Carbonic anhydrase inhibitors. Inhibition studies of a coral secretory isoform by sulfonamides

The inhibition of a newly cloned coral carbonic anhydrase (CA, EC 4.2.1.1) has been investigated with a series of sulfonamides, including some clinically used derivatives (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, benzolamide, and sulpiride, or indisulam, a compound in clinical development as antitumor drug), as well as the sulfamate antiepileptic topiramate. Some simple amino-/hydrazine-/hydroxy-substituted aromatic/heterocyclic sulfonamides have also been included in the study. All types of activity have been detected, with low potency inhibitors (K_Is in the range of 163–770 nM), or with medium potency inhibitors (K_Is in the range of 75.1–105 nM), whereas ethoxzolamide, several clinically used sulfonamides and heterocyclic compounds showed stronger potency, with K_Is in the range of 16–48.2 nM. These inhibitors may be useful to better understand the physiological role of the Stylophora pistillata CA (STPCA) in corals and its involvement in biomineralisation in this era of global warming.     

Carbonic anhydrase inhibitors. Inhibition studies of the human secretory isoform VI with anions

The unique secretory isozyme of human carbonic anhydrase (hCA, EC 4.2.1.1), hCA VI, has been cloned, expressed, and purified. The kinetic parameters for the CO(2) hydration reaction proved hCA VI to possess a k(cat) of 3.4x10(5)s(-1) and k(cat)/K(M) of 4.9x10(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 isoforms CA I or CA IX. A series of anions (such as bicarbonate, chloride, nitrate, etc.) were shown to inhibit the activity of the enzyme, with inhibition constants typically in the range of 0.60-0.90mM. The best hCA VI inhibitors were cyanide, azide, sulfamide, and sulfamate, with inhibition constants in the range of 70-90microM.     

Carbonic anhydrase inhibitors: The membrane-associated isoform XV is highly inhibited by inorganic anions

The membrane-associated mouse isozyme of carbonic anhydrase XV (mCA XV), has been investigated for its interaction with anion inhibitors. mCA XV is an isoforms possessing a very particular inhibition profile by anions, dissimilar to that of all other mammalian CAs investigated earlier. Many simple inorganic anions (thiocyanate, cyanide, azide, bicarbonate, hydrogen sulfide, bisulfite and sulfate) showed low micromolar inhibition constants against mCA XV (K_Is of 8.2–10.1 μM), whereas they acted as much weaker (usually millimolar) inhibitors of other isoforms. Halides, nitrate, nitrite, carbonate, sulfamate, sulfamide and phenylboronic/arsonic acid were weaker inhibitors, with inhibition constants in the range of 27.6–288 μM. Our data may be useful for the design of more potent inhibitors of mCA XV (considering various zinc binding groups present in the anions investigated here, e.g., the sulfonate one) and for understanding some physiologic/pharmacologic consequences of mCA XV inhibition by anions such as bicarbonate or sulfate which show quite high affinity for it.     

Carbonic anhydrase inhibitors. Inhibition studies with anions and sulfonamides of a new cytosolic enzyme from the scleractinian coral Stylophora pistillata

The catalytic activity and the inhibition of a new coral carbonic anhydrase (CA, EC 4.2.1.1), from the scleractinian coral Stylophora pistillata, STPCA-2, has been investigated. STPCA-2 has high catalytic activity for the physiological reaction being less sensitive to anion and sulfonamide inhibitors compared to STPCA, a coral enzyme previously described. The best STPCA-2 anion inhibitors were sulfamide, sulfamic acid, phenylboronic acid, and phenylarsonic acid (K_Is of 5.7-67.2 μM) whereas the best sulfonamide inhibitors were acetazolamide and dichlorophenamide (K_Is of 74-79 nM). Because this discriminatory effect between these two coral CAs, sulfonamides may be useful to better understand the physiological role of STPCA and STPCA-2 in corals and biomineralization processes.     

Carbonic anhydrase inhibitors: Inhibition of the new membrane-associated isoform XV with phenols

Inhibition of the newest isoform of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), CA XV, with a series of phenols was investigated. Murine CA XV showed an inhibition profile by phenols distinct of those of the cytosolic human isoforms CA I and II. Phenol and some of its 2-, 3-, and 4-substituted derivatives incorporating hydroxy, fluoro, carboxy, and acetamido moieties were effective CA XV inhibitors, with inhibition constants in the range of 7.20-11.30 microM, whereas compounds incorporating 4-amino-, 4-cyano, or 3-hydroxy groups were less effective (K(I)s of 335-434 microM). The best phenol inhibitor was clioquinol (K(I) of 2.33 microM). Phenols show a different inhibition mechanism as compared to sulfonamides and their isosteres, and may lead to the design of compounds with selectivity for inhibiting different CA isozymes with medicinal chemistry applications.     

