CAIs). Seven inhibitors belonging to the sulphonamide type and possessing a diverse scaffold have been explored for their in vitro inhibition of the whole diatom CAs and the in vivo inhibitory effect on the growth of P. tricornutum. Interesting, inhibition of growth was observed, in vivo, demonstrating that this diatom is a good model for testing the cell wall penetrability of this class of pharmacological agents. Considering that many pathogens are difficult and dangerous to grow in the laboratory, the growth inhibition of P. tricornutum with different such CAIs may be subsequently used to design inhibition studies of CAs from pathogenic organisms.

The use of natural systems, such as outer membrane protein A (OmpA), phosphoporin E (PhoE), ice nucleation protein (INP), etc., has been proved very useful for the surface exposure of proteins on the outer membrane of Gram-negative bacteria. These strategies have the clear advantage of unifying in a one-step the production, the purification and the in vivo immobilisation of proteins/biocatalysts onto a specific biological support. Here, we introduce the novel Anchoring-and-Self-Labelling-protein-tag (ASLtag), which allows the in vivo immobilisation of enzymes on E. coli surface and the labelling of the neosynthesised proteins with the engineered alkylguanine-DNA-alkyl-transferase (H5) from Sulfolobus solfataricus. Our results demonstrated that this tag enhanced the overexpression of thermostable enzymes, such as the carbonic anhydrase (SspCA) from Sulfurihydrogenibium yellowstonense and the β-glycoside hydrolase (SsβGly) from S. solfataricus, without affecting their folding and catalytic activity, proposing a new tool for the improvement in the utilisation of biocatalysts of biotechnological interest.

[Image: see text] A novel series of of 4-[(3-phenyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulfonamides (EMAC10111a–g) was synthesized and assayed toward both human carbonic anhydrase isozymes I, II, IX, and XII and cyclooxygenase isoforms. The majority of these derivatives preferentially inhibit hCA isoforms II and XII and hCOX-2 isozyme, indicating that 2,3,4-trisubstituted 2,3-dihydrothiazoles are a promising scaffold for the inhibition of hCA isozymes and of hCOX-2 enzyme. The nature of the substituent at the dihydrothiazole ring position 4 influenced the activity and selectivity toward both enzyme families. EMAC10111g resulted as the best performing compound toward both enzyme families and exhibited preferential activity toward hCA XII and hCOX-2 isozymes.

The γ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the pathogenic bacterium Vibrio cholerae, VchCAγ, was investigated for its activation with a panel of natural and non-natural amino acids and amines. The enzyme was effectively activated by l -tryptophan, 1-(2-minoethyl)-piperazine and 4-(2-aminoethyl)-morpholine, in the low nanomolar range (KAs 8–71 nM). In contrast, l -/ d -Phe, l -/ d -DOPA, d -Trp, l -/ d -Tyr, 4-amino- l -Phe, histamine, dopamine, serotonin, some pyridyl-alkylamines, as well as l -adrenaline were submicromolar activators (KAs between 0.10 and 0.73 µM). l - and d -His were the least effective VchCAγ activators (KAs of 1.01–14.2 µM). The activation of CAs from bacteria have not been considered to date for possible biomedical applications. It would be of interest to study in more details the role of CA activators in processes connected with the virulence and colonization of the host by pathogenic bacteria, such as Vibrio cholerae, which is highly dependent on the concentration of bicarbonate in tissues.

The activation of a β-class carbonic anhydrase (CAs, EC 4.2.1.1) from Mycobacterium tuberculosis, encoded by the gene Rv3273 (mtCA 3), was investigated using a panel of natural and non-natural amino acids and amines. mtCA 3 was effectively activated by D-DOPA, L-Trp, dopamine and serotonin, with KAs ranging between 8.98 and 12.1 µM. L-His and D-Tyr showed medium potency activating effects, with KAs in the range of 17.6–18.2 µM, whereas other amines and amino acids were relatively ineffective activators, with KAs in the range of 28.9–52.2 µM. As the physiological roles of the three mtCAs present in this pathogen are currently poorly understood and considering that inhibition of these enzymes has strong antibacterial effects, discovering molecules that modulate their enzymatic activity may lead to a better understanding of the factors related to the invasion and colonisation of the host during Mycobacterium tuberculosis infection.

The inhibition of the δ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the diatom Thalassiosira weissflogii, TweCAδ, was investigated using a panel of 36 mono- and di-thiocarbamates chemotypes that have recently been shown to inhibit mammalian and pathogenic CAs belonging to the α- and β-classes. TweCAδ was not significantly inhibited by most of such compounds (KI values above 20 µM). However, some aliphatic, heterocyclic, and aromatic mono and di-thiocarbamates inhibited TweCAδ in the low micromolar range. For some compounds incorporating the piperazine ring, TweCAδ was effectively inhibited (KIs from 129 to 791 nM). The most effective inhibitors identified in this study were 3,4-dimethoxyphenyl-ethyl-mono-thiocarbamate (KI of 67.7 nM) and the R-enantiomer of the nipecotic acid di-thiocarbamate (KI of 93.6 nM). Given that the activity and inhibition of this class of enzyme have received limited attention until now, this study provides new molecular probes and information for investigating the role of δ-CAs in the carbon fixation processes in diatoms, which are responsible for significant amounts of CO2 taken from the atmosphere by these marine organisms.

