The extremo-α-carbonic anhydrase from the thermophilic bacterium Sulfurihydrogenibium azorense is highly inhibited by sulfonamides

The α-carbonic anhydrase (CA, EC 4.2.1.1) from the newly discovered extremophilic bacterium Sulfurihydrogenibium azorense (SazCA) is the most effective CA known to date. Here we investigated the inhibition profile of this enzyme with a series of aromatic and heterocyclic sulfonamides, and one sulfamate. Many clinically used sulfonamides, such as acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, topiramate, celecoxib and sulpiride were low nanomolar/subnanomolar SazCA inhibitors (K_Is in the range of 0.9–10.8 nM) whereas simple aromatic derivatives were less effective as SazCA inhibitors. The inhibition profile of SazCA is slightly different from that of the related enzyme from S. yellostonense (SspCA), investigated earlier by our groups.     

The alpha-carbonic anhydrase from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 is highly susceptible to inhibition by sulfonamides

The α-carbonic anhydrase (CA, EC 4.2.1.1) from the newly discovered thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 (SspCA) was investigated for its inhibition with a large series of sulfonamides and a sulfamate, the classical inhibitors of these zinc enzymes. SspCA showed an inhibition profile with these compounds very similar to that of the predominant human cytosolic isoform hCA II, and not to that of the bacterial α-CA from Helicobacter pylori. Some clinically used drugs such as acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, topiramate, celecoxib and sulthiame were low nanomolar SspCA/hCA II inhibitors (K_Is in the range of 4.5–12.3 nM) whereas simple aromatic/heterocyclic sulfonamides were less effective, micromolar inhibitors. As this highly catalytically active and thermostable enzyme may show biotechnological applications, its inhibition studies may be relevant for designing on/off systems to control its activity.     

Synthesis 4-[2-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-ethyl]-benzenesulfonamides with subnanomolar carbonic anhydrase II and XII inhibitory properties

Condensation of substituted anthranilic acids with 4-isothiocyanatoethyl-benzenesulfonamide led to series of heterocyclic benzenesulfonamides incorporating 2-mercapto-quinazolin-4-one tails. These sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA XII (a transmembrane, tumor-associated enzyme also involved in glaucoma-genesis). The new sulfonamides acted as medium potency inhibitors of hCA I (K_Is of 28.5–2954 nM), being highly effective as hCA II (K_Is in the range of 0.62–12.4 nM) and XII (K_Is of 0.54–7.11 nM) inhibitors. All substitution patterns present in these compounds (e.g., halogens, methyl and methoxy moieties, in positions 6, 7 and/or 8 of the 2-mercapto-quinazolin-4-one ring) led to highly effective hCA II/XII inhibitors. These compounds should thus be of interest as preclinical candidates in pathologies in which the activity of these enzymes should be inhibited, such as glaucoma (CA II and XII as targets) or some tumors in which the activity of isoforms CA II and XII is dysregulated.     

Microwave assisted synthesis of novel acridine–acetazolamide conjugates and investigation of their inhibition effects on human carbonic anhydrase isoforms hCA I,II, IV and VII

4-Amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide was condensed with cyclic-1,3-diketones (dimedone and cyclohexane-1,3-dione) and aromatic aldehydes under microwave irradiation, leading to a series of acridine–acetazolamide conjugates. The new compounds were investigated as inhibitors of carbonic anhydrases (CA, EC 4.2.1.1), and more precisely cytosolic isoforms hCA I, II, VII and membrane-bound one hCA IV. All investigated isoforms were inhibited in low micromolar and nanomolar range by the new compounds. hCA IV and VII were inhibited with K_Is in the range of 29.7–708.8 nM (hCA IV), and of 1.3–90.7 nM (hCA VII). For hCA I and II the K_Is were in the range of 6.7–335.2 nM (hCA I) and of 0.5–55.4 nM (hCA II). The structure–activity relationships (SAR) for the inhibition of these isoforms with the acridine–acetazolamide conjugates reported here were delineated.     

Development of 3-(4-aminosulphonyl)-phenyl-2-mercapto-3H-quinazolin-4-ones as inhibitors of carbonic anhydrase isoforms involved in tumorigenesis and glaucoma

A series of heterocyclic benzenesulfonamides incorporating 2-mercapto-3H-quinazolin-4-one tails were prepared by condensation of substituted anthranilic acids with 4-isothiocyanato-benzenesulfonamide. These sulfonamides were investigated as inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isozymes), as well as hCA IX and XII (trans-membrane, tumor-associated enzymes). They acted as medium potency inhibitors of hCA I (K_Is of 81.0–3084 nM), being highly effective as hCA II (K_Is in the range of 0.25–10.8 nM), IX (K_Is of 3.7–50.4 nM) and XII (K_Is of 0.60–52.9 nM) inhibitors. These compounds should thus be of interest as preclinical candidates in pathologies in which the activity of these enzymes should be inhibited, such as glaucoma (CA II and XII as targets) or some tumors in which the activity of three isoforms (CA II, IX and XII) is dysregulated.     

