Carbonic anhydrase inhibitors. Inhibition of the fungal β-carbonic anhydrases from Candida albicans and Cryptococcus neoformans with boronic acids

Inhibition of the β-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Cryptococcus neoformans (Can2) and Candida albicans (Nce103) with a series of aromatic, arylalkenyl- and arylalkylboronic acids was investigated. Aromatic, 4-phenylsubstituted- and 2-naphthylboronic acids were the best Can2 inhibitors, with inhibition constants in the range of 8.5–11.5 μM, whereas arylalkenyl and aryalkylboronic acids showed K_Is in the range of 428–3040 μM. Nce103 showed a similar inhibition profile, with the 4-phenylsubstituted- and 2-naphthylboronic acids possessing K_Is in the range of 7.8–42.3 μM, whereas the arylalkenyl and aryalkylboronic acids were weaker inhibitors (K_Is of 412–5210 μM). The host human enzymes CA I and II were also effectively inhibited by these boronic acids. The B(OH)₂ moiety is thus a new zinc-binding group for designing effective inhibitors of the α- and β-CAs.     

Carbonic anhydrase inhibitors. Inhibition of the β-class enzymes from the fungal pathogens Candida albicans and Cryptococcus neoformans with aliphatic and aromatic carboxylates

The inhibition of the β-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Cryptococcus neoformans (Can2) and Candida albicans (Nce103) with carboxylates such as the C1–C5 aliphatic carboxylates, oxalate, malonate, maleate, malate, pyruvate, lactate, citrate and some benzoates has been investigated. The best Can2 inhibitors were acetate and maleate (K_Is of 7.3–8.7 μM), whereas formate, acetate, valerate, oxalate, maleate, citrate and 2,3,5,6-tetrafluorobenzoate showed less effective inhibition, with K_Is in the range of 42.8–88.6 μM. Propionate, butyrate, malonate, l-malate, pyruvate, l-lactate and benzoate, were weak Can2 inhibitors, with inhibition constants in the range of 225–1267 μM. Nce103 was more susceptible to inhibition with carboxylates compared to Can2, with the best inhibitors (maleate, benzoate, butyrate and malonate) showing K_Is in the range of 8.6–26.9 μM. l-Malate and pyruvate together with valerate were the less efficient Nce103 inhibitors (K_Is of 87.7–94.0 μM), while the remaining carboxylates showed a compact behavior of efficient inhibitors (K_Is in the range of 35.1–61.6 μM). Notably the inhibition profiles of the two fungal β-CAs was very different from that of the ubiquitous host enzyme hCA II (belonging to the α-CA family), with maleate showing selectivity ratios of 113.6 and 115 for Can2 and Nce103, respectively, over hCA II inhibition. Therefore, maleate is a promising starting lead molecule for the development of better, low nanomolar, selective β-CA inhibitors.     

Carbonic anhydrase inhibitors. Inhibition of the β-class enzymes from the fungal pathogens Candida albicans and Cryptococcus neoformans with branched aliphatic/aromatic carboxylates and their derivatives

The inhibition of the β-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Cryptococcus neoformans (Can2) and Candida albicans (Nce103) with a series of 25 branched aliphatic and aromatic carboxylates has been investigated. Human isoforms hCA I and II were also included in the study for comparison. Aliphatic carboxylates were generally millimolar hCA I and II inhibitors and low micromolar/submicromolar β-CA inhibitors. Aromatic carboxylates were micromolar inhibitors of the four enzymes but some of them showed low nanomolar activity against the fungal pathogenic enzymes. 4-Hydroxy- and 4-methoxy-benzoate inhibited Can2 with K_Is of 9.5-9.9 nM. The methyl esters, hydroxamates, hydrazides and carboxamides of some of these derivatives were also effective inhibitors of the α- and β-CAs investigated here.     

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).     

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.     

