[Show abstract][Hide abstract] ABSTRACT: A series of novel 5-substituted 2,4-dichlorobenzenesulfonamides 5a-c, 6a-d, 7a-j and 10a-i have been synthesized and investigated as inhibitors of four isoforms of zinc enzyme carbonic anhydrase (CA.EC 184.108.40.206), that is the cytosolic CA I and II, and tumor-associated isozymes CA IX and XII. Against the human CA I investigated compounds displayed KI values from 349 to 7355 nM, toward hCA II at range of 6.9 to 164 nM, while against hCA IX ranging from 2.8 to 76 nM and against hCA XII in the range of 2.7 to 95 nM. The excellent inhibitory activity against tumor-associated hCA IX was found. The twenty one new compounds displayed a powerful inhibitory potency toward hCA IX (KI = 2.8-21.7 nM) in comparison with the clinically used CAIs AAZ, MZA, EZA, DCP and IND (24-50 nM). Among them the most potent hCA IX inhibitor 7b (KI = 2.8 nM) was 8.5-fold stronger than IND (KI = 24 nM). Toward tumor-associated hCA XII compounds 6c and 10a (KI = 2.7 and 2.8 nM, respectively) showed a better inhibitory potency than reference sulfonamides MZA and IND (KI = 3.4 nM).
European journal of medicinal chemistry. 05/2014; 82C:47-55.
[Show abstract][Hide abstract] ABSTRACT: Carbonic anhydrases (CAs) are metalloenzymes catalyzing the reversible hydration of carbon dioxide to bicarbonate (hydrogen carbonate) and protons. CAs have been identified in archaea, bacteria and eukaryotes and can be classified into five groups (α, β, γ, δ, ζ) that are unrelated in sequence and structure. The fungal β-class has only recently attracted attention. In this study, we investigated the structure and function of the plant-like β-CA proteins CAS1 and CAS2 from the filamentous ascomycete Sordaria macrospora. We demonstrated that both proteins can substitute for the Saccharomyces cerevisiae β-CA Nce103 and exhibit an in vitro CO2 hydration activity (kcat /Km of CAS1: 1.30 × 10(6) M(-1) s(-1) ; CAS2: 1.21 × 10(6 ) M(-1) s(-1) ). To further investigate the structural properties of CAS1 and CAS2 we determined their crystal structures to a resolution of 2.7 Å and 1.8 Å, respectively. The oligomeric state of both proteins is tetrameric. With exception of the active site composition, no further major differences have been found. In both enzymes the Zn(2+) -ion is tetrahedrally coordinated, in CAS1 by Cys45, His101 and Cys104 and a water molecule and in CAS2 by the side chains of four residues (Cys56, His112, Cys115 and Asp58). Both CAs are only weakly inhibited by anions making them good candidates for industrial applications. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: A series of benzene sulfonamides incorporating 1,3,5-triazinyl moieties were synthesized using cyanuric chloride as starting material. Inhibition studies against human carbonic anhydrase (hCA, EC 220.127.116.11) isoforms I, II (cytosolic) and IX, XII (transmembrane, tumor-associated) isoforms were performed with the new compounds. hCA I was modestly inhibited (KIs in the range of 87nM-4.35μM), hCA II was moderately inhibited by most of the new compounds (KIs in the range of 12.5-130nM), whereas the tumor associated isoforms were potently inhibited, with KIs in the range of 1.2-34.1nM against hCA IX and of 2.1-33.9 against hCA XII, respectively. Docking studies of some of the new compounds showed an effective binding mode within the enzyme active site, as demonstrated earlier by X-ray crystallography for structurally-related sulfonamides incorporating 1,3,5-triazinyl functionalities.
[Show abstract][Hide abstract] ABSTRACT: Abstract Human carbonic anhydrase XII (CA XII) is a single-pass transmembrane protein with an extracellular catalytic domain. This enzyme is being recognized as a potential biomarker for different tumours. The current study was aimed to generate monoclonal antibodies (MAbs) neutralizing the enzymatic activity of CA XII. Bioinformatics analysis of CA XII structure revealed surface-exposed sequences located in a proximity of its catalytic centre. Two MAbs against the selected antigenic peptide spanning 167-180 aa sequence of CA XII were generated. The MAbs were reactive with recombinant catalytic domain of CA XII expressed either in E. coli or mammalian cells. Inhibitory activity of the MAbs was demonstrated by a stopped flow CO2 hydration assay. The study provides new data on the surface-exposed linear CA XII epitope that may serve as a target for inhibitory antibodies with a potential immunotherapeutic application.
