Carbonic anhydrase inhibitors. Inhibition and homology modeling studies of the fungal β-carbonic anhydrase from Candida albicans with sulfonamides

The β-carbonic anhydrase (CA, EC 4.2.1.1) from the fungal pathogen Candida albicans (Nce103) is involved in a CO₂ sensing pathway critical for the pathogen life cycle and amenable to drug design studies. Herein we report an inhibition study of Nce103 with a library of sulfonamides and one sulfamate, showing that Nce103, similarly to the related enzyme from Cryptococcus neoformans Can2, is inhibited by these compounds with K_Is in the range of 132 nM–7.6 μM. The best Nce103 inhibitors were acetazolamide, methazolamide, bromosulfanilamide, and 4-hydroxymethylbenzenesulfonamide (K_Is < 500 nM). A homology model was generated for Nce103 based on the crystal structure of Can2. The model shows that compounds with zinc-binding groups incorporating less polar moieties and compact scaffolds generate stronger Nce103 inhibitors, whereas highly polar zinc-binding groups and bulkier compounds appear more promising for the specific inhibition of Can2. Such compounds may be useful for the design of antifungal agents possessing a new mechanism of action.     

Carbonic anhydrase activators: Activation of the β-carbonic anhydrase from the pathogenic yeast Candida glabrata with amines and amino acids

The protein encoded by the NCE103 gene of Candida glabrata, a β-carbonic anhydrase (CA, EC 4.2.1.1) designated as CgCA, was investigated for its activation with amines and amino acids. CgCA was weakly activated by amino acids such as l-/d-His, l-Phe, l-DOPA, and l-Trp and by histamine or dopamine (K_As of 21.2–37 μM) but more effectively activated by d-Phe, d-DOPA, d-Trp as well as serotonin, pyridyl-alkylamines, aminoethyl-piperazine/morpholine (K_As of 10.1–16.7 μM). The best activators were l-/d-Tyr, with activation constants of 7.1–9.5 μM. This study may bring a better understanding of the catalytic/activation mechanisms of β-CAs from pathogenic fungi.     

Inhibition of the α-carbonic anhydrase from Vibrio cholerae with amides and sulfonamides incorporating imidazole moieties

We discovered novel and selective sulfonamides/amides acting as inhibitors of the α-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Vibrio cholerae (VchCA). This Gram-negative bacterium is the causative agent of cholera and colonises the upper small intestine where sodium bicarbonate is present at a high concentration. The secondary sulfonamides and amides investigated here were potent, low nanomolar VchCA inhibitors whereas their inhibition of the human cytosolic isoforms CA I and II was in the micromolar range or higher. The molecules represent an interesting lead for antibacterial agents with a possibly new mechanism of action, although their CA inhibition mechanism is unknown for the moment.     

Inhibition of the R1 fragment of the cadmium-containing ζ-class carbonic anhydrase from the diatom Thalassiosira weissflogii with anions

We investigated the catalytic activity and inhibition of both the zinc and cadmium-containing R1 fragment of the ζ-class carbonic anhydrase (CA, EC 4.2.1.1) from the marine diatom Thalassiosira weissflogii. Our data prove that these enzymes are not only very efficient catalysts for the physiological reaction, but also sensitive to sulfonamide and anion inhibitors, with inhibition constants from the nanomolar to millimolar range. Acetazolamide inhibited the two enzymes with K_Is in the range of 58–92 nM. The best anion inhibitors of Cd-R1 were thiocyanate, sulfamate and sulfamide, with K_Is of 10–89 μM, whereas the best Zn-R1 anion inhibitors were sulfamate and sulfamide with K_Is of 60–72 μM. These enzymes were only weakly inhibited by chloride, bromide or sulfate, main anion components of sea water, with inhibition constants in the range of 0.24–0.85 mM. Thus, similarly to CAs belonging to other classes, the ζ-class CA (with either cadmium or zinc ions at the active site) was inhibited by both anions and sulfonamides.     

