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.     

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.     

Sulfonamide inhibition studies of the δ-carbonic anhydrase from the diatom Thalassiosira weissflogii

The δ-carbonic anhydrase (CA, EC 4.2.1.1) TweCA from the marine diatom Thalassiosira weissflogii has recently been cloned, purified and its activity/inhibition with anions investigated. Here we report the first sulfonamide/sulfamate inhibition study of a δ-class CA. Among the 40 such compounds investigated so far, 3-bromosulfanilamide, acetazolamide, ethoxzolamide, dorzolamide and brinzolamide were the most effective TweCA inhibitors detected, with K_Is of 49.6–118 nM. Many simple aromatic sulfonamides as well as dichlorophenamide, benzolamide, topiramate, zonisamide, indisulam and valdecoxib were medium potency inhibitors, (K_Is of 375–897 nM). Saccharin and hydrochlorothiazide were ineffective inhibitors of the δ-class enzyme, with K_Is of 4.27–9.20 μM. The inhibition profile of the δ-CA is very different from that of α-, β- and γ-CAs from different organisms. Although no X-ray crystal structure of this enzyme is available, we hypothesize that as for other CA classes, the sulfonamides inhibit the enzymatic activity by binding to the Zn(II) ion from the δ-CA active site.     

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.     

Anion inhibition studies of the α-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae

An α-carbonic anhydrase (CA, EC 4.2.1.1) has been recently cloned and characterized in the human pathogenic bacterium Vibrio cholerae, denominated VchCA (Del Prete et al. J. Med. Chem. 2012, 55, 10742). This enzyme shows a good catalytic activity for the CO₂ hydration reaction, comparable to that of the human (h) isoform hCA I. Many inorganic anions and several small molecules were investigated as VchCA inhibitors. Inorganic anions such as cyanate, cyanide, hydrogen sulfide, hydrogen sulfite, and trithiocarbonate were effective VchCA inhibitors with inhibition constants in the range of 33–88 μM. Other effective inhibitors were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, with K_Is of 7–43 μM. Halides (bromide, iodide), bicarbonate and carbonate were much less effective VchCA inhibitors, with K_Is in the range of 4.64–28.0 mM. The resistance of VchCA to bicarbonate inhibition may represent an evolutionary adaptation of this enzyme to living in an environment rich in this ion, such as the gastrointestinal tract, as bicarbonate is a virulence enhancer of this bacterium.     

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.     

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.     

Biochemical characterization of the δ-carbonic anhydrase from the marine diatom Thalassiosira weissflogii,TweCA

Abstract

Diatom genome sequences clearly reveal the presence of different systems for HCO₃^? uptake. Carbon-concentrating mechanisms (CCM) based on HCO₃^? transport and a plastid-localized carbonic anhydrase (CA, EC 4.2.1.1) appear to be more probable than the others because CAs have been identified in the genome of many diatoms. CAs are key enzymes involved in the acquisition of inorganic carbon for photosynthesis in phytoplankton, as they catalyze efficiently the interconversion between carbon dioxide and bicarbonate. Five genetically distinct classes of CAs exist, α-, β-, γ-, δ- and ζ and all of them are metalloenzymes. Recently we investigated for the first time the catalytic activity and inhibition of the δ-class CA from the marine diatom Thalassiosira weissflogii, named TweCA. This enzyme is an efficient catalyst for the CO₂ hydration and its inhibition profile with sulfonamide/sulfamate and anions have also been investigated. Here, we report the detailed biochemical characterization and chemico-physical properties of the δ-CA of T. weissflogii. The δ-CA encoding gene was cloned and expressed in Artic Express cells and the recombinant protein purified to homogeneity. Interesting to note that TweCA has no intrinsic esterase activity with 4-nitrophenyl acetate (pNpA) as substrate although the phylogenetic analysis showed that δ-CAs are closer to the α-CAs than to the other classes of such enzymes.     