Journal of Enzyme Inhibition and Medicinal Chemistry 01/2014; · 1.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 1. Among synthesized N4-substituted 4-(2-aminoethyl)benzenesulfonamides the most potent hCAIX and XII inhibitors possess cationic character. 2. The most promising inhibitors presented significant affinity to tumor associated hCAIX and hCAXII than to physiologically dominat hCAI and hCAII.
European Journal of Medicinal Chemistry. 01/2014; 84:59–67.
[Show abstract][Hide abstract] ABSTRACT: A series of novel 5-substituted 2,4-dichlorobenzenesulfonamides have been synthesized. Some of them showed good carbonic anhydrase CA I, II and XII inhibitory efficiency. The twenty one new compounds displayed a powerful inhibitory potency toward hCA IX.
European Journal of Medicinal Chemistry. 01/2014; 82:47–55.
[Show abstract][Hide abstract] ABSTRACT: A series of novel N-substituted N'-(2-arylmethylthio-4-chloro-5-methylbenzenesulfonyl)guanidines 9-41 have been synthesized and investigated as inhibitors of four isoforms of zinc enzyme carbonic anhydrase (CA.EC 18.104.22.168), that is the cytosolic CA I and II, and cancer-associated isozymes CA IX and XII. Against the human CA I investigated compounds showed KI in the range of 87-6506 nM, toward hCA II ranging from 7.8 to 4500 nM, against hCA IX in the range of 4.7-416 nM and against hCA XII at range of 0.96-540 nM. Compounds 10, 12-14, 16, 18-20, 24-26, 31 and 32 exhibited a powerful inhibitory potency toward hCA IX (KI = 4.7-21 nM) in comparison to the reference sulfonamides AAZ, MZA, EZA, DCP and IND (KI = 24-50 nM). Compound 14 was the most potent inhibitor of hCA I (KI = 87 nM), hCA IX (KI = 4.7 nM) and hCA XII (KI = 0.96 nM), while 26 was the most effective inhibitor of hCA II (KI = 7.8 nM). The most promising compound 32 exerted the highest selectivity ratios toward hCA IX versus hCA I (hCA I/hCA IX = 261) and hCA II (hCA II/hCA IX = 26). The in vitro antitumor activity of compounds 10, 13, 14, 21, 22, 25, 32, 38 and 41 was evaluated at the US National Cancer Institute (NCI) against a panel of 60 human tumor cell lines. The most active antitumor agents 21 and 25, inhibiting 32-35 human tumor cell lines with GI50 in the range of 2.1-5.0 μM also showed relatively high inhibitory activity toward hCA IX and XII with KI from 18 to 40 nM.
European journal of medicinal chemistry 11/2013; 71C:135-147. · 3.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Among the different mammalian isoforms of Carbonic Anhydrase, the hCA VII is mainly expressed in the brain where it is involved in several neurological diseases. Thereby hCA VII has been validated as an attractive target for the discovery of selective inhibitors for the treatment of epilepsy and neurological pain. To identify new chemical entities as carbonic anhydrase inhibitors (CAIs) targeting hCA VII, we used a structure-based approach. By means of LigandScout software we built pharmacophore models from crystal structures of two well-known CAIs in complex with hCA VII. A merged pharmacophore hypothesis has been obtained. Subsequently, a focused library of compounds was screened against pharmacophore model and the most interesting hits were docked into the crystal structure of hCA VII. As a result, we identified new compounds displaying significant CA inhibitory effects in the nanomolar range.