The inhibition of α-, β-, γ-, and δ-class carbonic anhydrases (CAs, EC 4.2.1.1) from bacteria (Vibrio cholerae and Porphyromonas gingivalis) and diatoms (Thalassiosira weissflogii) with a panel of N’-aryl-N-hydroxy-ureas is reported. The α-/β-CAs from V. cholerae (VchCAα and VchCAβ) were effectively inhibited by some of these derivatives, with KIs in the range of 97.5 nM – 7.26 µM and 52.5 nM – 1.81 µM, respectively, whereas the γ-class enzyme VchCAγ was less sensitive to inhibition (KIs of 4.75 – 8.87 µM). The β-CA from the pathogenic bacterium Porphyromonas gingivalis (PgiCAβ) was not inhibited by these compounds (KIs > 10 µM) whereas the corresponding γ-class enzyme (PgiCAγ) was effectively inhibited (KIs of 59.8 nM – 6.42 µM). The δ-CA from the diatom Thalassiosira weissflogii (TweCAδ) showed effective inhibition with these derivatives (KIs of 33.3 nM – 8.74 µM). As most of these N-hydroxyureas are also ineffective as inhibitors of the human (h) widespread isoforms hCA I and II (KIs > 10 µM), this class of derivatives may lead to the development of CA inhibitors selective for bacterial/diatom enzymes over their human counterparts and thus to anti-infectives or agents with environmental applications.

The α- and β-class carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacterium Vibrio cholerae, VchCAα, and VchCAβ, were investigated for their activation with natural and non-natural amino acids and amines. The most effective VchCAα activators were L-tyrosine, histamine, serotonin, and 4-aminoethyl-morpholine, which had KAs in the range of 8.21–12.0 µM. The most effective VchCAβ activators were D-tyrosine, dopamine, serotonin, 2-pyridyl-methylamine, 2-aminoethylpyridine, and 2-aminoethylpiperazine, which had KAs in the submicromolar – low micromolar range (0.18–1.37 µM). The two bacterial enzymes had very different activation profiles with these compounds, between each other, and in comparison to the human isoforms hCA I and II. Some amines were selective activators of VchCAβ, including 2-pyridylmethylamine (KA of 180 nm for VchCAβ, and more than 20 µM for VchCAα and hCA I/II). The activation of CAs from bacteria, such as VchCAα/β has not been considered previously for possible biomedical applications. It would be of interest to study in more detail the extent that CA activators are implicated in the virulence and colonisation of the host by such pathogenic bacteria, which for Vibrio cholerae, is highly dependent on the bicarbonate concentration and pH in the surrounding tissue.

We report the kinetic properties and sulfonamide inhibition profile of an a-carbonic anhydrase (CA, EC 4.2.1.1), named CruCA4, identified in the red coral Corallium rubrum. This isoform is involved in the biomineralization process leading to the formation of a calcium carbonate skeleton. Experiments performed on the recombinant protein show that the enzyme has a "moderate activity" level. Our results are discussed compared to values obtained for other CA isoforms involved in biomineralization. This is the first study describing the biochemical characterization of an octocoral CA. (C) 2017 Elsevier Ltd. All rights reserved.

We cloned, expressed, purified, and determined the kinetic constants of the recombinant alpha-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the alpha-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32 kDa. Moreover, the determined kinetic parameters for the CO2 hydration reaction were k(cat) = 4.2 x 10(5) s(-1) and k(cat)/K-m of 3.5 x 10(7) M-1 x s(-1). Curiously, the rec-MgaCA showed a very similar kinetic and acetazolamide inhibition features when compared to those of the native enzyme (MgaCA), which has a molecular weight of 50 kDa. Analysing the SDS-PAGE, the protonography, and the kinetic analysis performed on the native and recombinant enzyme, we hypothesised that probably the native MgaCA is a multidomain protein with a single CA domain at the N-terminus of the protein. This hypothesis is corroborated by the existence in mollusks of multidomain proteins with a hydratase activity. Among these proteins, nacrein is an example of alpha-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.

A panel of 22 phenols was investigated as inhibitors of the 8-class carbonic anhydrase (CAs, EC 4.2.1.1) from the fungal parasite Malassezia globosa (MgCA), a validated anti-dandruff drug target. The displayed inhibitory activities were compared to the ones previously reported against the off-target widely distributed human (h) isoforms hCA I and II. All tested phenols possessed a better efficacy in inhibiting MgCA than the clinically used sulfonamide acetazolamide, with Kis in the range of 2.5 and 65.0 mu M. A homology-built model of MgCA was also used for understanding the binding mode of phenols to the fungal enzyme. Indeed, a wide network of hydrogen bonds and hydrophobic interactions between the phenol and active site residues were evidenced. The OH moiety of the inhibitor was observed anchored to the zinc-coordinated water, also making hydrogen bonds with Ser48 and Asp49. The diverse substituents at the phenolic scaffold were observed to interact with different portions of the hydrophobic pocket according to their nature and position. Considering the effective MgCA inhibitory properties of phenols, beside to the rather low inhibition against the off-target hCA I and II, this class of compounds might be of considerable interest in the cosmetics field as potential anti-dandruff drugs. (C) 2017 Elsevier Ltd. All rights reserved.

