Comparison of the amine/amino acid activation profiles of the β- and γ-carbonic anhydrases from the pathogenic bacterium Burkholderia pseudomallei

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 K_As 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 K_As 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.     

Activation studies with amines and amino acids of the β-carbonic anhydrase from the pathogenic protozoan Leishmania donovani chagasi

The activation of a β-class carbonic anhydrase (CAs, EC 4.2.1.1) from Leishmania donovani chagasi (LdcCA) was investigated using a panel of natural and non-natural amino acids and amines. The most effective activators belonged to the amine class, with histamine, dopamine, serotonin, 2-pyridyl-methylamine and 4-(2-aminoethyl)-morpholine with activation constants in the range of 0.23–0.94 µM. In addition, 2-(2-aminoethyl)pyridine and 1-(aminoethyl)-piperazine were even more effective activators (K_As of 9–12 nM). Amino acids such as L-/D-His, L-/D-Phe, L-/D-DOPA, L-/D-Trp and L-/D-Tyr were slightly less effective activators compared to the amines, but showed activation constants in the low micromolar range (1.27–9.16 µM). Many of the investigated activators are autacoids that are present in rather high concentrations in different tissues of the host mammals infected by these parasites. As CA activators have not yet been investigated for protozoan CAs, this study may be relevant for an improved understanding of the role of this enzyme in the life cycle of Leishmania.     

Carbonic anhydrase activators. Activation of the membrane-associated isoform XV with amino acids and amines

An activation study of the membrane-associated carbonic anhydrase (CA, EC 4.2.1.1) isoform XV with a series of natural and non-natural amino acids and aromatic/heterocyclic amines is reported. Murine CA XV was strongly activated by some amino acids (d-Phe, l-/d-DOPA, d-Trp, l-Tyr) and amines (dopamine, serotonin, l-adrenaline and 4-(2-aminoethyl)-morpholine) with activation constants in the range of 4.0–9.5 μM. l-/d-His, l-Phe, histamine and several other heterocyclic amines showed less efficient activation (K_As in the range of 11.6–33.4 μM). The activation profile of CA XV is quite different from that of the cytosolic isoforms CA I and II or the membrane-associated CA IV. All mammalian isoforms CA I–XV are thus characterized for their interaction with this set of amino acid and amine activators, some of which are biogenic amines or neurotransmitters present in sufficiently high amounts in various tissues for exerting significant biologic responses.     

Carbonic anhydrase activators: activation of the archaeal beta-class (Cab) and gamma-class (Cam) carbonic anhydrases with amino acids and amines

Activation of the archaeal beta-class (Cab) and gamma-class (Cam) carbonic anhydrases (CAs, EC 4.2.1.1) with a series of natural and non-natural amino acids and aromatic/heterocyclic amines has been investigated. Cab, Zn-Cam and Co-Cam showed an activation profile with natural, L- and D-amino acids very different of those of the alpha-class enzymes CA I, II and III. Most of these compounds showed medium efficacy as archaeal CA activators, except for D-Phe and L-Tyr which were effective Cab activators (K(A)s of 10.3-10.5 microM), 2-pyridylmethylamine and 1-(2-aminoethyl)-piperazine which effectively activated Zn-Cam (K(A)s of 10.1-11.4 microM) and serotonin, L-adrenaline and 2-pyridylmethylamine which were the best Co-Cam activators (K(A)s of 0.97-8.9 microM). We prove here that the activation mechanisms of the alpha-, beta-, and gamma-class CAs are similar, although the activation profiles with various compounds differ dramatically between these diverse enzymes.     