European journal of medicinal chemistry 11/2013; 71C:105-111. · 3.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A series of novel heterocyclic 4-substituted pyridine-3-sulfonamides 2-13, 15-20 have been synthesized and investigated as inhibitors of four isoforms of zinc enzyme carbonic anhydrase (CA.EC 22.214.171.124), that is the cytosolic CA I and II, and tumor-associated isozymes CA IX and XII. Against the human isozymes hCA I the new compounds showed KI values in the range 169-5400 nM, toward hCA II in range 58.5-1238 nM, against hCA IX in range 19.5-652 nM and against hCA XII in the range of 16.8-768 nM. Compounds 15-19 representing 4-(1H-pyrazol-1-yl)-3-pyridinesulfonamide derivatives showed good hCA IX inhibitory efficacy with KI = 19.5-48.6 nM comparable or more effective than clinically used sulfonamides: AAZ, MZA, EZA, DCP, IND (KI = 24-50 nM). Anticancer evaluation at a single dose 10 μM, against a panel of 60 human tumor cell lines, was performed at the US National Cancer Institute, on compounds 2, 3, 5-13, 16, 17, 19, 20. Among them 6 bearing 4-(3,4,-dichlorophenyl)piperazine moiety showed broad spectrum of growth inhibition in the range 25-89% over 26 cell lines representing all tumors subpanels.
European journal of medicinal chemistry 09/2013; 69C:701-710. · 3.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A series of benzenesulfonamides incorporating cyanoacrylamide moieties (tyrphostine analogs) were assayed as inhibitors of the β-carbonic anhydrase (CA, EC 126.96.36.199) 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 0.73-6.54μM.
[Show abstract][Hide abstract] ABSTRACT: A series of novel sulfamides incorporating the dopamine scaffold were synthesized. Reaction of amines and tert-butyl-alcohol/benzyl alcohol in the presence of chlorosulfonyl isocyanate (CSI) afforded sulfamoyl carbamates, which were converted to the title compounds by treatment with trifluoroacetic acid or by palladium-catalyzed hydrogenolysis. Inhibition of six α-carbonic anhydrases (CAs, EC 188.8.131.52), that is, CA I, CA II, CA VA, CA IX, CA XII and CA XIV, and two β-CAs from Candida glabrata (CgCA) and Mycobacterium tuberculosis (Rv3588) with these sulfamides was investigated. All CA isozymes were inhibited in the low micromolar to nanomolar range by the dopamine sulfamide analogues. Kis were in the range of 0.061-1.822μM for CA I, 1.47-2.94nM for CA II, 2.25-3.34μM for CA VA, 0.041-0.37μM for CA IX, 0.021-1.52μM for CA XII, 0.007-0.219μM for CA XIV, 0.35-5.31μM for CgCA and 0.465-4.29μM for Rv3588. The synthesized sulfamides may lead to inhibitors targeting medicinally relevant CA isoforms with potential applications as antiepileptic, antiobesity antitumor agents or anti-infective.
[Show abstract][Hide abstract] ABSTRACT: Several β-carbonic anhydrases (CAs, EC 184.108.40.206) are present in all land plants examined thus far. Here we report the first detailed biochemical characterization of one such isoform, FbiCA 1, from the C plant Flaveria bidentis, which was cloned, purified and characterized as recombinant protein. FbiCA 1 has an interesting CO hydrase catalytic activity (k of 1.2×10 and k/K of 7.5×10M×s) and was moderately inhibited by most simple/complex inorganic anions. Potent FbiCA 1 inhibitors were also detected, such as trithiocarbonate, diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid (Ks in the range of 4-60μM). Such inhibitors may be used as tools to better understand the role of various β-CA isoforms in photosynthesis.
[Show abstract][Hide abstract] ABSTRACT: An α-carbonic anhydrase (CA, EC 220.127.116.11) 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 CO 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 33-88μM. Other effective inhibitors were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, with Ks of 7-43μM. Halides (bromide, iodide), bicarbonate and carbonate were much less effective VchCA inhibitors, with Ks in the range of 4.64-28.0mM. 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.
[Show abstract][Hide abstract] ABSTRACT: By using phthalimido-substituted aromatic sufonamides as lead molecules, a series of new sulfonamides incorporating ortho-benzenedisulfonimide moieties have been synthesized and tested against the human (h) cytosolic carbonic anhydrase (CA, EC 18.104.22.168) isozymes hCA I and hCA II and the transmembrane, tumor-associated isozymes hCA IX and hCA XII. All these compounds showed K(i) values lower than 100nM and many of them showed better K(i)s than the reference compound acetazolamide, a clinically used sulfonamide. The tumor-associated isozymes were better inhibited than the cytosolic ones. A molecular docking within the active site of some CA isoforms, such as hCA I, explained these findings, as the benzenedisulfonimide moiety makes favorable interactions (hydrogen bonds) with amino acid residues involved in binding of inhibitors, such as Gln92, His67, and His64.