5-Substituted-(1,2,3-triazol-4-yl)thiophene-2-sulfonamides strongly inhibit human carbonic anhydrases I,II, IX and XII: Solution and X-ray crystallographic studies

We report here a series of 2-thiophene-sulfonamides incorporating 1-substituted aryl-1,2,3-triazolyl moieties, prepared by click chemistry from 5-ethynylthiophene-2-sulfonamide and substituted aryl azides. The new sulfonamides were investigated as inhibitors of the zinc metalloenzyme CA (EC 4.2.1.1), and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated ones hCA IX and XII: The new compounds were medium–weak hCA I inhibitors (K_Is in the range of 224–7544 nM), but were compactly, highly effective, low nanomolar hCA II inhibitors (K_Is of 2.2–7.7 nM). The tumor-associated hCA IX was inhibited with K_Is ranging between 5.4 and 811 nM, whereas hCA XII with inhibition constants in the range of 3.4–239 nM. The X-ray crystal structure of the adducts of two such compounds bound to hCA II (one incorporating 1-naphthyl, the other one 3-cyanophenyl moieties) evidenced the reasons of the high affinity for hCA II. Highly favorable, predominantly hydrophobic interactions between the sulfonamide scaffold and the hCA II active site were responsible for the binding, in addition to the coordination of the sulfamoyl moiety to the zinc ion. The tails of the two inhibitors adopted very diverse orientations when bound to the active site, with the naphthyltriazolyl moiety orientated towards the hydrophobic half of the active site, and the 3-cyanophenyl one pointing towards the hydrophilic half. These data may be used for the structure-based drug design of even more effective hCA II inhibitors, with potential use as antiglaucoma agents or as diuretics.     

The first activation study of a bacterial carbonic anhydrase (CA). The thermostable α-CA from Sulfurihydrogenibium yellowstonense YO3AOP1 is highly activated by amino acids and amines

The α-carbonic anhydrase (CA, EC 4.2.1.1) from the newly discovered thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1 (SspCA) was investigated for its activation with a series of amino acids and amines. d-His, l-Phe, l-Tyr, l- and d-Trp were the most effective SspCA activators, with activation constants in the range of 1–12 nM, whereas l-His, l/d-DOPA, d-Tyr, and several biogenic amines/catecholamines were slightly less effective activators (K_A in the range of 37 nM–0.97 μM). The least effective SspCA activator was d-Phe (K_A of 5.13 μM). The thermal stability, robustness and very high catalytic activity of SspCA make this enzyme an ideal candidate for biomimetic CO₂ capture processes.     

Carbonic anhydrase inhibitors. Aromatic/heterocyclic sulfonamides incorporating phenacetyl,pyridylacetyl and thienylacetyl tails act as potent inhibitors of human mitochondrial isoforms VA and VB

A series of aromatic/heterocyclic sulfonamides incorporating phenyl(alkyl), halogenosubstituted-phenyl- or 1,3,4-thiadiazole-sulfonamide moieties and thienylacetamido; phenacetamido and pyridinylacetamido tails were prepared and assayed as inhibitors of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic human (h) hCA I and hCA II, and the mitochondrial hCA VA and hCA VB. The new compounds showed moderate inhibition of the two cytosolic isoforms (K_Is of 50–390 nM) and excellent inhibitory activity against the two mitochondrial enzymes, with many low nanomolar inhibitors detected (K_Is in the range of 5.9–10.2 nM). All substitution patterns explored here lead to effective hCA VA/VB inhibitors. Some hCA VA/VB selective inhibitors were also detected, with selectivity ratios for inhibiting the mitochondrial over the cytosolic isozymes of around 55.5–56.9. As hCA VA/VB are involved in several biosynthetic processes catalyzed by pyruvate carboxylase, acetyl CoA carboxylase, and carbamoyl phosphate synthetases I and II, providing the bicarbonate substrate to these carboxylating enzymes involved in fatty acid biosynthesis, their selective inhibition may lead to the development of antiobesity agents possessing a new mechanism of action.     

Coumarinyl-substituted sulfonamides strongly inhibit several human carbonic anhydrase isoforms: solution and crystallographic investigations

We investigated a series of coumarinyl-substituted aromatic sulfonamides as inhibitors of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms with medical applications, the cytosolic hCA I, and II, and the transmembrane, tumor-associated hCA IX and XII. Compounds incorporating 7-methoxy-coumarin-4-yl-acetamide-tails and benzenesulfonamide and benzene-1,3-disulfonamide scaffolds showed medium potency inhibition of hCA I (K_Is of 73–131 nM), effective hCA II inhibition (K_Is of 9.1–36 nM) and less effective hCA IX and XII inhibition (K_Is of 55–128 nM). Only one compound, the derivatized 4-amino-6-trifluoromethyl-benzene-1,3-disulfonamide with the coumarinyl tail, showed effective inhibition of the transmembrane isoforms, with K_Is of 5.9–14.2 nM, although it was less effective as hCA I and II inhibitor (K_Is of 36–120 nM). An X-ray crystal structure of hCA II in complex with 4-(7-methoxy-coumarin-4-yl-acetamido)-benzenesulfonamide (K_I of 9.1 nM against hCA II) showed the intact inhibitor coordinated to the zinc ion from the enzyme active site by the sulfonamide moiety, and participating in a edge-to-face stacking with Phe131, in addition to other hydrophobic and hydrophilic interactions with water molecules and amino acid residues from the active site. Thus, sulfonamides incorporating coumarin rings have a distinct inhibition mechanism compared to the coumarins, and may lead to compounds with interesting inhibition profiles against various α-CAs found in mammals or parasites, such as Plasmodium falciparum.     