Carbonic anhydrase activators. The first activation study of a coral secretory isoform with amino acids and amines

The activity of the coral Stylophora pystillata secretory carbonic anhydrase STPCA has been tested in presence of amino acids and amines. All the investigated compounds showed a positive, activating effect on k_cat and have been separated in weak (K_A in the range of 21–126 μM), medium (10.1–19 μM) and strong enzyme activators (K_A of 0.18–3.21 μM). D-DOPA was found to be the best coral enzyme activator, with an activation constant K_A of 0.18 μM. This enhancement of STPCA activity, as well as previous enzyme inhibition results, might now be tested on living organisms to better understand the role played by these enzymes in the coral calcification processes.     

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.     

Carbonic anhydrase inhibitors. Inhibition of the beta-class enzyme from the yeast Saccharomyces cerevisiae with anions

The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a beta-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, has been cloned, purified, characterized kinetically, and investigated for its inhibition with a series simple, inorganic anions such as halogenides, pseudohalogenides, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, perchlorate, sulfate, and some of its isosteric species. The enzyme showed high CO(2) hydrase activity, with a k(cat) of 9.4x10(5) s(-1) and k(cat)/K(m) of 9.8x10(7) M(-1) s(-1). scCA was weakly inhibited by metal poisons (cyanide, azide, cyanate, thiocyanate, K(I)s of 16.8-55.6 mM) and strongly inhibited by bromide, iodide, and sulfamide (K(I)s of 8.7-10.8 microM). The other investigated anions showed inhibition constants in the low millimolar range.     

Carbonic anhydrase inhibitors. Inhibition of transmembrane isozymes XII (cancer-associated) and XIV with anions

Metal complexing anions represent an important class of inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The first inhibition study of the transmembrane isozymes CA XII (tumor-associated) and XIV with anions is reported. These isozymes showed inhibition profiles with physiologic/non-physiologic anions quite distinct from any other cytosolic (CA I and II) or transmembrane isoforms (e.g., CA IX) investigated earlier. hCA XII has a good affinity for fluoride and bicarbonate but is not inhibited by heavier halides, perchlorate, nitrate, and nitrite. The best hCA XII inhibitors were cyanide (K(I) of 1 microM) and azide (K(I) of 80 microM). hCA XIV was on the other hand weakly inhibited by fluoride and not at all inhibited by perchlorate, but showed good affinity for most other anions investigated here. Chloride and bicarbonate showed K(I)s in the range of 0.75-0.77 mM for this isoform. The best hCA XIV anion inhibitors were sulfate, phenylarsonic, and phenylboronic acid (K(I) in the range of 10-92 microM).     

Carbonic anhydrase inhibitors: inhibition of the membrane-bound human isozyme IV with anions

The membrane-associated human isozyme of carbonic anhydrase, hCA IV, has been investigated for its interaction with anion inhibitors, for the CO(2) hydration reaction catalyzed by this enzyme. Surprisingly, halides were observed to act as potent hCA IV inhibitors, with inhibition constants in the range of 70-90 microM, although most of these ions, and especially fluoride, the best hCA IV inhibitor among the halides, are weak inhibitors of other isozymes, such as hCA I, II and V. The metal poisons cyanate, cyanide and hydrogen sulfide were weaker hCA IV inhibitors (K(i)'s in the range of 0.6-3.9 mM), whereas thiocyanate, azide, nitrate and nitrite showed even weaker inhibitory properties (K(i)'s in the range of 30.8-65.1 mM). Sulfate was a good hCA IV inhibitor (K(i) of 9 mM), although it is a much weaker inhibitor of isozymes I, II, V and IX. Excellent hCA IV inhibitory properties showed sulfamic acid, sulfamide, phenylboronic acid and phenylarsonic acid, with K(i)'s in the range of 0.87-0.93 microM, whereas their affinities for the other investigated isozymes were in the millimolar range. The interaction of some anions with the mitochondrial isozyme hCA V has also been investigated for the first time here. It has been observed that among all these isozymes, hCA V has the lowest affinity for bicarbonate and carbonate (K(i)'s in the range of 82-95 mM), which may represent an evolutionary adaptation of this isozyme to the rather alkaline environment (pH 8.5) within the mitochondria, where hCA V plays important functions in some biosynthetic reactions involving carboxylating enzymes (pyruvate carboxylase and acetyl coenzyme A carboxylase). There are important differences of affinity for anions between the two membrane-associated isozymes, hCA IV and hCA IX.     