We report the cloning, purification and characterization of Bps beta CA, a beta-class carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei, the etiological agent of melioidosis, and compare its activity and inhibition with those of the gamma-CA from the same organism, Bps gamma CA, recently investigated by our groups. Bps beta CA showed a significant catalytic activity for the physiologic, CO2 hydration reaction, with the following kinetic parameters, k(cat) of 1.6 x 10(5) s(-1) and k(cat)/K-m of 3.4 x 10(7) M-1 x s(-1). The inhibition of Bps beta CA with a group of anions and small molecules was also investigated. The best inhibitors were sulfamide, sulfamic acid and phenylarsonic acid, which showed K(I)s in the range of 83-92 mu M, whereas phenylboronic acid, fluoride, cyanide, azide, bisulfite, tetraborate, perrhenate, perruthenate, peroxydisulfate, perchlorate, tetrafluoroborate, fluorosulfonate and hexafluorophosphate showed K(I)s > 100 mM. Other inhibitors of this new enzyme were bicarbonate, trithiocarbonate, some complex inorganic anions and N,N-diethyldithiocarbamate, which had inhibition constants of 0.32-8.6 mM. As little is known of the life cycle and virulence of this bacterium, this type of study may bring information of interest for the development of novel strategies to fight bacterial infection and drug resistance to commonly used antibiotics. (C) 2017 Elsevier Ltd. All rights reserved.

Carbonic anhydrases from Vibrio cholerae (VchCAs) play a significant role in bacterial pathophysiological processes. Therefore, their inhibition leads to a reduction of gene expression virulence and bacterial growth impairment. Herein, we report the first ligand-based pharmacophore model as a computational tool to study selective inhibitors of the beta-class of VchCA. By a virtual screening on a collection of sulfonamides, we retrieved 9 compounds that were synthesized and evaluated for their inhibitory effects against VchCA beta as well as alpha- and gamma-classes of VchCAs and selectivity over human ubiquitous isoforms hCA I and IL Notably, all tested compounds were active inhibitors of VchCAs. The N-(4-sulfamoylbenzyl)-[1,1'-biphenyl]-4-carboxamide (20e) stood out as the most exciting inhibitor toward the beta-class (K-i = 95.6 nM), also showing a low affinity against the tested human isoforms. By applying docking procedures, we described the binding mode of the inhibitor 20e within the catalytic cavity of the modeled open conformation of VchCA beta.

There is significant interest in increasing the microalgal efficiency for producing high-quality products that are commonly used as food additives in nutraceuticals. Some natural substances that can be extracted from algae include lipids, carbohydrates, proteins, carotenoids, long-chain polyunsaturated fatty acids, and vitamins. Generally, microalgal photoautotrophic growth can be maximised by optimising CO2 biofixation, and by adding sodium bicarbonate and specific bacteria to the microalgal culture. Recently, to enhance CO2 biofixation, a thermostable carbonic anhydrase (SspCA) encoded by the genome of the bacterium Sulfurihydrogenibium yellowstonense has been heterologously expressed and immobilised on the surfaces of bacteria. Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes, which catalyse the physiologically reversible reaction of carbon dioxide hydration to bicarbonate and protons: CO2 + H2O ⇄ HCO3 − + H+. Herein, we demonstrate for the first time that the fragments of bacterial membranes containing immobilised SspCA (M-SspCA) on their surfaces can be doped into the microalgal culture of the green unicellular alga, Chlorella sorokiniana, to significantly enhance the biomass, photosynthetic activity, carotenoids production, and CA activity by this alga. These results are of biotechnological interest because C. sorokiniana is widely used in many different areas, including photosynthesis research, human pharmaceutical production, aquaculture-based food production, and wastewater treatment. © 2020, © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

Acidity, hypoxia and increased release of exosomes are severe phenotypes of tumours. The regulation of pH in tumours involves the interaction of several proteins, including the carbonic anhydrases which catalyze the formation of bicarbonate and protons from carbon dioxide and water. Among CA isoforms, CA IX is over-expressed in a large number of solid tumours, conferring to cancer cells a survival advantage in hypoxic and acidic microenvironment, but there isn’t evidence that CA IX expression could have a real clinical impact. Therefore, in this study for the first time the expression and activity of CA IX have been investigated in the plasmatic exosomes obtained from patients with prostate carcinoma (PCa). For this purpose, the study was performed through different methodological approaches, such as NTA, western blot analysis, enzyme activity assay, Nanoscale flow cytometry, ELISA, confocal microscopy. The results showed that PCa exosomes significantly overexpressed CA IX levels and related activity as compared to healthy donors. Furthermore, CA IX expression and activity were correlated to the exosome intraluminal pH, demonstrating for the first time that PCa exosomes are acidic. Our data suggest the possible use of the exosomal CA IX expression and activity as a biomarker of cancer progression in PCa.

A series of urea/thiourea substituted benzoxaboroles was investigated for the inhibition of the three carbonic anhydrases encoded by Vibrio cholerae (VchCA alpha, VchCA beta, and VchCA gamma). In particular, benzoxaborole derivatives were here first assayed for the inhibition of a gamma-class CA, extending the panel of CA classes that benzoxaboroles efficiently target beyond alpha and beta. Inhibition profiles demonstrated that VchCA alpha was significantly more inhibited compared to VchCAy and, in turn, more efficientl gamma modulated than VchCA beta. Among the many selective benzoxaborole ligands detected against VchCA alpha over the off-target hCA II, compound 18, a p-NO2-phenylthiourea derivative, even exhibited a fully selective inhibition profile against the three VchCAs over hCA IL A comprehensive ligand/target interaction study was performed in silico for all three VchCA isoforms providing the first molecular modeling investigation with inhibitors of a gamma-class CA to the best of our knowledge. The present study reinforces the rationale behind the use of benzoxaboroles as innovative antibacterial agents with a new mechanism of action, furnishing suggestions for the rational design of new potent and selective inhibitors targeting V. cholerae CAs over human off-target ones.