[Show abstract][Hide abstract] ABSTRACT: A series of benzenesulfonamides incorporating cyanoacrylamide moieties (tyrphostine analogues) have been obtained by reaction of sulfanilamide with ethylcyanoacetate followed by condensation with aromatic/heterocyclic aldehydes, isothiocyanates or diazonium salts. The new compounds have been investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4. 2.1.1), and more specifically against the cytosolic human (h) isoforms hCA I and II, as well as the transmembrane, tumor-associated ones CA IX and XII, which are validated antitumor targets. Most of the new benzenesulfonamides were low nanomolar or subnanomolar CA IX/XII inhibitors whereas they were less effective as inhibitors of CA I and II. The structure-activity relationship for this class of effective CA inhibitors is also discussed. Generally, electron donating groups in the starting aldehyde reagent favored CA IX and XII inhibition, whereas halogeno, methoxy and dimethylamino moieties led to very potent CA XII inhibitors.
[Show abstract][Hide abstract] ABSTRACT: The α-carbonic anhydrase (CA, EC 22.214.171.124) from the extremophilic bacterium Sulfurihydrogenibium azorense (SazCA) was recently shown to be the fastest CA known. Here we investigated this enzyme for its activation with a series of amino acids and amines. The best SazCA activators were d-Phe, l-DOPA, l- and d-Trp, dopamine and serotonin, which showed activation constants in the range of 3-23nM. l- and d-His, l-Phe, l-Tyr, 2-pyridyl-methylamine and L-adrenaline were also effective activators (K(A)s in the range of 62-90nM), whereas d-Dopa, d-Tyr and several heterocyclic amines showed activity in the micromolar range. The good thermal stability, robustness, very high catalytic activity and propensity to be activated by simple amino acids and amines, make SazCA a very interesting candidate for biomimetic CO(2) capture processes.
[Show abstract][Hide abstract] ABSTRACT: Coumarins were recently shown to constitute a novel class of mechanism-based carbonic anhydrase (CA, EC 4..2.1.1) inhibitors. We demonstrate that sulfocoumarins (1,2-benzoxathiine 2,2-dioxides) possess a similar mechanism of action, acting as effective CA inhibitors. The sulfocoumarins were hydrolyzed by the esterase CA activity to 2-hydroxyphenyl-vinylsulfonic acids which thereafter bind to the enzyme, in a region rarely occupied by other classes of inhibitors. The X-ray structure of one of these compounds in adduct with a modified CA II enzyme possessesing two amino acid residues from the CA IX active site, allowed us to decipher the inhibition mechanism. The sulfonic acid was observed anchored to the zinc-coordinated water molecule, making favorable interactions with Thr200 and Pro201. Some other sulfocoumarins incorporating substituted-1,2,3-triazole moieties, were prepared by using click chemistry, and showed low nanomolar inhibitory action against the tumor-associated isoforms CA IX and XII, being less effective against the cytosolic CA I and II.
Journal of Medicinal Chemistry 12/2012; · 5.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have cloned, purified and characterized an α-carbonic anhydrase (CA, EC 126.96.36.199) from the human pathogenic bacterium Vibrio cholerae, VchCA. The new enzyme has significant catalytic activity and an inhibition study with sulfonamides and sulfamates led to the detection of a large number of low nanomolar inhibitors, among which methazolamide, acetazolamide, ethoxzolamide, dorzolamide, brinzolamide, benzolamide and indisulam (KIs in the range of 0.69 - 8.1 nM). As bicarbonate is a virulence factor of this bacterium and since ethoxzolamide was shown to inhibit the in vivo virulence, we propose that VchCA may be a target for antibiotic development, exploiting a mechanism of action rarely considered up until now.
Journal of Medicinal Chemistry 11/2012; · 5.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report the biochemical characterization of a new carbonic anhydrase (CA, EC 188.8.131.52), 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.