Ozen Ozensoy Guler

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 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 of sulfonamides and one sulfamate. 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). 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(I)s in the range of 0.976-18.45 microM. Better inhibition (K(I)s 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(I)s 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 beta-CAs in yeast and some pathogenic fungi which encode orthologues of the yeast enzyme and eventually for designing novel antifungal therapies.

Carbonic anhydrase inhibitors. Inhibition of the β-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.

A magnificent enzyme superfamily: carbonic anhydrases, their purification and characterization

Abstract In this paper, we reviewed the purification and characterization methods of the α-carbonic anhydrase (CA, EC 4.2.1.1) class. Six genetic families (α-, β-, γ-, δ-, ζ- and η-CAs) all know to date, all encoding such enzymes in organisms widely distributed over the phylogenetic tree. Starting from the manuscripts published in the 1930s on the isolation and purification of α-CAs from blood and other tissues, and ending with the recent discovery of the last genetic family in protozoa, the η-CAs, considered for long time an α-CA, we present historically the numerous and different procedures which were employed for obtaining these catalysts in pure form. α-CAs possess important application in medicine (as many human α-CA isoforms are drug targets) as well as biotechnological processes, in which the enzymes are ultimately used for CO2 capture in order to mitigate the global warming effects due to greenhouse gases. Recently, it was discovered an involvement of CAs in cancerogenesis as well as infection caused by pathogenic agents such as bacteria, fungi and protozoa. Inhibition studies of CAs identified in the genome of the aforementioned organisms might lead to the discovery of innovative drugs with a novel mechanism of action.

A new affinity gel for the purification of α-carbonic anhdrases

Abstract The new affinity gel reported in this study was prepared using EUPERGIT C250L as a chromatographic bed material, to which etylenediamine spacer arms were attached to prevent steric hindrance between the matrix and ligand, and to facilitate effective binding of the CA-specific ligand, of the aromatic sulfonamide type for the purification of α-carbonic anhydrases (Cas; EC 4.2.1.1). Indeed, the aminoethyl moieties of the affinity gel were derivatized by reaction with 4-isothiocyanatobenzenesulfonamide, with the formation of a thiourea-based gel, having inhibitory effects against CAs. Both bovine erythrocyte carbonic anhydrase BCA and human (h) erythrocyte CA isoforms I, II (hCA I and II) have been purified from hemolysates, by using this affinity gel. The greatest purification fold and column yields for BCA and for cytosolic (hCA I + II) enzymes were of 181-fold (21.07%) and 184-fold (9.49%), respectively. Maximum binding was achieved at 15 °C and I = 0.3 ionic strength for α-carbonic anhydrases.

Synthesis, carbonic anhydrase inhibition and cytotoxic activity of novel chromone-based sulfonamide derivatives

Four series of sulfonamides incorporating chromone moieties were synthesized and assessed for their cytotoxic activity against MCF-7 and A-549 cell lines, considering the fact that some of these tumors overexpress isoforms of carbonic anhydrase (CA, EC 4.2.1.1) which is inhibited by sulfonamides. Most new sulfonamides showed weak inhibitory activity against the offtarget, cytosolic isoforms hCA I, II but effectively inhibited the tumor-associated hCA IX and XII. The most active compounds featured a primary SO2NH2 group and were active in the low micromolar range against MCF-7 and A-549 cell lines. Compound 4a showed IC50 of 0.72 and 0.50 μM against MCF-7 and A-549 cell lines, respectively, and was further evaluated for its proapoptotic activity which proved enhanced in both tumor types.

Carbonic anhydrase activators: Activation of the β-carbonic anhydrase Nce103 from the yeast Saccharomyces cerevisiae with amines and amino acids

The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a beta-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, was investigated for its activation with amines and amino acids. scCA was poorly activated by amino acids such as l-/d-His, Phe, DOPA, Trp (K(A)s of 82-90 microM) and more effectively activated by amines such as histamine, dopamine, serotonin, pyridyl-alkylamines, aminoethyl-piperazine/morpholine (K(A)s of 10.2-21.3 microM). The best activator was l-adrenaline, with an activation constant of 0.95 microM. This study may help to better understand the catalytic/activation mechanisms of the beta-CAs and eventually to design modulators of CA activity for similar enzymes present in pathogenic fungi, such as Candida albicans and Cryptococcus neoformans.