Anion inhibition profiles of the complete domain of the η-carbonic anhydrase from Plasmodium falciparum

We have cloned, purified and investigated the catalytic activity and anion inhibition profiles of a full catalytic domain (358 amino acid residues) carbonic anhydrase (CA, EC 4.2.1.1) from Plasmodium falciparum, PfCAdom, an enzyme belonging to the η-CA class and identified in the genome of the malaria-producing protozoa. A truncated such enzyme, PfCA1, containing 235 residues was investigated earlier for its catalytic and inhibition profiles. The two enzymes were efficient catalysts for CO₂ hydration: PfCAdom showed a k_cat of 3.8 × 10⁵ s⁻¹ and k_cat/K_m of 7.2 × 10⁷ M⁻¹ × s⁻¹, whereas PfCA showed a lower activity compared to PfCAdom, with a k_cat of 1.4 × 10⁵ s⁻¹ and k_cat/K_m of 5.4 × 10⁶ M⁻¹ × s⁻¹. PfCAdom was generally less inhibited by most anions and small molecules compared to PfCA1. The best PfCAdom inhibitors were sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid, which showed K_Is in the range of 9–68 μM, followed by bicarbonate, hydrogensulfide, stannate and N,N-diethyldithiocarbamate, which were submillimolar inhibitors, with K_Is in the range of 0.53–0.97 mM. Malaria parasites CA inhibition was proposed as a new strategy to develop antimalarial drugs, with a novel mechanism of action.     

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.     

Tritium NMR studies of the human carbonic anhydrase I–benzenesulfonamide complex

Tritium NMR spectroscopy has been used to examine the complexformed by [4-³H]benzenesulfon-amide and human carbonicanhydrase I. The results show that in solution the inhibitor forms a 1:1complex with the enzyme. A 100-spin computational model of the system,constructed with reference to crystallographic results, was used tointerpret tritium relaxation behavior and ³H{¹H}NOEs. The analysis shows that the rate of dissociation of theenzyme–sulfonamide complex is 0.35 s^–1 and thatthe aromatic ring of the inhibitor undergoes rapid rotation while complexed.     

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.     

Biochemical characterization of the native α-carbonic anhydrase purified from the mantle of the Mediterranean mussel,Mytilus galloprovincialis

A α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified and characterized biochemically from the mollusk Mytilus galloprovincialis. As in most mollusks, this α-CA is involved in the biomineralization processes leading to the precipitation of calcium carbonate in the mussel shell. The new enzyme had a molecular weight of 50?kDa, which is roughly two times higher than that of a monomeric α-class enzyme. Thus, Mytilus galloprovincialis α-CA is either a dimer, or similar to the Tridacna gigas CA described earlier, may have two different CA domains in its polypeptide chain. The Mytilus galloprovincialis α-CA sequence contained the three His residues acting as zinc ligands and the gate-keeper residues present in all α-CAs (Glu106-Thr199), but had a Lys in position 64 and not a His as proton shuttling residue, being thus similar to the human isoform hCA III. This probably explains the relatively low catalytic activity of Mytilus galloprovincialis α-CA, with the following kinetic parameters for the CO₂ hydration reaction: k_cat =?4.1?×?10⁵ s^?1 and k_cat/K_m of 3.6?×?10⁷ M^?1 × s^?1. The enzyme activity was poorly inhibited by the sulfonamide acetazolamide, with a K_I of 380?nM. This study is one of the few describing in detail the biochemical characterization of a molluskan CA and may be useful for understanding in detail the phylogeny of these enzymes, their role in biocalcification processes and their potential use in the biomimetic capture of the CO₂.     

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.     

Cloning,expression and purification of the complete domain of the η-carbonic anhydrase from Plasmodium falciparum

Abstract

The antimalarial drugs are of fundamental importance in the control of malaria, especially for the lack of efficient treatments and acquired resistance to the existing drugs. For this reason, there is a continuous work in identifying novel, less toxic and effective chemotherapies as well as new therapeutic targets against the causative agents of malaria. In this context, a superfamily of metalloenzymes named carbonic anhydrases (CAs, EC 4.2.1.1) has aroused a great interest as druggable enzymes to limit the development of Plasmodium falciparum gametocytes. CAs catalyze a common reaction in all life domains, the carbon dioxide hydration to bicarbonate and protons (CO₂?+?H₂O ? HCO₃^-?+?H⁺). P. falciparum synthesizes pyrimidines de novo starting from HCO₃^-, which is generated from CO₂ through the action of the η-CA identified in the genome of the protozoan. Here, we propose a procedure for the preparation of a wider portion of the protozoan η-CA, named PfCAdom (358 amino acid residues), with respect to the truncated form prepared by Krungkrai et al. (PfCA1, 235 amino acid residues). The results evidenced that the recombinant PfCAdom, produced as a His-tag fusion protein, was 2.7 times more active with respect the truncated form PfCA1.     