Carbonic anhydrase inhibitors: inhibition of the cytosolic human isozyme VII with anions

An inhibition study of the cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozyme VII (hCA VII) with anions has been conducted. Cyanate, cyanide, and hydrogensulfite were weak hCA VII inhibitors (K(I)s in the range of 7.3-15.2 mM). Cl- and HCO3- showed good inhibitory activity against hCA VII (K(I)s of 0.16-1.84 mM), suggesting that this enzyme is not involved in metabolons with anion exchangers or sodium bicarbonate cotransporters. The best inhibitors were sulfamate, sulfamide, phenylboronic, and phenylarsonic acid (K(I)s of 6.8-12.5 microM).     

Carbonic anhydrase inhibitors: inhibition of human and murine mitochondrial isozymes V with anions

In addition to sulfonamides, metal complexing anions represent the second class of inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The first inhibition study of the mitochondrial isozyme CA V (of murine and human origin) with anions is reported here. Inhibition data of the cytosolic isozymes CA I and CA II as well as the membrane-bound isozyme CA IV with a large number of anionic species such as halides, pseudohalides, bicarbonate, nitrate, hydrosulfide, arsenate, sulfamate, and sulfamidate and so on, are also provided for comparison. Isozyme V has an inhibition profile by anions completely different to those of CA I and IV, but similar to that of hCA II, which may have interesting physiological consequences. Similarly to hCA II, the mitochondrial isozymes show micro-nanomolar affinity for sulfonamides such as sulfanilamide and acetazolamide.     

Carbonic anhydrase inhibitors. Cloning, characterization and inhibition studies of the cytosolic isozyme III with anions

The cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozyme III (hCA III) has been cloned and purified by the GST-fusion protein method. Recombinant pure hCA III had the following kinetic parameters for the CO(2) hydration reaction at 20 degrees C and pH 7.5: k(cat) of 1.3 x 10(4) s(- 1) and k(cat)/K(M) of 2.5.10(5) M(- 1) s(- 1). The first detailed inhibition study of this enzyme with anions is reported. Inhibition data of the cytosolic isozymes hCA I - hCA III with a large number of anions (halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, sulfate, sulfamic acid, sulfamide, etc.), were determined and these values are comparatively discussed for these three cytosolic isoforms. Fluoride, nitrate, nitrite, phenylboronic acid and phenylarsonic acid (as anions) were weak hCA III inhibitors (K(I)s of 21-78.5 mM), whereas bicarbonate, chloride, bromide, sulfate and several other simple anions showed K(I)s around 1 mM. The best hCA III inhibitors were carbonate, cyanide, thiocyanate, azide and hydrogensulfide, which showed K(I)s in the range of 10-90 microM. It is difficult to explain the inhibitory activity of carbonate (K(I) of 10 microM) against hCA III, also considering the fact that this ion has an affinity of 15-73 mM for hCA I and II and is in equilibrium with one of the substrates of this enzyme, i.e., bicarbonate, which is a much weaker inhibitor (K(I) of 0.74 mM against hCA III, of 12 mM against hCA I and of 85 mM against hCA II).     

Carbonic anhydrase inhibitors. Inhibition of the zinc and cobalt gamma-class enzyme from the archaeon Methanosarcina thermophila with anions

Anions represent the second class of inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), in addition to sulfonamides, which possess clinical applications. The first inhibition study of the zinc and cobalt gamma-class enzyme from the archaeon Methanosarcina thermophila (Cam) with anions is reported here. Inhibition data of the alpha-class human isozymes hCA I and hCA II (cytosolic) as well as the membrane-bound isozyme hCA IV with a large number of anionic species such as halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, bisulfite, and sulfate, etc., are also provided for comparison. The best Zn-Cam anion inhibitors were hydrogen sulfide and cyanate, with inhibition constants in the range of 50-90 microM, whereas thiocyanate, azide, carbonate, nitrite, and bisulfite were weaker inhibitors (K(I)s in the range of 5.8-11.7 mM). Fluoride, chloride, and sulfate do not inhibit this enzyme appreciably up to concentrations of 200 mM, whereas the substrate bicarbonate behaves as a weak inhibitor (K(I)s of 42 mM). The best Co-Cam inhibitor was carbonate, with an inhibition constant of 9 microM, followed by nitrate and bicarbonate (K(I)s in the range of 90-100 microM). The metal poisons were much more ineffective inhibitors of this enzyme, with cyanide possessing an inhibition constant of 51.5mM, whereas cyanate, thiocyanate, azide, iodide, and hydrogen sulfide showed K(I)s in the range of 2.0-6.1mM. As for Zn-Cam, fluoride, chloride, and sulfate are not inhibitors of Co-Cam. These major differences between the two gamma-CAs investigated here can be explained only in part by the different geometries of the metal ions present within their active sites.     