The carbonic anhydrases (CAs, EC 4.2.1.1) catalyse a simple but physiologically crucial reversible reaction, the carbon dioxide hydration with the production of bicarbonate and protons. In the last years, and especially, to the rapid emergence of the bacterial antibiotic resistance that is occurring worldwide, the understanding of the function of bacterial CAs has increased significantly. Recently, a new CA-class (iota-CA) was discovered in the marine diatom T. pseudonana. It has been reported that bacterial genomes may contain genes with relevant homology to the diatom iota-class CA. Still, the catalytic activity of the enzyme encoded by the gene was not investigated. Thus, herein, for the first time, we cloned, expressed, and purified the recombinant bacterial iota-CA (acronym BteCA iota) identified in the genome of Burkholderia territorii. The recombinant BteCA iota resulted in a good catalyst for the hydration of CO2 to bicarbonate and protons, with a k(cat) of 3.0 x 10(5) s (-1) and k(cat)/K-M of 3.9 x 10(7) M (-1) s (-1), and is also sensitive to inhibition by the sulphonamide acetazolamide. Furthermore, with the aid of the protonography, it has been demonstrated that BteCA iota can be present as a dimer. This result is corroborated by the construction of a molecular model of BteCA iota, which showed that the enzyme is formed by two equivalent monomers having a structure similar to a butterfly.

A beta-carbonic anhydrase (CA, EC 4.2.1.1) from the widespread bacteriumEscherichia coli(EcoCA beta), encoded by the CynT2 gene, has been investigated for its catalytic properties and enzymatic activation by a panel of amino acids and amines. EcoCA beta showed a significant catalytic activity for the hydration of CO(2)to bicarbonate and a proton, with a kinetic constantk(cat)of 5.3 x 10(5)s(-)and a Michaelis-Menten constantK(M)of 12.9 mM. The most effective EcoCA beta activators were L- and D-DOPA, L-Tyr, 4-amino-Phe, serotonin and L-adrenaline, withK(A)s from 2.76 to 10.7 mu M. L-His, 2-pyridyl-methylamine, L-Asn and L-Gln were relatively weak activators (K(A)s from 36.0 to 49.5 mu M). D-His, L- and D-Phe, L- and D-Trp, D-Tyr, histamine, dopamine, 2-(aminoethyl)pyridine/piperazine/morpholine, L-Asp, L- and D-Glu haveK(A)s from 11.3 to 23.7 mu M. Endogenous CA activators may play a role in bacterial virulence and colonisation of the host.

Carbonic anhydrase (CA) is a zinc enzyme that catalyzes the reversible conversion of carbon dioxide to bicarbonate and proton. Currently, CA inhibitors are widely used as antiglaucoma, anticancer, and anti-obesity drugs and for the treatment of neurological disorders. Recently, the potential use of CA inhibitors to fight infections caused by protozoa, fungi, and bacteria has emerged as a new research line. In this article, the X-ray crystal structure of &beta, CA from Burkholderia pseudomallei was reported. The X-ray crystal structure of this new enzyme was solved at 2.7 Å resolution, revealing a tetrameric type II &beta, CA with a &ldquo, closed&rdquo, active site in which the zinc is tetrahedrally coordinated to Cys46, Asp48, His102, and Cys105. B. pseudomallei is known to encode at least two CAs, a &beta, CA, and a &gamma, CA. These proteins, playing a pivotal role in its life cycle and pathogenicity, offer a novel therapeutic opportunity to obtain antibiotics with a different mechanism of action. Furthermore, the new structure can provide a clear view of the &beta, CA mechanism of action and the possibility to find selective inhibitors for this class of CAs.

A series of 2-thio- and 2-seleno-acetamides bearing the benzenesulfonamide moiety were evaluated as Carbonic Anhydrase (CA, EC 4.2.1.1) inhibitors against different pathogenic bacteria such as the Vibrio cholerae (VchCA-&alpha, and VchCA-&beta, ), Burkholderia pseudomallei (BpsCA-&beta, and BpsCA-&gamma, ), Mycobacterium tuberculosis (Rv3723-&beta, ) and the Salmonella enterica serovar Typhimurium (StCA2-&beta, ). The molecules represent interesting leads worth developing as innovative antibacterial agents since they possess new mechanism of action and isoform selectivity preferentially against the bacterial expressed CAs. The identification of potent and selective inhibitors of bacterial CAs may lead to tools also useful for deciphering the physiological role(s) of such proteins.

A series of sixteen benzenesulfonamide derivatives has been synthesised and tested as inhibitors of Vibrio cholerae carbonic anhydrase (CA) enzymes, belonging to α-CA, β-CA, and γ-CA classes (VchCAα, VchCAβ, and VchCAγ). The determined Ki values were compared to those of selected human CA isoforms (hCA I and hCA II). Structure-affinity relationship analysis highlighted that all tested compounds proved to be active inhibitors of VchCAα at nanomolar concentration. The VchCAβ activity was lower to respect inhibitory efficacy toward VchCAα, whereas, these benzenesulfonamide derivatives failed to inhibit VchCAγ. Interestingly, compound 7e combined the best activity toward VchCAα and VchCAβ. In order to obtain a model for binding mode of our inhibitors toward bacterial CAs, we carried out docking simulations by using the available crystal structures of VchCAβ., GRAPHICAL ABSTRACT

Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes, which catalyze the crucial physiological CO2 hydration/dehydration reaction (CO2 + H2O ⇌ HCO3&minus, + H+) balancing the equilibrium between CO2, H2CO3, HCO3&minus, and CO32&minus, It has been demonstrated that their selective inhibition alters the equilibrium of the metabolites above affecting the biosynthesis and energy metabolism of the organism. In this context, our interest has been focalized on the fungus Malassezia restricta, which may trigger dandruff and seborrheic dermatitis altering the complex bacterial and fungal equilibrium of the human scalp. We investigated a rather large number of inorganic metal-complexing anions (a well-known class of CA inhibitors) for their interaction with the &beta, CA (MreCA) encoded by the M. restricta genome. The results were compared with those obtained for the two human ?-CA isoforms (hCAI and hCAII) and the &beta, CA from Malassezia globosa. The most effective MreCA inhibitors were diethyldithiocarbamate, sulfamide, phenyl arsenic acid, stannate, tellurate, tetraborate, selenocyanate, trithiocarbonate, and bicarbonate. The different KI values obtained for the four proteins investigated might be attributed to the architectural features of their catalytic site. The anion inhibition profile is essential for better understanding the inhibition/catalytic mechanisms of these enzymes and for designing novel types of inhibitors, which may have clinical applications for the management of dandruff and seborrheic dermatitis.

The cloning, purification, and initial characterization of the &beta, carbonic anhydrase (CA, EC 4.2.1.1) from the genome of the opportunistic pathogen Malassezia restricta (MreCA), which a fungus involved in dandruff and seborrheic dermatitis (SD), is reported. MreCA is a protein consisting of 230 amino acid residues and shows high catalytic activity for the hydration of CO2 into bicarbonate and protons, with the following kinetic parameters: kcat of 1.06 &times, 106 s&minus, 1 and kcat/KM of 1.07 &times, 108 M&minus, 1 s&minus, 1. It is also sensitive to inhibition by the sulfonamide acetazolamide (KI of 50.7 nM). Phylogenetically, MreCA and other CAs from various Malassezia species seem to be on a different branch, distinct from that of other &beta, CAs found in fungi, such as Candida spp., Saccharomyces cerevisiae, Aspergillus fumigatus, and Sordaria macrospora, with only Cryptococcus neoformans and Ustilago maydis enzymes clustering near MreCA. The further characterization of this enzyme and the identification of inhibitors that may interfere with its life cycle might constitute new strategies for fighting dandruff and SD.

The genome of Helicobacter pylori encodes for carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α- and β-CA classes, which together with urease, have a pivotal role in the acid acclimation of the microorganism within the human stomach. Recently, in the exoproteome of H. pylori, a CA with no indication of the corresponding class was identified. Here, using the protonography and the mass spectrometry, a CA belonging to the α-class was detected in the outer membrane vesicles (OMVs) generated by planktonic and biofilm phenotypes of four H. pylori strains. The amount of this metalloenzyme was higher in the planktonic OMVs (pOMVs) than in the biofilm OMVs (bOMVs). Furthermore, the content of α-CA increases over time in the pOMVs. The identification of the α-CA in pOMVs and bOMVs might shed new light on the role of this enzyme in the colonization, survival, persistence, and pathogenesis of H. pylori.

Carbonic anhydrases (CAs; EC 4.2.1.1) are metalloenzymes with a pivotal potential role in the biomimetic CO2 capture process (CCP) because these biocatalysts catalyse the simple but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons in all life kingdoms. The CAs are among the fastest known enzymes, with kcat values of up to 106 s−1 for some members of the superfamily, providing thus advantages when compared with other CCP methods, as they are specific for CO2. Thermostable CAs might be used in CCP technology because of their ability to perform catalysis in operatively hard conditions, typical of the industrial processes. Moreover, the improvement of the enzyme stability and its reuse are important for lowering the costs. These aspects can be overcome by immobilising the enzyme on a specific support. We report in this article that the recombinant thermostable SspCA (α-CA) from the thermophilic bacterium Sulfurihydrogenibium yellowstonense can been heterologously produced by a high-density fermentation of Escherichia coli cultures, and covalently immobilised onto the surface of magnetic Fe3O4 nanoparticles (MNP) via carbodiimide activation reactions. Our results demonstrate that using a benchtop bioprocess station and strategies for optimising the bacterial growth, it is possible to produce at low cost a large amount SspCA. Furthermore, the enzyme stability and storage greatly increased through the immobilisation, as SspCA bound to MNP could be recovered from the reaction mixture by simply using a magnet or an electromagnetic field, due to the strong ferromagnetic properties of Fe3O4.

The carbonic anhydrase superfamily (CA, EC 4.2.1.1) of metalloenzymes is present in all three domains of life (Eubacteria, Archaea, and Eukarya), being an interesting example of convergent/divergent evolution, with its seven families (alpha-, beta-, gamma-, delta-, zeta-, eta-, and theta-CAs) described so far. CAs catalyse the simple, but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons. Recently, our groups characterised the a- CA from the thermophilic bacterium, Sulfurihydrogenibium yellowstonense finding a very high catalytic activity for the CO2 hydration reaction (k(cat) = 9.35 x 10(5) s(-1) and k(cat)/K-m = 1.1 x 10(8)M(-1) s(-1)) which was maintained after heating the enzyme at 80 degrees C for 3 h. This highly thermostable SspCA was covalently immobilised within polyurethane foam and onto the surface of magnetic Fe3O4 nanoparticles. Here, we describe a one-step procedure for immobilising the thermostable SspCA directly on the surface membrane of Escherichia coli, using the INPN domain of Pseudomonas syringae. This strategy has clear advantages with respect to other methods, which require as the first step the production and the purification of the biocatalyst, and as the second step the immobilisation of the enzyme onto a specific support. Our results demonstrate that thermostable SspCA fused to the INPN domain of P. syringae ice nucleation protein (INP) was correctly expressed on the outer membrane of engineered E. coli cells, affording for an easy approach to design biotechnological applications for this highly effective thermostable catalyst.