Structure of a dimeric fungal α-type carbonic anhydrase

The crystal structure of Aspergillus oryzae carbonic anhydrase (AoCA) was determined at 2.7 Å resolution and it revealed a dimer, which only has precedents in the α class in two membrane and cancer-associated enzymes. α carbonic anhydrases are underrepresented in fungi compared to the β class, this being the first structural representative. The overall fold and zinc binding site resemble other well studied carbonic anhydrases. A major difference is that the histidine, thought to be the major proton shuttle residue in most mammalian enzymes, is replaced by a phenylalanine in AoCA. This finding poses intriguing questions as to the biological functions of fungal α carbonic anhydrases, which are promising candidates for biotechnological applications.Structured summaryAoCA binds to AoCA by molecular sieving (View interaction)AoCA binds to AoCA X-ray crystallography (View interaction)     

The molecular evolution of β-carbonic anhydrase in Flaveria

Limited information exists regarding molecular events that occurred during the evolution of C(4) plants from their C(3) ancestors. The enzyme β-carbonic anhydrase (CA; EC 4.2.1.1), which catalyses the reversible hydration of CO(2), is present in multiple forms in C(3) and C(4) plants, and has given insights into the molecular evolution of the C(4) pathway in the genus Flaveria. cDNAs encoding three distinct isoforms of β-CA, CA1-CA3, have been isolated and examined from Flaveria C(3) and C(4) congeners. Sequence data, expression analyses of CA orthologues, and chloroplast import assays with radiolabelled CA precursor proteins from the C(3) species F. pringlei Gandoger and the C(4) species F. bidentis (L.) Kuntze have shown that both contain chloroplastic and cytosolic forms of the enzyme, and the potential roles of these isoforms are discussed. The data also identified CA3 as the cytosolic isoform important in C(4) photosynthesis and indicate that the C(4) CA3 gene evolved as a result of gene duplication and neofunctionalization, which involved mutations in coding and non-coding regions of the ancestral C(3) CA3 gene. Comparisons of the deduced CA3 amino acid sequences from Flaveria C(3), C(4), and photosynthetic intermediate species showed that all the C(3)-C(4) intermediates investigated and F. brownii, a C(4)-like species, have a C(3)-type CA3, while F. vaginata, another C(4)-like species, contains a C(4)-type CA3. These observations correlate with the photosynthetic physiologies of the intermediates, suggesting that the molecular evolution of C(4) photosynthesis in Flaveria may have resulted from a temporally dependent, stepwise modification of protein-encoding genes and their regulatory elements.     

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.     

Inhibition of human carbonic anhydrase isoforms I–XIV with sulfonamides incorporating fluorine and 1,3,5-triazine moieties

Reaction of cyanuryl fluoride with sulfanilamide or 4-aminoethylbenzenesulfonamide afforded triazinyl-substituted benzenesulfonamides incorporating fluorine, which were further derivatized by reaction with amines, amino alcohols, amino acids or amino acid esters. Inhibition studies of all the human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms, hCA I–XIV with these compounds revealed that they show moderate-weak inhibition of hCA III, IV, VA and XIII, rather moderate inhibition against hCA I, VI, and IX, and excellent inhibition of the physiologically relevant hCA II, VII and XII. The inhibition profile of these fluorine containing triazinyl sulfonamides is thus very different from the corresponding analogs incorporating chlorine, which were previously investigated as inhibitors of some of these enzymes.     

Carbonic anhydrase inhibitors. Inhibition of mammalian isoforms I–XIV with a series of natural product polyphenols and phenolic acids

A series of phenolic acids and phenol natural products, such as p-hydroxybenzoic acid, p-coumaric acid, caffeic acid, ferulic acid, gallic acid, syringic acid, quercetin, and ellagic acid, were investigated for their inhibitory effects against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). All mammalian isozymes of human (h) or murine (m) origin hCA I–hCA XII, mCA XIII and hCA XIV were inhibited in the low micromolar or submicromolar range by these (poly)phenols (K_Is in the range of 0.87–7.79 μM). p-Hydroxybenzoic acid was the best inhibitor of all isozymes (K_Is of 0.87–35.4 μM) and the different isozymes showed very variable inhibition profiles with these derivatives. Phenols like the ones investigated here possess a CA inhibition mechanism distinct of that of the sulfonamides/sulfamates used clinically or the coumarins. Unlike the sulfonamides, which bind to the catalytic zinc ion, phenols are anchored at the Zn(II)-coordinated water molecule and bind more externally within the active site cavity, making contacts with various amino acid residues. As this is the region with the highest variability between the many CA isozymes found in mammals, this class of compounds may lead to isoform-selective inhibitors targeting just one or few of the medicinally relevant CAs.     