Carbonic anhydrase activators: activation of the human tumor-associated isozymes IX and XII with amino acids and amines

The first activation study of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms associated to tumors, hCA IX and XII, with a small library of natural and non-natural amino acids as well as aromatic/heterocyclic amines is reported. hCA IX was activated efficiently by dopamine, adrenaline and heterocyclic amines possessing aminoethyl-/aminomethyl-moieties (K(A)s of 9 nM-1.07 microM), whereas the best hCA XII activators were serotonin, L-adrenaline, 4-(2-aminoethyl)-morpholine and d-Phe (K(A) of 0.24-0.41 microM). Precise steric and electronic requirements are needed to be present in the molecules of effective hCA IX/hCA XII activators, in order to assure an adequate fit within the enzyme active site cavity for the formation of the enzyme-activator complex, and for an efficient proton transfer process within this complex, leading to the release of a proton and formation of the catalytically active, zinc-hydroxide species of the enzyme. Selective activation of these CA isoforms might be useful to develop pharmacologic tools or to understand whether some of these biogenic amines/amino acids may influence the progression of tumors overexpressing CA IX and/or CA XII.     

Burkholderia pseudomallei γ-carbonic anhydrase is strongly activated by amino acids and amines

Activation of the γ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei (BpsγCA) with a series of natural and non-natural amino acids and aromatic/heterocyclic amines has been investigated. The best BpsγCA activators were d-His, l-DOPA, d-Trp, 4-amino-l-Phe, dopamine, 2-(2-aminoethyl)pyridine, 2-aminoethyl-piparazine/morpholine and l-adrenaline, which showed activation constants ranging between 9 and 86 nM. The least effective activators were l-His, l-Phe and 2-pyridyl-methylamine, with K_As in the range of 1.73–24.7 μM. As little is known about the role of γ-CAs in the lifecycle and virulence of this saprophytic bacterium, this study may shed some light on such phenomena. This is the first CA activation study of a γ-CA from a pathogenic bacterium, the only other such study being on the enzyme discovered in the archaeon Methanosarcina thermophila, Cam.     

Carbonic anhydrase activators: Activation of the human cytosolic isozyme III and membrane-associated isoform IV with amino acids and amines

An activation study of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA III (cytosolic) and IV (membrane-associated) with a series of natural and non-natural amino acids and aromatic/heterocyclic amines is reported. hCA III was efficiently activated by d-His, serotonin, pyridyl-alkylamines, and aminoethyl-piperazine/morpholine (K_As of 91nM–1.12 μM), whereas the best hCA IV activators were 4-amino-phenylalanine, serotonin, and 4-(2-aminoethyl)-morpholine (K_As of 79 nM–3.14 μM). Precise steric and electronic requirements are needed to be present in the molecules of effective CA III/IV activators, in order to assure an adequate fit within the enzyme active site for the formation of the enzyme-activator complex, and for efficient proton transfer processes between the active site and the reaction medium. The activation profiles of CA III and IV are distinct from those of all other mammalian CA isoforms investigated so far for their interaction with amino acids and amines.     

Carbonic anhydrase activators: the first activation study of the human secretory isoform VI with amino acids and amines

The secretory isozyme of human carbonic anhydrase (hCA, EC 4.2.1.1), hCA VI, has been cloned, expressed, and purified in a bacterial expression system. The kinetic parameters for the CO(2) hydration reaction proved hCA VI to possess a k(cat) of 3.4 x 10(5)s(-1) and k(cat)/K(M) of 4.9 x 10(7)M(-1)s(-1) (at pH 7.5 and 20 degrees C). hCA VI has a significant catalytic activity for the physiological reaction, of the same order of magnitude as the ubiquitous isoform CA I or the transmembrane, tumor-associated isozyme CA IX. A series of amino acids and amines were shown to act as CA VI activators, with variable efficacies. l-His, l-Trp, and dopamine showed weak CA VI activating effects (K(A)s in the range of 21-42 microM), whereas d-His, d-Phe, l-DOPA, l-Trp, serotonin, and some pyridyl-alkylamines were better activators, with K(A)s in the range of 13-19 microM. The best CA VI activators were l-Phe, d-DOPA, l-Tyr, 4-amino-l-Phe, and histamine, with K(A)s in the range of 1.23-9.31 microM. All these activators enhance k(cat), having no effect on K(M), participating thus in the rate determining step in the catalytic cycle, the proton transfer reactions between the enzyme active site and the environment.     