Carbonic anhydrases (CA, EC 4.2.1.1) are ubiquitous metalloenzymes, which catalyze the conversion of carbon dioxide (CO2) to bicarbonate (HCO3) and protons (H+). In prokaryotes, the existence of genes encoding for alpha-, beta- and gamma-classes suggests that these enzymes play an important role in the prokaryotic physiology. It has been demonstrated, in fact, that their inhibition in vivo leads to growth impairment or growth defects of the microorganism. Ultimately, we started to investigate the biochemical properties and the inhibitory profiles of the alpha- and beta-CAs identified in the genome of Vibrio cholerae, which is the causative agent of cholera. The genome of this pathogen encodes for CAs belonging to alpha, beta and gamma classes. Here, we report a sulfonamide inhibition study of the gamma-CA (named VchCA gamma) comparing it with data obtained for the alpha- and beta-CA enzymes. VchCA gamma activity (k(cat) = 7.39 x 10(5) s(-1)) was significantly higher than the other gamma-CAs. The inhibition study with a panel of sulfonamides and one sulfamate led to the detection of a large number of nanomolar VchCA gamma inhibitors, including simple aromatic/heterocyclic sulfonamides (compounds 2-9, 11, 13-15, 24) as well as EZA, DZA, BRZ, BZA, TPM, ZNS, SLP, IND (K(I)s in the range of 66.2-95.3 nM). As it was proven that bicarbonate is a virulence factor of this bacterium and since ethoxzolamide was shown to inhibit this virulence in vivo, we propose that VchCA, VchCA beta and VchCA gamma may be a target for antibiotic development, exploiting a mechanism of action rarely considered up until now, i.e., interference with bicarbonate supply as a virulence factor. (C) 2016 Elsevier Ltd. All rights reserved.

We have cloned, purified and characterized the gamma-carbonic anhydrase (CA, EC 4.2.1.1) present in the genome of the Antarctic bacterium Colwellia psychrerythraea, which is an obligate psychrophile. The enzyme shows a significant catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with the following kinetic parameters: k(cat) of 6.0 x 10(5) s(-1) and a k(cat)/K-m of 4.7 x 10(6) M-1 x s(-1). This activitywas inhibited by the sulfonamide CA inhibitor (CAI) acetazolamide, with a K-I of 502 nM. A range of anions was also investigated for their inhibitory action against the new enzyme CpsCA. Perchlorate, tetrafluoroborate, fluoride and bromide were not inhibitory, whereas cyanate, thiocyanate, cyanide, hydrogensulfide, carbonate and bicarbonate showed K(I)s in the range of 1.4-4.4 mM. Diethyldithiocarbamate was a better inhibitor (K-I of 0.58 mM) whereas sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid were the most effective inhibitors detected, with K(I)s ranging between 8 and 38 mu M. The present study may shed some more light regarding the role that gamma-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here. (c) 2016 Elsevier Ltd. All rights reserved.

The Antarctic bacterium Colwellia psychrerythraea encodes for a gamma-class carbonic anhydrase (CA, EC 4.2.1.1), which was cloned, purified and characterized. The enzyme (CpsCA gamma) has a moderate catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with a k(cat) 6.0 x 10(5) s(-1) and a k(cat)/K-m of 4.7 x 10(6) M-1 s(-1). A series of sulfonamides and a sulfamate were investigated as inhibitors of the new enzyme. The best inhibitor was metanilamide (K-I of 83.5 nM) followed by indisulam, valdecoxib, celecoxib, sulthiame and hydrochlorothiazide (K(I)s ranging between 343 and 491 nM). Acetazolamide, methazolamide as well as other aromatic/heterocyclic derivatives showed inhibition constants between 502 and 7660 nM. The present study may shed some more light regarding the role that gamma-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here, by allowing the identification of inhibitors which may be useful as pharmacologic tools. (C) 2016 Elsevier Ltd. All rights reserved.

A series of 1,2,3-triazole-bearing benzenesulfonamides was assessed for the inhibition of carbonic anhydrases (CA, EC 4.2.1.1) from bacteria Vibrio cholerae (VchCAa and VchCA beta) and Mycobacterium tuberculosis (beta-mtCA3). Growing resistance phenomena against existing antimicrobial drugs are globally spreading and highlight a urgent need of agents endowed with alternative mechanisms of action. Two global WHO strategies aim to reduce cholera deaths by 90% and eradicate the tuberculosis epidemic by 2030. The derivatives here reported represent interesting leads towards the optimization of new antibiotic agents showing excellent inhibitory efficiency and selectivity for the target CAs over the human (h) off-target isoform hCA I. In detail, the first subset of derivatives potently inhibits VchCA alpha in a low nanomolar range (K(I)s between 0.72 and 22.6 nM). Compounds of a second subset, differing from the first one for the position of the spacer between benzenesulfonamide and triazole, preferentially inhibit VchCA beta (K(I)s in the range 54.8-102.4 nM) and beta-mtCA3 (K(I)s in the range 28.2-192.5 nM) even more than the clinically used AAZ, used as the standard.