Structural studies of β-carbonic anhydrase from the green alga Coccomyxa: inhibitor complexes with anions and acetazolamide

The β-class carbonic anhydrases (β-CAs) are widely distributed among lower eukaryotes, prokaryotes, archaea, and plants. Like all CAs, the β-enzymes catalyze an important physiological reaction, namely the interconversion between carbon dioxide and bicarbonate. In plants the enzyme plays an important role in carbon fixation and metabolism. To further explore the structure-function relationship of β-CA, we have determined the crystal structures of the photoautotroph unicellular green alga Coccomyxa β-CA in complex with five different inhibitors: acetazolamide, thiocyanate, azide, iodide, and phosphate ions. The tetrameric Coccomyxa β-CA structure is similar to other β-CAs but it has a 15 amino acid extension in the C-terminal end, which stabilizes the tetramer by strengthening the interface. Four of the five inhibitors bind in a manner similar to what is found in complexes with α-type CAs. Iodide ions, however, make contact to the zinc ion via a zinc-bound water molecule or hydroxide ion — a type of binding mode not previously observed in any CA. Binding of inhibitors to Coccomyxa β-CA is mediated by side-chain movements of the conserved residue Tyr-88, extending the width of the active site cavity with 1.5-1.8 Å. Structural analysis and comparisons with other α- and β-class members suggest a catalytic mechanism in which the movements of Tyr-88 are important for the CO₂-HCO₃ ^- interconversion, whereas a structurally conserved water molecule that bridges residues Tyr-88 and Gln-38, seems important for proton transfer, linking water molecules from the zinc-bound water to His-92 and buffer molecules.     

Carbonic anhydrase inhibitors. Inhibition of the β-class enzyme from the pathogenic yeast Candida glabrata with anions

A β-carbonic anhydrase (CA, EC 4.2.1.1), the protein encoded by the NCE103 gene of Candida glabrata which also present in Candida albicans and Saccharomyces cerevisiae, was cloned, purified, characterized kinetically and investigated for its inhibition by a series simple, inorganic anions such as halogenides, pseudohalogenides, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, perchlorate, sulfate and some isosteric species. The enzyme showed significant CO₂ hydrase activity, with a k_cat of 3.8 × 10⁵ s^?1 and k_cat/K_M of 4.8 × 10⁷ M^?1 s^?1. The Cà glabrata CA (CgCA) was moderately inhibited by metal poisons (cyanide, azide, cyanate, thiocyanate, K_Is of 0.60–1.12 mM) but strongly inhibited by bicarbonate, nitrate, nitrite and phenylarsonic acid (K_Is of 86–98 μM). The other anions investigated showed inhibition constants in the low millimolar range, with the exception of bromide and iodide (K_Is of 27–42 mM).     

Dithiocarbamates with potent inhibitory activity against the Saccharomyces cerevisiae β-carbonic anhydrase

Dithiocarbamates (DTCs) prepared from primary or secondary amines, which incorporated amino/hydroxyl-alkyl, mono-/bicyclic aliphatic/heterocyclic rings based on the quinuclidine, piperidine, hydroxy-/carboxy-/amino-substituted piperidine, morpholine and piperazine scaffolds, were investigated for the inhibition of α- and β-carbonic anhydrases (CAs, EC 4.2.1.1) of pharmacologic relevance, such as the human (h) isoform hCA I and II, as well as the Saccharomyces cerevisiae β-CA, scCA. The yeast and its β-CA were shown earlier to be useful models of pathogenic fungal infections. The DTCs investigated here were medium potency hCA I inhibitors (K_Is of 66.5–910?nM), were more effective as hCA II inhibitors (K_Is of 8.9–107?nM) and some of them showed excellent, low nanomolar activity against the yeast enzyme, with inhibition constants ranging between 6.4 and 259?nM. The detailed structure activity relationship for inhibition of the yeast and human enzymes is discussed. Several of the investigated DTCs showed excellent selectivity ratios for inhibiting the yeast over the human cytosolic CA isoforms.     