Carbonic anhydrase inhibitors. Characterization and inhibition studies of the most active β-carbonic anhydrase from Mycobacterium tuberculosis,Rv3588c

The Rv3588c gene product of Mycobacterium tuberculosis, a β-carbonic anhydrase (CA, EC 4.2.1.1) denominated here mtCA 2, shows the highest catalytic activity for CO₂ hydration (k_cat of 9.8 × 10⁵ s^?1, and k_cat/K_m of 9.3 × 10⁷ M^?1 s^?1) among the three β-CAs encoded in the genome of this pathogen. A series of sulfonamides/sulfamates was assayed for their interaction with mtCA 2, and some diazenylbenzenesulfonamides were synthesized from sulfanilamide/metanilamide by diazotization followed by coupling with amines or phenols. Several low nanomolar mtCA 2 inhibitors have been detected among which acetazolamide, ethoxzolamide and some 4-diazenylbenzenesulfonamides (K_Is of 9–59 nM). As the Rv3588c gene was shown to be essential to the growth of M. tuberculosis, inhibition of this enzyme may be relevant for the design of antituberculosis drugs possessing a novel 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 studies of the β-carbonic anhydrases from the bacterial pathogen Salmonella enterica serovar Typhimurium with sulfonamides and sulfamates

The two β-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Salmonella enterica serovar Typhimurium, stCA 1 and stCA 2, were investigated for their inhibition with a large panel of sulfonamides and sulfamates. Unlike inorganic anions, which are weak, millimolar inhibitors of the two enzymes [Vullo et al., Bioorg. Med. Chem. Lett.2011, 21, 3591], sulfonamides and sulfamates are effective micro-to nanomolar inhibitors of the two enzymes. Various types of inhibitors have been detected among the 38 investigated sulfonamides/sulfamates, with K(I)s in the range of 31 nM-5.87 μM. The best stCA 1 inhibitors were acetazolamide and benzolamide-based compounds, whereas the best stCA 2 inhibitors were sulfonylated benzenesulfonamides and amino-benzolamide derivatives (K(I)s in the range of 31-90 nM). 3-Fluoro-5-chloro-4-aminobenzolamide showed an inhibition constant of 51 nM against stCA 1 and of 38 nM against stCA 2, being the best inhibitor detected so far for these enzymes. As many strains of S. enterica show extensive resistance to classical antibiotics, inhibition of the β-CAs investigated here may be useful for developing novel antibacterials, targeting β-CAs which may be involved in pathogenicity and invasion of some bacteria.     

Inhibition of the β-carbonic anhydrase from the dandruff-producing fungus Malassezia globosa with monothiocarbamates

A series of monothiocarbamates (MTCs) was investigated for the inhibition of the β-class carbonic anhydrase (CAs, EC 4.2.1.1) from the fungal parasite Malassezia globosa, MgCA. These MTCs incorporate various scaffolds, among which aliphatic amine with 1–4 carbons atom in their molecule, morpholine, piperazine, as well as phenethylamine and benzylamine derivatives. All the reported MTCs displayed a better efficacy in inhibiting MgCA compared to the clinically used sulphonamide drug acetazolamide (K_I of 74?μM), with K_Is spanning between 1.85 and 18.9?μM. The homology model of the enzyme previously reported by us was used to rationalize the results by docking some of these MTCs within the fungal CA active site. This study might be useful to enrich the knowledge of the MgCA inhibition profile, eliciting novel ideas pertaining the design of modulators with potential efficacy in combatting dandruff or other fungal infections.     

Carbonic anhydrase inhibitors. Inhibition of Plasmodium falciparum carbonic anhydrase with aromatic sulfonamides: towards antimalarials with a novel mechanism of action?

The malarial parasite Plasmodium falciparum encodes for an alpha-carbonic anhydrase (CA) enzyme possessing catalytic properties distinct of that of the human host, which was only recently purified. A series of aromatic sulfonamides, most of which were Schiff's bases derived from sulfanilamide/homosulfanilamide/4-aminoethylbenzenesulfonamide and substituted-aromatic aldehydes, or ureido-substituted such sulfonamides, were investigated for in vitro inhibition of the malarial parasite enzyme (pfCA) and the growth of P. falciparum. Several inhibitors with affinity in the micromolar range (K(I)'s in the range of 0.080-1.230 microM) were detected, whereas the most potent such derivatives were the clinically used sulfonamide CA inhibitor acetazolamide, and 4-(3,4-dichlorophenyl-ureidoethyl)-benzenesulfonamide, which showed an inhibition constant of 80 nM against pfCA, being four times more effective an inhibitor as compared to acetazolamide (K(I) of 315 nM). The lipophilic 4-(3,4-dichlorophenylureido-ethyl)-benzenesulfonamide was also an effective in vitro inhibitor for the growth of P. falciparum (IC50 of 2 microM), whereas acetazolamide achieved the same level of inhibition at 20 microM. This is the first study proving that antimalarials possessing a novel mechanism of action can be obtained, by inhibiting a critical enzyme for the life cycle of the parasite. Indeed, by inhibiting pfCA, the synthesis of pyrimidines mediated by carbamoylphosphate synthase is impaired in P. falciparum but not in the human host. Sulfonamide CA inhibitors have the potential for the development of novel antimalarial drugs.     