Carbonic anhydrase activators: an activation study of the human mitochondrial isoforms VA and VB with amino acids and amines

The mitochondrial isozymes of human carbonic anhydrase (hCA, EC 4.2.1.1), hCA VA and hCA VB, were investigated for activation with a series of amino acids and amines. D-His, L-DOPA, histamine, dopamine, and 4-(2-aminoethyl)morpholine were excellent hCA VA activators, with KAs in the range of 10-130 nM. Good hCA VB activating effects were identified for L-His, D-Phe, D-DOPA, L-Trp, L-Tyr, serotonin, and 2-(2-aminoethyl)-pyridine, with KAs in the range of 44-110 nM. All these activators enhanced kcat, having no effect on KM, favoring thus the rate-determining step in the catalytic cycle, the proton transfer reactions between the active site and environment. The activation pattern of the two mitochondrial isoforms is very different from each other and as compared to those of the cytosolic isoforms hCA I and II.     

Characterization of CA XIII, a novel member of the carbonic anhydrase isozyme family

The carbonic anhydrase (CA) gene family has been reported to consist of at least 11 enzymatically active members and a few inactive homologous proteins. Recent analyses of human and mouse databases provided evidence that human and mouse genomes contain genes for still another novel CA isozyme hereby named CA XIII. In the present study, we modeled the structure of human CA XIII. This model revealed a globular molecule with high structural similarity to cytosolic isozymes, CA I, II, and III. Recombinant mouse CA XIII showed catalytic activity similar to those of mitochondrial CA V and cytosolic CA I, with k(cat)/K(m) of 4.3 x 10(7) m(-1) s(-1), and k(cat) of 8.3 x 10(4) s(-1). It is very susceptible to inhibition by sulfonamide and anionic inhibitors, with inhibition constants of 17 nm for acetazolamide, a clinically used sulfonamide, and of 0.25 microm, for cyanate, respectively. Using panels of cDNAs we evaluated human and mouse CA13 gene expression in a number of different tissues. In human tissues, positive signals were identified in the thymus, small intestine, spleen, prostate, ovary, colon, and testis. In mouse, positive tissues included the spleen, lung, kidney, heart, brain, skeletal muscle, and testis. We also investigated the cellular and subcellular localization of CA XIII in human and mouse tissues using an antibody raised against a polypeptide of 14 amino acids common for both human and mouse orthologues. Immunohistochemical staining showed a unique and widespread distribution pattern for CA XIII compared with the other cytosolic CA isozymes. In conclusion, the predicted amino acid sequence, structural model, distribution, and activity data suggest that CA XIII represents a novel enzyme, which may play important physiological roles in several organs.     

QSAR studies on the activation of the human carbonic anhydrase cytosolic isoforms I and II and secretory isozyme VI with amino acids and amines

The first QSAR study on the activation of the human secretory isoform of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), CA VI, with a series of amines and amino acids is reported. A large set of topological indices have been used to obtain several tri-/tetra-parametric models. We compared the CA VI activating QSAR models with those calculated for activation of the cytosolic human isozymes hCA I and hCA II. In addition, the effect of D- and L-amino acids as activators of hCA I, hCA II and of hCA VI as compared to those of structurally related biogenic amines was investigated for obtaining statistically significant and predictive QSAR equations. The obtained models are discussed using a variety of statistical parameters. The best models were obtained for hCA II activation, followed by hCA I, whereas the QSAR models for the activation of hCA VI were statistically weaker.     

Carbonic anhydrase inhibitors. Inhibition of the newly isolated murine isozyme XIII with anions