Analysis of saponins and phenolic compounds as inhibitors of α-carbonic anhydrase isoenzymes

A series of phenolic and saponin type natural products such as quercetin, rutin, catechin, epicatechin, silymarin, trojanoside H, astragaloside IV, astragaloside VIII and astrasieversianin X, were investigated for their inhibitory effects against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). We here report inhibitory effects of these compounds against five α-CA isozymes (hCA I, hCA II, bCA III, hCA IV and hCA VI). Most of the phenolic and saponin type compounds inhibited the isoenzymes quite effectively at low micromolar K_I-s ranging between 0.1 and 4 µM, whereas a few derivatives were ineffective (K_I-s > 100 µM). The results were remarkable which might lead to design of novel CAIs with a diverse inhibition mechanism compared to sulfonamide/sulfamate inhibitors.     

Carbonic anhydrase — a universal enzyme of the carbon-based life

Carbonic anhydrase (CA) is a metalloenzyme that performs interconversion between CO₂ and the bicarbonate ion (HCO₃ ^?). CAs appear among all taxonomic groups of three domains of life. Wide spreading of CAs in nature is explained by the fact that carbon, which is the major constituent of the enzyme’s substrates, is a key element of life on the Earth. Despite the diversity of CAs, they all carry out the same reaction of CO₂/HCO₃ ^? interconversion. Thus, CA obviously represents a universal enzyme of the carbon-based life. Within the classification of CAs, here we proposed the existence of an extensive family of CA-related proteins (γCA-RPs)–the inactive forms of γ-CAs, which are widespread among the Archaea, Bacteria, and, to a lesser extent, in Eukarya. This review focuses on the history of CAs discovery and integrates the most recent data on their classification, catalytic mechanisms, and physiological roles at various organisms.     

Kinetic and in silico analysis of thiazolidin-based inhibitors of α-carbonic anhydrase isoenzymes

Carbonic anhydrases (CAs, EC 4.2.1.1) are inhibited by sulfonamides, inorganic anions, phenols, salicylic acid derivatives (acting as drug or prodrugs). A novel class of CA inhibitors (CAIs), interacting with the CA isozymes I and II (cytosolic) in a different manner, is reported here. Kinetic measurements allowed us to identify thiazolidin-based compounds as submicromolar-low micromolar inhibitors of these two CA isozymes. Molecular docking studies of a set of such inhibitors within CA I and II active site allowed us to understand the inhibition mechanism. This new class of inhibitors bind differently compared to other classes of inhibitors known to date: they were found between the phenol-binding site, filling thus the middle of the enzyme cavity.     

Purification and inhibition studies with anions and sulfonamides of an α-carbonic anhydrase from the Antarctic seal Leptonychotes weddellii

A high activity α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified from various tissues of the Antarctic seal Leptonychotes weddellii. The new enzyme, denominated lwCA, has a catalytic activity for the physiologic CO(2) hydration to bicarbonate reaction, similar to that of the high activity human isoform hCA II, with a k(cat) of 1.1×10(6) s(-1), and a k(cat)/K(m) of 1.4×10(8) M(-1) s(-1). The enzyme was highly inhibited by cyanate, thiocyanate, cyanide, bicarbonate, carbonate, as well as sulfamide, sulfamate, phenylboronic/phenylarsonic acids (K(I)s in the range of 46-100 μM). Many clinically used sulfonamides, such as acetazolamide, methazolamide, dorzolamide, brinzolamide and benzolamide were low nanomolar inhibitors, with K(I)s in the range of 5.7-67 nM. Dichlorophenamide, zonisamide, saccharin and hydrochlorothiazide were weaker inhibitors, with K(I)s in the range of 513-5390 nM. The inhibition profile with anions and sulfonamides of the seal enzyme was rather different from those of the human isoforms hCA I and II. The high sensitivity to bicarbonate inhibition of lwCA, unlike that of the human enzymes, may reflect an evolutionary adaptation to the deep water, high CO(2) partial pressure and hypoxic conditions in which Weddell seals spend much of their life.     

Carbonic anhydrase inhibitors: Inhibition of the β-class enzyme from the yeast Saccharomyces cerevisiae with sulfonamides and sulfamates