Famotidine, an antiulcer drug belonging to the H2 antagonists class of pharmacological agents, was recently shown to potently inhibit human (h) and bacterial carbonic anhydrases (CAs, EC 4.2.1.1). We investigated the inhibitory effects of famotidine against all classes of CAs from the pathogenic bacteria Vibrio cholerae, Burkholderia pseudomallei and Mycobacterium tuberculosis Rv3273 β-CA, as well as the CAs from the nonpathogenic bacteria/cyanobacteria Sulfurihydrogenibium yellowstonensis, S. azorense, Pseudoalteromonas haloplanktis, Colwellia psychrerythraea and Nostoc commune. The δ- and ζ-CAs from the diatom Thalassiosira weissflogii, the fungal enzymes from Cryptococcus neoformans, Candida glabrata and Malassezia globosa, as well as the protozoan enzymes from Trypanosoma cruzi and Plasmodium falciparum, were also investigated. Anopheles gambiae β-CA was also investigated. All these enzymes were effectively inhibited by famotidine, with affinities between the low nanomolar to the micromolar range. The best inhibition was observed against C. glabrata β-CA and TweCAζ, with KIs ranging between 13.6 and 22.1 nM.

gamma-Class carbonic anhydrases (CAs; EC 4.2.1.1) lack of extended inhibition characterization in comparison to alpha- and beta-class isozymes. For this reason, a panel of 22 phenols was investigated here for the inhibition of the gamma-CAs from the pathogenic bacteria Burkholderia pseudomallei (BpsCA gamma), Porphyromonas gingivalis (PgiCA), Vibrio cholerae (VchCA gamma) and from the antarctic bacteria Pseudoalteromonas haloplanktis (PhaCA gamma) and Colwellia psychrerythraea (CpsCA gamma). The exploration of the chemical space around the main phenolic group led to the discovery of a number of such derivatives showing effective, sometimes sub-micromolar inhibition against BpsCA gamma (K(I)s 0.45-8.6 mu M), PgiCA (K(I)s 0.36-9.8 mu M) and VchCA gamma (K(I)s 0.47-9.6 mu M). A subset of compounds even demonstrated a significant selectivity for the target gamma-CAs over the human physiologically most relevant isoform CA II. This study enriches the inhibitory profiles database for gamma-class CAs and promotes the identification of new potent and selective inhibitors against bacterial isoforms over human off-target ones. These agents are of remarkable interest and importance in the search of novel, worldwide required, antibiotic agents possessing alternative mechanisms of action as a strategy to overcome the spread to antimicrobic resistance.

Acidity and hypoxia are crucial phenotypes of tumour microenvironment both contributing to the selection of malignant cells under a micro evolutionistic pressure. During the tumour progression, nanovesicles, called exosomes and the metalloenzyme carbonic anhydrase IX (CA IX) affect the tumour growth and proliferation. Exosomes are released into the tumour microenvironment and spilt all over the body, while CA IX is a tumour-associated protein overexpressed in many different solid tumours. In the present study, to better understand the relationships between exosomes and CA IX, it has been used an in vitro cellular model of cells cultured in different pH conditions. The results showed that the acidic microenvironment induced upregulation of both expression and activity of CA IX in cancer cells and their exosomes, together with increasing the number of released exosomes. These data strongly support the importance of CA IX as a cancer biomarker and as a valuable target of new anticancer therapies.

A series of benzenesulfonamides incorporating selenazoles with diverse substitution patterns were investigated as inhibitors of six bacterial carbonic anhydrases (CAs, EC 4.2.1.1) from bacterial pathogens, such as Helicobacter pylori (hpCAα was the investigated enzyme), Vibrio cholerae (all the three CAs from this pathogen were considered, VchCAα, VchCAβ and VchCAγ) and Burkholderia pseudomallei (with its two CAs, BpsCAβ and BpsCAγ). All these sulfonamides were effective CA inhibitors, with potencies in the low micromolar or submicromolar range, making them attractive as lead compounds for designing antibacterials with a novel mechanism of action, which could counteract the extensive resistance problem observed with many clinically used antibiotics.

Thirty novel sulfonamide derivatives incorporating dipeptide were synthesized by facile acylation through benzotriazole mediated reactions and their structures were identified by 1H NMR, 13C NMR, MS and FT-IR spectroscopic techniques and elemental analysis. The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity of the new compounds was assessed against four human (h) isoforms, hCA I, hCA II, hCA IV and hCA XII. Most of the synthesized compounds showed excellent in vitro carbonic anhydrase inhibitory properties comparable to those of the clinically used drug acetazolamide (AAZ). The new unprotected dipeptide-sulfonamide conjugates showed very effective inhibitory activity, in the low nanomolar range against II and XII, being less effective as hCA I and IV inhibitors. Four of the thirty compounds also showed strong inhibitory activity against hCA XII compared to AAZ.

A series of selenides bearing benzenesulfonamide moieties was evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors against the pathogenic bacteria Vibrio cholerae (VchCAα and VchCAβ) and Burkholderia pseudomallei (BpsCAβ) enzymes. The molecules represent an interesting lead for antibacterial agents with a possibly new mechanism of action showing excellent inhibitory action and selectivity for inhibiting VchCAα and BpsCAβ over the human (h) off-target isoforms hCA I and II. Identification of potent and possibly selective inhibitors of bacteria CAs over the human counterparts may lead to pharmacological tools useful for understanding the physiological role(s) of these under-investigated proteins.

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the CO2 hydration/dehydration reversible reaction: CO2 + H2O ? (Formula presented.) + H+. Living organisms encode for at least six distinct genetic families of such catalyst, the ?-, ?-, ?-, ?-, ?- and ?-CAs. The main function of the CAs is to quickly process the CO2 derived by metabolic processes in order to regulate acid-base homeostasis, connected to the production of protons (H+) and bicarbonate. Few data are available in the literature on Antarctic CAs and most of the scientific information regards CAs isolated from mammals or prokaryotes (as well as other mesophilic sources). It is of great interest to study the biochemical behavior of such catalysts identified in organism living in the Antarctic sea where temperatures average -1.9 °C all year round. The enzymes isolated from Antarctic organisms represent a useful tool to study the relations among structure, stability and function of proteins in organisms adapted to living at constantly low temperatures. In the present paper, we report in detail the cloning, purification, and physico-chemical properties of NcoCA, a ?-CA isolated from the Antarctic cyanobacterium Nostoc commune. This enzyme showed a higher catalytic efficiency at lower temperatures compared to mesophilic counterparts belonging to ?-, ?-, ?-classes, as well as a limited stability at moderate temperatures.