The inhibition of the newly discovered cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozyme XIII of murine origin (mCA XIII) has been investigated with a series of anions, such as the physiological ones (bicarbonate, chloride), or the metal complexing anions (cyanate, cyanide, azide, hydrogen sulfide, etc), nitrate, nitrite, sulfate, sulfamate, sulfamide as well as with phenylboronic and phenylarsonic acids. The best mCA XIII inhibitors were cyanate, thiocyanate, cyanide and sulfamide, with K(I)-s in the range of 0.25microM-0.74 mM, whereas fluoride, iodide, azide, carbonate and hydrogen sulfide were less effective (K(I)-s in the range of 3.0-5.5mM). The least effective inhibitors were sulfate, chloride and bicarbonate (K(I)-s in the range of 138-267 mM). The affinity of mCA XIII for anions is very different from that of the other cytosolic isozymes (hCA I and II) or the mitochondrial isozyme hCA V. This resistance to inhibition by the physiological anions bicarbonate and chloride suggests an evolutionary adaptation of CA XIII to the presence of high concentrations of such anions (e.g., in the reproductive tract of both female and male), and the possible participation of this isozyme (similarly to CA II, CA IV and CA V) in metabolons with proteins involved in the anion exchange and transport, such as the anion exchangers (AE1-3) or the sodium bicarbonate co-transporter (NBC1 and NBC3) proteins, which remain to be identified.     

Type IV carbonic anhydrase is present in the gills of spiny dogfish (Squalus acanthias)

Physiological and biochemical studies have provided indirect evidence for a membrane-associated carbonic anhydrase (CA) isoform, similar to mammalian type IV CA, in the gills of dogfish (Squalus acanthias). This CA isoform is linked to the plasma membrane of gill epithelial cells by a glycosylphosphatidylinositol anchor and oriented toward the plasma, such that it can catalyze the dehydration of plasma HCO(3)(-) ions. The present study directly tested the hypothesis that CA IV is present in dogfish gills in a location amenable to catalyzing plasma HCO(3)(-) dehydration. Homology cloning techniques were used to assemble a 1,127 base pair cDNA that coded for a deduced protein of 306 amino acids. Phylogenetic analysis suggested that this protein was a type IV CA. For purposes of comparison, a second cDNA (1,107 base pairs) was cloned from dogfish blood; it encoded a deduced protein of 260 amino acids that was identified as a cytosolic CA through phylogenetic analysis. Using real-time PCR and in situ hybridization, mRNA expression for the dogfish type IV CA was detected in gill tissue and specifically localized to pillar cells and branchial epithelial cells that flanked the pillar cells. Immunohistochemistry using a polyclonal antibody raised against rainbow trout type IV CA revealed a similar pattern of CA IV immunoreactivity and demonstrated a limited degree of colocalization with Na(+)-K(+)-ATPase immunoreactivity. The presence and localization of a type IV CA isoform in the gills of dogfish is consistent with the hypothesis that branchial membrane-bound CA with an extracellular orientation contributes to CO(2) excretion in dogfish by catalyzing the dehydration of plasma HCO(3)(-) ions.     

Investigations of the esterase, phosphatase, and sulfatase activities of the cytosolic mammalian carbonic anhydrase isoforms I, II, and XIII with 4-nitrophenyl esters as substrates

The esterase, phosphatase, and sulfatase activities of carbonic anhydrase (CA, EC 4.2.1.1) isozymes, CA I, II, and XIII with 4-nitrophenyl esters as substrates was investigated. These enzymes show esterase activity with 4-nitrophenyl acetate as substrate, with second order rate constants in the range of 753-7706M(-1)s(-1), being less effective as phosphatases (k(cat)/K(M) in the range of 14.89-1374.40M(-1)s(-1)) and totally ineffective sulfatases. The esterase/phosphatase activities were inhibited by sulfonamide CA inhibitors, proving that the zinc-hydroxide mechanism responsible for the CO(2) hydrase activities of CAs is also responsible for their esterase/phosphatase activity. CA XIII was the most effective esterase and phosphatase. CA XIII might catalyze other physiological reactions than CO(2) hydration, based on its relevant phosphatase activity.     