The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a β-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 of sulfonamides and one sulfamate. The enzyme showed high CO₂ hydrase activity, with a k_cat of 9.4 × 10⁵ s^?1, and k_cat/K_M of 9.8 × 10⁷ M^?1 s^?1. Simple benzenesulfonamides substituted in 2-, 4- and 3,4-positions of the benzene ring with amino, alkyl, halogeno and hydroxyalkyl moieties were weak scCA inhibitors with K_Is in the range of 0.976–18.45 μM. Better inhibition (K_Is in the range of 154–654 nM) was observed for benzenesulfonamides incorporating aminoalkyl/carboxyalkyl moieties or halogenosulfanilamides; benzene-1,3-disulfonamides; simple heterocyclic sulfonamides and sulfanilyl-sulfonamides. The clinically used sulfonamides/sulfamate (acetazolamide, ethoxzolamide, methazolamide, dorzolamide, topiramate, celecoxib, etc.) generally showed effective scCA inhibitory activity, with K_Is in the range of 82.6–133 nM. The best inhibitor (K_I of 15.1 nM) was 4-(2-amino-pyrimidin-4-yl)-benzenesulfonamide. These inhibitors may be useful to better understand the physiological role of β-CAs in yeast and some pathogenic fungi which encode orthologues of the yeast enzyme and eventually for designing novel antifungal therapies.     

Anion inhibition studies of a β-carbonic anhydrase from Clostridium perfringens

A β-carbonic anhydrases (CAs, EC 4.2.1.1) was recently cloned, purified and characterized kinetically in the pathogen Clostridium perfringens. We report here the first inhibition study of this enzyme (CpeCA). CpeCA was poorly inhibited by iodide and bromide, and was inhibited with K_Is in the range of 1–10 mM by a range of anions such as (thio)cyanate, azide, bicarbonate, nitrate, nitrite, hydrogensulfite, hydrogensulfide, stannate, tellurate, pyrophosphate, divanadate, tetraborate, peroxydisulfate, sulfate, iminodisulfonate and fluorosulfonate. Better inhibitory power, with K_Is of 0.36–1.0 mM, was observed for cyanide, carbonate, selenate, selenocyanide, trithiocarbonate and diethyldithiocarbamate, whereas the best CpeCA inhibitors were sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, which had K_Is in the range of 7–75 μM. This study thus provides the basis for developing better clostridial enzyme inhibitors with potential as antiinfectives with a new mechanism of action.     

Carbonic anhydrase inhibitors: X-ray crystallographic structure of the adduct of human isozyme II with a bis-sulfonamide-two heads are better than one?

The X-ray crystal structure for the adduct of human carbonic anhydrase II (hCA II) with 4-(4-sulfamoylphenylcarboxamidoethyl)benzenesulfonamide, a topically acting antiglaucoma sulfonamide has been resolved at a resolution of 1.8 A. Its binding to the enzyme is similar with that of other sulfonamides, considering the interactions of the sulfonamide zinc anchoring group, but differs considerably when the organic part of the inhibitor is analyzed. This part of the inhibitor interacts only within the hydrophobic half of the CA active site, leaving the hydrophilic half able to accomodate several water molecules not present in the uncomplexed enzyme. Furthermore, the second head (sulfonamide moiety) participates in two strong hydrogen bonds with amino acid residues (Gly 132 and Gln 136) situated on the rim of the entrance to the active site cleft. Thus, the answer to the question in the title of this paper is that two heads are better than one, since the two sulfamoyl moieties of the inhibitor allow its proper orientation within the active site, with only one head binding in ionized form to the zinc ion, the organic part lying within the hydrophobic half of the active site, and the terminal, carboxamido containing phenylsulfamoyl head participating in strong hydrogen bonds with amino acid residues located at the entrance of it. All these findings are important for the design of better carboxamido CA inhibitors with applications in clinical medicine.     

Sulfonamide inhibition studies of the β carbonic anhydrase from Drosophila melanogaster

An inibition study of the β-carbonic anhydrase (CA, EC 4.2.1.1) DmBCA from the insect Drosophila melanogaster with sulfonamides and sulfamates is reported. Among the panel of 40 investigated compounds, the best DmBCA inhibitors were the sulfonylated benzenesulfonamides and ethoxzolamide, which showed inhibition constants in the range of 65.3–138 nM. Methazolamide and sulthiame were also effective inhibitors with K_Is ranging between 237 and 249 nM, whereas most of the simple aromatic/heterocyclic sulfonamides showed inhibition constants in the range of 0.47–6.40 μM. Topiramate, zonisamide and saccharine did not inhibit DmBCA. As orthologs of this mitochondrial CA are found in many insect species involved in the spread of various diseases, inhibitors interfering with their activity may be of interest for developing insecticides with an alternative mechanism of action to the presently used agents, for which many insects developed extensive resistance.