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. Benzenesulfonamides incorporating cyanoacrylamide moieties strongly inhibit Saccharomyces cerevisiae β-carbonic anhydrase

A series of benzenesulfonamides incorporating cyanoacrylamide moieties (tyrphostine analogs) were assayed as inhibitors of the β-carbonic anhydrase (CA, EC 4.2.1.1) from Saccharomyces cerevisiae, ScCA. Some of these compounds were low nanomolar or subnanomolar ScCA inhibitors and showed selectivity ratios in the range of 4.91–69.86 for inhibiting the yeast enzyme over the offtarget human (h) isoforms hCA I and of 6.46–13.52 for inhibiting ScCA over hCA II. The model organism S. cerevisiae and this particular enzyme may be useful for detecting antifungals with a novel mechanism of action compared to the classical azole drugs to which significant drug resistance emerged. Indeed, some of these sulfonamides inhibited the growth of the yeast with CC₅₀-s in the range of 0.73–6.54 μM.     

Carbonic anhydrase inhibitors. Inhibition of the transmembrane isozyme XII with sulfonamides—a new target for the design of antitumor and antiglaucoma drugs?

The inhibition of a newly cloned human carbonic anhydrase (CA, EC 4.2.1.1), isozyme XII (hCA XII), 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 drug 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 several medium potency inhibitors (K(I)s in the range of 34-220 nM), whereas ethoxzolamide and several halogenated sulfanilamides showed stronger potency, with K(I)s in the range of 11-22 nM. The antiglaucoma sulfonamides used clinically, except dichlorophenamide, which is a moderate inhibitor (K(I) of 50 nM), as well as topiramate, indisulam, and sulpiride behave as very potent hCA XII inhibitors, with K(I)s in the range of 3.0-5.7 nM. Several subnanomolar inhibitors (K(I)s in the range of 0.30-0.85 nM) have also been detected. Compounds with excellent selectivity against hCA XII over hCA II have been found, showing selectivity ratios in the range of 177.7-566.7. Apparently, hCA XII is a target of the antiglaucoma sulfonamides, and potent hCA XII inhibitors may be developed/used for the management of hypoxic tumors, together with inhibitors of the other tumor-associated isozyme, CA IX.     

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.     

Sulfonamide inhibition studies of the β-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae

The genome of the pathogenic bacterium Vibrio cholerae encodes for three carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α-, β- and γ-classes. VchCA, the α-CA from this species was investigated earlier, whereas the β-class enzyme, VchCAβ was recently cloned, characterized kinetically and its X-ray crystal structure reported by this group. Here we report an inhibition study with sulfonamides and one sulfamate of this enzyme. The best VchCAβ inhibitors were deacetylated acetazolamide and methazolamide and hydrochlorothiazide, which showed inhibition constants of 68.2–87.0 nM. Other compounds, with medium potency against VchCAβ, (K_Is in the range of 275–463 nM), were sulfanilamide, metanilamide, sulthiame and saccharin whereas the clinically used agents such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, zonisamide and celecoxib were micromolar inhibitors (K_Is in the range of 4.51–8.57 μM). Identification of potent and possibly selective inhibitors of VchCA and VchCAβ over the human CA isoforms, may lead to pharmacological tools useful for understanding the physiological role(s) of this under-investigated enzymes.     

Sulphonamide inhibition studies of the β-carbonic anhydrase from the bacterial pathogen Clostridium perfringens

The β-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacterium Clostridium perfringens (CpeCA) was recently characterised kinetically and for its anion inhibition profile. In the search of effective CpeCA inhibitors, possibly useful to inhibit the growth/pathogenicity of this bacterium, we report here an inhibition study of this enzyme with a panel of aromatic, heterocyclic and sugar sulphonamides/sulphamates. Some sulphonamides, such as acetazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, sulthiame and 4-(2-hydroxymethyl-4-nitrophenyl-sulphonamido)ethylbenzenesulphonamide were effective CpeCA inhibitors, with K_Is in the range of 37.4–71.6?nM. Zonisamide and saccharin were the least effective such inhibitors, whereas many other aromatic and heterocyclic sulphonamides were moderate – weak inhibitors with K_Is ranging between 113 and 8755?nM. Thus, this study provides the basis for developing better clostridial enzyme inhibitors with potential as antiinfectives with a new mechanism of action.     

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.     

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.