Around 50 % of the worldwide population is affected by dandruff, which is triggered by a variety of factors. The yeast Malassezia globosa has been labeled as the most probable causative agent for the onset of dandruff. The β-carbonic anhydrase (CA) of MgCA was recently validated as an anti-dandruff target, with its inhibition being responsible for in vivo growth defects in the fungus. As classical CA inhibitors of the sulfonamide type give rise to permeability problems through biological membranes, finding non-sulfonamide alternatives for MgCA inhibition is of considerable interest in the cosmetic field. We recently screened a large library of human (h) CA inhibitors for MgCA inhibition, including different chemotypes, such as monothiocarbamates, dithiocarbamates, phenols, and benzoxaboroles. Herein, we expanded the research toward new MgCA inhibitors by considering a set of natural polyphenols (including flavones, flavonols, flavanones, flavanols, isoflavones, and depsides) that exhibited MgCA inhibitory activity in the micromolar range, as well as selectivity for the fungal isozyme over off-target human isoforms. The binding mode of representative derivatives within the MgCA catalytic cleft was investigated by docking studies using a homology-built model.

The synthesis of a new series of sulfamides incorporating ortho-, meta, and para-benzenesulfamide moieties is reported, which were investigated for the inhibition of two human (h) isoforms of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), hCA I and II, and two Vibrio cholerae enzymes, belonging to the α- and β-CA classes (VchCAα, VchCAβ). The compounds were prepared by using the “tail approach”, aiming to overcome the scarcity of selective inhibition profiles associated to CA inhibitors belonging to the zinc binders. The built structure–activity relationship showed that the incorporation of benzhydryl piperazine tails on a phenyl sulfamide scaffold determines rather good efficacies against hCA I and VchCAα, with several compounds showing KIs

CruCA4 is a secreted isoform of the α-carbonic anhydrase (CA, EC 4.2.1.1) family, which has been identified in the octocoral Corallium rubrum. This enzyme is involved in the calcification process leading to the formation of the coral calcium carbonate skeleton. We report here experiments performed on the recombinant CruCA4 with the technique of protonography that can be used to detect in a simple way the enzyme activity. We have also investigated the inhibition profile of CruCA4 with one major class of CA inhibitors, the inorganic anions. A range of weak and moderate inhibitors have been identified having KI in the range of 1-100 mM, among which the halides, pseudohalides, bicarbonate, sulfate, nitrate, nitrite, and many complex inorganic anions. Stronger inhibitors were sulfamide, sulfamate, phenylboronic acid, phenylarsonic acid, and diethylditiocarbamate, which showed a better affinity for this enzyme, with KI in the range of 75 μM-0.60 mM. All these anions/small molecules probably coordinate to the Zn(II) ion within the CA active site as enzyme inhibition mechanism.

Coumarin derivatives are a peculiar class of inhibitors of the family of metalloenzymes carbonic anhydrases (CA, EC 4.2.1.1). Several coumarins display higher affinity and selectivity toward most relevant and druggable CA isoforms. By decorating the natural compound umbelliferone (1) we have identified a new series of coumarin-based compounds demonstrating high CA inhibitory effects with nanomolar affinity for hCA IX and hCA XII isoforms that were considered a target amenable to develop antitumor agents. The most active tested compounds proved to be potent inhibitors with Ki values equal to that of the well-known inhibitor acetazolamide (AAZ), that lacks selectivity over ubiquitous hCA I and hCA II. As suggested by docking studies the coumarins, that are lacking of the canonical metal binding groups, do not interact with Zinc ion within the catalytic site as found for classical sulfonamide type inhibitors of CAs. Thus, the studied inhibitors might possess a non-classical inhibitory mode of action preventing the carbon dioxide to entry into catalytic cavity and its conversion into bicarbonate ion. Specifically, the most active inhibitor of hCA XII compound 18i (Ki value of 5.5 nM) and its supposed hydrolytic products could establish a web of H-bond interactions within the enzymatic cavity.

The β-class carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei, BpsCAβ, that is responsible for the tropical disease melioidosis was investigated for its activation with natural and non-natural amino acids and amines. Previously, the γ-CA from this bacterium has been investigated with the same library of 19 amines/amino acids, which show very potent activating effects on both enzymes. The most effective BpsCAβ activators were L- and D-DOPA, L- and D-Trp, L-Tyr, 4-amino-L-Phe, histamine, dopamine, serotonin, 2-pyridyl-methylamine, 1-(2-aminoethyl)-piperazine and L-adrenaline with KAs of 0.9–27 nM. Less effective activators were D-His, L- and D-Phe, D-Tyr, 2-(2-aminoethyl)pyridine and 4-(2-aminoethyl)-morpholine with KAs of 73 nM–3.42 µM. The activation of CAs from bacteria, such as BpsCAγ/β, has not been considered previously for possible biomedical applications. It would be of interest to perform studies in which bacteria are cultivated in the presence of CA activators, which may contribute to understanding processes connected with the virulence and colonization of the host by pathogenic bacteria.