In vitro efficacy of some cattle drugs on bovine serum paraoxonase 1 (PON1) activity

Serum paraoxonase 1 (EC 3.1.8.1, PON1), a calcium-associated enzyme, has an ability to hydrolyze organophosphate compounds. Related to this property, PON1 has a critical role in antioxidant mechanisms. It is well-known that the enzyme protects LDL from oxidation. In this study we investigated the in vitro inhibitory effects of some drugs. These drugs are oxytocin, dexamethasone, atropine sulphate, gentamicin sulphate, sulfadoxine-trimethoprim, furosemid, metamizole sodium and toldimfos sodium. The IC50 values obtained varied markedly from 0.014 to 507.72 mg/mL. According to our findings, most potent and significant inhibition was displayed by dexamethasone, atropine sulphate and furosemid.

Inhibition of carbonic anhydrase isoforms I, II, IX and XII with secondary sulfonamides incorporating benzothiazole scaffolds

Abstract Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the fundamental reaction of CO2 hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological conditions. A series of benzothiazole-based sulfonamides were synthesized and tested as possible CA inhibitors. Their inhibitory activity was assessed against the cytosolic human isoforms hCA I and hCA II and the transmembrane hCA IX and hCA XII. Several of the investigated derivatives showed interesting inhibition activity and selectivities for inhibiting hCA IX and hCA XII over the off-target ones hCA I and hCA II. Furthermore, computational procedures were used to investigate the binding mode of this class of compounds, within the active site of hCA IX.

Saccharomyces cerevisiae β-Carbonic Anhydrase: Inhibition and Activation Studies

The β-carbonic anhydrase from Saccharomyces cerevisiae (CA, EC 4.2.1.1), scCA, which is encoded by the Nce103 gene, is an effective catalyst for CO(2) hydration to bicarbonate and protons, with a k(cat) of 9.4 x 10(5) s(-1), and k(cat)/K(M) of 9.8 x 10(7) M(-1).s(-1). Its inhibition with anions and sulfonamides has been investigated, as well as its activation with amines and amino acids. Bromide, iodide and sulfamide, were the best anion inhibitors, with K(I)s of 8.7 - 10.8 µM. Benzenesulfonamides substituted in 2-, 4- and 3,4-positions with amino, alkyl, halogeno and hydroxyalkyl moieties had K(I)s in the range of 0.976 - 18.45 µM. Better inhibition (K(I)s 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(I)s 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. L-adrenaline and some piperazines incorporating aminoethyl moieties were the most effective scCA activators. These studies may lead to a better understanding of the role of this enzyme in yeasts/fungi, and since the Nce103 gene is also present in many pathogenic organisms (Candida spp., Cryptococcus neoformans, etc) they may be useful to develop antifungal drugs.

Mutation of Phe91 to Asn in human carbonic anhydrase I unexpectedly enhanced both catalytic activity and affinity for sulfonamide inhibitors.

Site-directed mutagenesis has been used to change one amino acid residue considered non essential (Phe91Asn) to catalysis in carbonic anhydrase (CA, EC 4.2.1.1) isozyme I (hCA I), but which is near the substrate binding pocket of the enzyme. This change led to a steady increase of 16% of the catalytic activity of the mutant hCA I over the wild type enzyme, which is a gain of 50% catalytic efficiency if one compares hCA I and hCA II as catalysts for CO(2) hydration. This effect may be due to the bigger hydrophobic pocket in the mutant enzyme compared to the wild type one, which probably leads to the reorganization of the solvent molecules present in the cavity and to a diverse proton transfer pathway in the mutant over the non mutated enzyme. To our surprise, the mutant CA I was not only a better catalyst for the physiologic reaction, but in many cases also showed higher affinity (2.6-15.9 times) for sulfonamide/sulfamate inhibitors compared to the wild type enzyme. As the residue in position 91 is highly variable among the 13 catalytically active CA isoforms, this study may shed a better understanding of catalysis/inhibition by this superfamily of enzymes.