Benzenesulfonamides with pyrimidine moiety as inhibitors of human carbonic anhydrases I,II, VI,VII, XII,and XIII

Two groups of benzenesulfonamide derivatives, bearing pyrimidine moieties, were designed and synthesized as inhibitors of carbonic anhydrases (CA). Their binding affinities to six recombinant human CA isoforms I, II, VI, VII, XII, and XIII were determined by the thermal shift assay (TSA). The binding of several inhibitors was measured by isothermal titration calorimetry (ITC). Direct demonstration of compound inhibition was achieved by determining the inhibition constant by stopped-flow CO₂ hydration assay. The most potent compounds demonstrated selectivity towards isoform I and affinities of 0.5 nM. The crystal structures of selected compounds in complex with CA II, XII, and XIII were determined to atomic resolution. Compounds described here were compared with previously published pyrimidinebenzenesulfonamides.¹ Systematic structure–activity analysis of 40 compound interactions with six isoforms yields clues for the design of compounds with greater affinities and selectivities towards target CA isoforms.     

Carbonic anhydrase inhibitors. Inhibition of cytosolic isozyme XIII with aromatic and heterocyclic sulfonamides: a novel target for the drug design

The inhibition of the newly discovered cytosolic carbonic anhydrase isozyme XIII (CA XIII) has been investigated with a series of aromatic and heterocyclic sulfonamides, including some of the clinically used derivatives, such as acetazolamide, methazolamide, dichlorophenamide, dorzolamide, and valdecoxib. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and the tumor associated isozyme IX (transmembrane) were also provided for comparison. A very interesting and unusual inhibition profile against CA XIII with these sulfonamides has been observed. The clinically used compounds (except valdecoxib, which was a weak CA XIII inhibitor) potently inhibit CA XIII, with Ki's in the range of 17-23 nM, whereas sulfanilamide, halogenated sulfanilamides, homosulfanilamide, 4-aminoethylbenzenesulfonamide, and orthanilamide were slightly less effective, with Ki's in the range of 32-56 nM. Several low nanomolar (Ki's in the range of 1.3-2.4 nM) CA XIII inhibitors have also been detected, all of them belonging to the sulfanilyl-sulfonamide type of inhibitors, of which aminobenzolamide is the best known representative. Because CA XIII is an active isozyme predominantly expressed in salivary glands, kidney, brain, lung, gut, uterus, and testis, where it probably plays an important role in pH regulation, its inhibition by sulfonamides may lead to novel therapeutic applications for this class of pharmacological agents.     

A class of carbonic anhydrase I – selective activators

A series of ureido and bis-ureido derivatives were prepared by reacting histamine with alkyl/aryl-isocyanates or di-isocyanates. The obtained derivatives were assayed as activators of the enzyme carbonic anhydrase (CA, EC 4.2.1.1), due to the fact that histamine itself has this biological activity. Although inhibition of CAs has pharmacological applications in the field of antiglaucoma, anticonvulsant, anticancer, and anti-infective agents, activation of these enzymes is not yet properly exploited pharmacologically for cognitive enhancement or Alzheimer’s disease treatment, conditions in which a diminished CA activity was reported. The ureido/bis-ureido histamine derivatives investigated here showed activating effects only against the cytosolic human (h) isoform hCA I, having no effect on the widespread, physiologically dominant isoform hCA II. This is the first report in which CA I-selective activators were identified. Such compounds may constitute interesting tools for better understanding the physiological/pharmacological effects connected to activation of this widespread CA isoform, whose physiological function is not fully understood.     

Carbonic anhydrase activators: synthesis of high affinity isozymes I, II and IV activators, derivatives of 4-(arylsulfonylureido-amino acyl)ethyl-1H-imidazole