Cloning,expression and purification of the α-carbonic anhydrase from the mantle of the Mediterranean mussel,Mytilus galloprovincialis

We cloned, expressed, purified, and determined the kinetic constants of the recombinant α-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the α-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32?kDa. Moreover, the determined kinetic parameters for the CO₂ hydration reaction were k_cat?=??4.2?×?10⁵?s^?1 and k_cat/K_m of 3.5?×?10⁷?M^?1 ×s^?1. Curiously, the rec-MgaCA showed a very similar kinetic and acetazolamide inhibition features when compared to those of the native enzyme (MgaCA), which has a molecular weight of 50?kDa. Analysing the SDS-PAGE, the protonography, and the kinetic analysis performed on the native and recombinant enzyme, we hypothesised that probably the native MgaCA is a multidomain protein with a single CA domain at the N-terminus of the protein. This hypothesis is corroborated by the existence in mollusks of multidomain proteins with a hydratase activity. Among these proteins, nacrein is an example of α-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.     

Anion inhibition studies of an α-carbonic anhydrase from the living fossil Astrosclera willeyana

An α-carbonic anhydrase (CA, EC 4.2.1.1) isolated from the living fossil sponge Astrosclera willeyana, Astrosclerin, was investigated for its inhibition profile with simple inorganic anions, complex anions and other small molecules known to interact with these zinc enzymes. Astrosclerin is a catalytically highly efficient enzyme, and is inhibited in the low micromolar range by sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid, and in the submillimolar range by a variety of anions including fluoride, chloride, cyanate, thiocyanate, cyanide, hydrogen sulfide, bisulfate, stannate, perosmate, divanadate, perrhenate, perruthenate, selenocyanide, trithiocarbonate, diethyldithiocarbamate and iminodisulfonate. Less efficient Astrosclerin inhibitors were sulfate, bromide, iodide, azide, bicarbonate, carbonate, tetraborate and perchlorate (K(I)s of 5.11-30.6mM) whereas tetrafluoroborate was not at all inhibitory. Because Astrosclerin is involved in calcification processes in vivo, its anion inhibition profile may be important for future studies designed to shed light on the physiologic functions of α-CAs in marine organisms.     

DNA polymorphism in the 5′ flanking region of the human carbonic anhydrase II gene on chromosome 8

Summary A restriction-fragment-length polymorphism (RFLP) is described which is associated with the human carbonic anhydrase II gene (CA2) that codes for one of the three genetically distinct carbonic anhydrase isozymes, CA I, CA II, and CA III. The isolated DNA was cleaved with several restriction enzymes and subjected to Southern blot hybridization analysis using a DNA probe containing the 5 end of the human CA II gene. A two allele RFLP which was detected with the restriction endonuclease, Taq I, is expressed phenotypically on Southern blots as either a 5.4 kilobase (kb) fragment or as 4.0 and 1.4 kb fragments. These fragments result from the presence or absence of a Taq I recognition site in the 5 flanking region approximately 1.0kb from the initiation codon of the CA II gene. Segregation analysis showed that the alleles are inherited in a Mendelian fashion, with a frequency of 50%.     

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).     

An α-carbonic anhydrase from the thermophilic bacterium Sulphurihydrogenibium azorense is the fastest enzyme known for the CO2 hydration reaction

We report the biochemical characterization of a new carbonic anhydrase (CA, EC 4.2.1.1), named SazCA, identified by translated genome inspection in Sulfurihydrogenibium azorense, a thermophilic bacterium from terrestrial hot springs of the Azores. SazCA is an α-CA showing kinetic parameters that make it the fastest enzyme of the CA family described so far. The biochemical properties, thermostability and inhibition of SazCA were compared with those of the thermophilic and mesophilic counterparts, demonstrating the special features of this unique enzyme.