Carbonic anhydrase inhibitors. Inhibition of the beta-class enzyme from the methanoarchaeon Methanobacterium thermoautotrophicum (Cab) with anions

The first inhibition study of the beta-class carbonic anhydrase (CA, EC 4.2.1.1) from the methanoarchaeon Methanobacterium thermoautotrophicum (Cab) with anions is reported here. Inhibition data of the alpha-class human isozymes hCA I and hCA II (cytosolic) as well as the membrane-bound isozyme hCA IV and the gamma-class enzyme from another archaeon, Methanosarcina thermophila (Cam) with a large number of anionic species such as halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, bisulfite, sulfate, etc., are also provided for comparison. The best Cab anion inhibitors were thiocyanate and hydrogen sulfide, with inhibition constants in the range of 0.52-0.70 mM, whereas cyanate, iodide, carbonate, and nitrate were weaker inhibitors (Ki's in the range of 7.8-13.2 mM). Fluoride, chloride, and sulfate do not inhibit this enzyme appreciably, whereas the CA substrate bicarbonate, or other anions, such as bromide, nitrite, bisulfite, or sulfamate behave as weak inhibitors (Ki in the range of 40-45 mM). It is interesting to note that the metal poison, coordinating anions cyanide and azide are also rather weak Cab inhibitors (Ki in the range of 27-55 mM), whereas sulfamide is a very weak Cab inhibitor (Ki of 103 mM), although it strongly inhibits Cam (Ki of 70 microM). Surprisingly, phenylboronic and phenylarsonic acids, which have been investigated for the inhibition of all these CAs for the first time, showed very weak activity against the alpha-CA isozymes, but were effective Cab and Cam inhibitors. The best Cab inhibitors were just these two compounds (Ki's of 0.20-0.33 mM), whereas the best Cam inhibitor was sulfamic acid (Ki of 96 nM). These major differences of behavior between the diverse CAs investigated here toward anion inhibitors can be difficultly explained considering the convergent evolution of so diverse enzymes for the binding and turnover of small molecules such as carbon dioxide and anions.     

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 inhibitors: cloning and sulfonamide inhibition studies of a carboxyterminal truncated alpha-carbonic anhydrase from Helicobacter pylori

A library of sulfonamides/sulfamates has been investigated for the inhibition of the carboxyterminal truncated form of the alpha-carbonic anhydrase (CA, EC 4.2.1.1) isolated from the gastric pathogen Helicobacter pylori (hpCA). This enzyme, incorporating 202 amino acid residues, showed a catalytic activity similar to that of the full length hpCA, with k(cat) of 2.35 x 10(5)s(-1) and k(cat)/K(M) of 1.56 x 10(7)M(-1)s(-1) at 25 degrees C and pH of 8.9, for the CO(2) hydration reaction. All types of activity for inhibition of the bacterial enzyme have been detected. Dorzolamide and simple 4-substituted benzenesulfonamides were weak hpCA inhibitors (inhibition constants, K(I)s, in the range of 830-4310 nM). Sulfanilamide, orthanilamide, some of their derivatives, and indisulam showed better activity (K(I)s in the range of 310-562 nM), whereas most of the clinically used CA inhibitors, such as methazolamide, ethoxzolamide, dichlorophenamide, brinzolamide, topiramate, zonisamide, etc., acted as medium potency hpCA inhibitors (K(I)s in the range of 124-287 nM). Some potent hpCA inhibitors were detected too (K(I)s in the range of 20-96 nM) such as acetazolamide, 4-amino-6-chloro-1,3-benzenedisulfonamide, 4-sulfanilyl-aminoethyl-benzenesulfonamide, and 4-(2-amino-pyrimidin-4-yl)-benzenesulfonamide. Most of the investigated derivatives acted as better inhibitors of the human isoform hCA II than as hpCA inhibitors. Since hpCA is essential for the survival of the pathogen in acid, its inhibition by compounds such as those investigated here might be used as a new pharmacologic tool in the management of drug resistant H. pylori.     

Carbonic anhydrase inhibitors: antisecretory and cytoprotective properties

Experimental studies located carbonic anhydrase (CA) in the parietal cells close to secretory canaliculi, in superficial epithelial cells and gastric microvasculature. The role of CA is CO2 hydration resulting H+ for acid secretion and conversion of OH into HCO3-. Our studies showed that the physiological secretagogue histamine, acetylcholine and gastrin are all CA activators, achieving potentiating interactions. Catecholamines are also strong enzymatic activators. Beside sulfonamides, other CA inhibitors are anticholinergics, PGE and PGI2, some calcium channel blockers, alpha 2- and beta 1-adrenoceptor blockers and Zn2+. Cytoprotective properties of CA inhibitors gained experimental evidence in the past years. These effects could be based on increase of gastric mucosal blood flow, proved experimentally, which might be mediated by increase of endogenous prostaglandin synthesis and sulfhydryls and, respectively, motility changes. The unique combination of strong antisecretory effect with the cytoprotective action explain the outstanding clinical efficacy of CA inhibitors in the healing of gastric and duodenal ulcers.