Based on the X-ray crystallographic structure of the adduct of human carbonic anhydrase II (hCA II) with the weak activator histamine (Briganti, F., Mangani, S., Orioli, P., Scozzafava, A., Vernaglione, G. and Supuran, C.T. (1997) Biochemistry, 36, 10,384-10,392), a novel class of tight-binding CA activators was designed by using histamine (Hst) as lead molecule. Thus, N-1-tritylsulfenyl Hst was synthesized by reaction of Hst with tetrabromophthalic anhydride followed by protection of its imidazole moiety with tritylsulfenyl chloride. After hydrazinolysis, it afforded a key intermediate which was derivatized at the aliphatic amino group. Reaction of the key intermediate with 4-fluorophenylsulfonylureido amino acids (fpu-AA) or 2-toluenesulfonylureido amino acids (ots-AA) in the presence of carbodiimides, afforded after deprotection, a series of compounds with the general formula fpu/ots-AA-Hst (fpu = 4-FC6H4SO2NHCO; ots = 2-MeC6H4SO2NHCO). Some structurally related dipeptides with the general formula fpu/ots-AA1-AA2-Hst (AA, AA1 and AA2 represent amino acyl moieties), were also prepared, by a strategy similar to that used for the simple amino acyl compounds above. The new derivatives proved to be efficient in vitro activators of three CA isozymes. Best activity was shown against hCA I and bCA IV, for which some of the new compounds (such as the Lys, Arg, His or the dipeptide derivatives) showed affinities in the 2-12 nm range (h = human; b = bovine isozymes). hCA II was on the other hand somehow less prone to activation by the new derivatives, which possessed affinities around 30-60 nM for this isozyme. Ex vivo experiments showed some of the new activators to strongly enhance red cell CA activity (180-230%) after incubation with human erythrocytes. This new class of CA activators might lead to the development of drugs/diagnostic tools for the CA deficiency syndrome, a genetic disease of bone, brain and kidneys.     

Inhibition of carbonic anhydrase isoforms I,II, IX and XII with novel Schiff bases: Identification of selective inhibitors for the tumor-associated isoforms over the cytosolic ones

A series of new Schiff bases was obtained from sulfanilamide, 3-fluorosulfanilamide or 4-(2-aminoethyl)-benzenesulfonamide and aromatic/heterocyclic aldehydes incorporating both hydrophobic and hydrophilic moieties. The obtained sulfonamides were investigated as inhibitors of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, as well as the transmembrane, tumor-associated CA IX and XII. Most derivatives were medium potency or weak hCA I/II inhibitors, but several of them showed nanomolar affinity for CA IX and/or XII, making them an interesting example of isoform-selective compounds. The nature of the aryl/hetaryl moiety present in the initial aldehyde was the main factor influencing potency and isoform selectivity. The best and most CA IX-selective compounds incorporated moieties such as 4-methylthiophenyl, 4-cyanophenyl-, 4-(2-pyridyl)-phenyl and the 4-aminoethylbenzenesulfonamide scaffold. The best hCA XII inhibitors, also showing selectivity for this isoform, incorporated 2-methoxy-4-nitrophenyl-, 2,3,5,6-tetrafluorophenyl and 4-(2-pyridyl)-phenyl functionalities and were also derivatives of 4-aminoethylbenzenesulfonamide. The sulfanilamide and 3-fluorosulfanilamide derived Schiff bases were less active compared to the corresponding 4-aminoethyl-benzenesulfonamide derivatives. As hCA IX/XII selective inhibition is attractive for obtaining antitumor agents/diagnostic tools with a new mechanism of action, compounds of the type described here may be considered interesting preclinical candidates.     

New chemotypes acting as isozyme-selective carbonic anhydrase inhibitors with low affinity for the offtarget cytosolic isoform II

Considering phenols and coumarins as lead molecules for obtaining non-sulfonamide inhibitors of carbonic anhydrases (CAs, EC 4.2.1.1), we screened a large number of compounds possessing diverse chemotypes, but structural features which resemble the two chemical classes. Here we report an investigation of such derivatives which do not significantly inhibit CA II, but show interesting inhibition profiles against other isozymes. Pyridine-N-oxide-2-thiophenol, thiobenzoic acid, thimerosal, two oximes derived from a six-membered-ring lactone and from coumarin; 2-hydroxyquinoline and coumaphos, were investigated as inhibitors of CA I-XIV. All these compounds did not inhibit CA II, whereas the two oximes and 2-hydroxyquinoline were low nanomolar inhibitors of CA I, IX, XII, XIII and XIV, showing a very different inhibition profile compared to sulfonamides and sulfamates. Some other compounds showed low micromolar inhibition of other isoforms of interest, such as CA VA/VB, CA VI and VII. This study demonstrates that a rather wide range of structures show low nanomolar-micromolar inhibitory activity against many CA isozymes, without inhibiting significantly the offtarget isoform CA II.