6-Triazolyl-substituted sulfocoumarins are potent,selective inhibitors of the tumor-associated carbonic anhydrases IX and XII

A series of 6-substituted sulfocoumarins incorporating substituted-1,2,3-triazol-4-yl-/5-yl moieties were synthesized by employing click chemistry. The new sulfocoumarins incorporated cycloalkyl, tert-butyl and substituted aryl moieties at the triazole ring, and were investigated for the inhibition of four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, the cytosolic hCA I and II; and the transmembrane, tumor-associated hCA IX and XII. The triazole-substituted sulfocoumarins did not inhibit the ubiquitous, off-target cytosolic isoforms hCA I and II (K_Is >10 μM) but showed effective inhibition against the two transmembrane CAs, with K_Is ranging from 7.2 to 10.5 nM against hCA IX, and between 5.5 and 17.7 nM against hCA XII. As hCA IX and XII are validated anti-tumor targets, such prodrug, isoform-selective inhibitors as the sulfocoumarins reported here, may be useful for identifying suitable drug candidates for clinical trials.     

Substituted benzene sulfonamides incorporating 1,3,5-triazinyl moieties potently inhibit human carbonic anhydrases II,IX and XII

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 4.2.1.1) isoforms I, II (cytosolic) and IX, XII (transmembrane, tumor-associated) isoforms were performed with the new compounds. hCA I was modestly inhibited (K_Is in the range of 87 nM–4.35 μM), hCA II was moderately inhibited by most of the new compounds (K_Is in the range of 12.5–130 nM), whereas the tumor associated isoforms were potently inhibited, with K_Is in the range of 1.2–34.1 nM 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.     

Carbonic anhydrase inhibitors. Phenacetyl-, pyridylacetyl- and thienylacetyl-substituted aromatic sulfonamides act as potent and selective isoform VII inhibitors

A series of aromatic/heterocyclic sulfonamides incorporating phenyl(alkyl), halogenosubstituted-phenyl- or 1,3,4-thiadiazole-sulfonamide moieties and thienylacetamido; phenacetamido- and pyridinylacetamido tails were prepared and assayed as inhibitors of cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms hCA I, II and VII. The new compounds showed moderate inhibition of the two ubiquitous isoforms I and II (K_Is of 50–390 nM) and excellent inhibitory activity against the brain associated hCA VII (K_Is in the range of 4.7–8.5 nM). Isoform VII highly selective inhibitors are being detected for the first time, with selectivity ratios for inhibiting CA VII over CA II of 11–75, and for inhibiting CA VII over CA I of 10–49, which may be useful for understanding the role of CA VII in epileptogenesis and other physiologic processes.     

Synthesis of 6-tetrazolyl-substituted sulfocoumarins acting as highly potent and selective inhibitors of the tumor-associated carbonic anhydrase isoforms IX and XII

A series of 6-substituted sulfocoumarins incorporating substituted-1,2,3,4-tetrazol-5-yl moieties were synthesized by reaction of 6-iodo-sulfocoumarin and the corresponding tetrazole via the CH activation reaction. The new sulfocoumarins incorporating alkyl and substituted aryl moieties at the 1-position of the tetrazole, were investigated for the inhibition of four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, the cytosolic hCA I and II; and the transmembrane, tumor-associated hCA IX and XII. The tetrazole-substituted sulfocoumarins did not inhibit the ubiquitous, off-target cytosolic isoforms (K_Is >10 μM) but showed effective inhibition against the two transmembrane CAs, with K_Is ranging from 6.5 to 68.6 nM against hCA IX, and between 4.3 and 59.8 nM against hCA XII. As hCA IX and XII are validated anti-tumor targets, such prodrug, isoform-selective inhibitors as the sulfocoumarins reported here, may be useful for identifying suitable drug candidates for clinical trials.     

A novel library of saccharin and acesulfame derivatives as potent and selective inhibitors of carbonic anhydrase IX and XII isoforms

Small libraries of N-substituted saccharin and N-/O-substituted acesulfame derivatives were synthesized and tested as atypical and selective inhibitors of four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). Most of them inhibited hCA XII in the low nanomolar range, hCA IX with K_Is ranging between 19 and 2482 nM, whereas they were poorly active against hCA II (K_Is >10 μM) and hCA I (K_Is ranging between 318 nM and 50 μM). Since hCA I and II are ubiquitous off-target isoforms, whereas the cancer-related isoforms hCA IX and XII were recently validated as drug targets, these results represent an encouraging achievement in the development of new anticancer candidates. Moreover, the lack of a classical zinc binding group in the structure of these inhibitors opens innovative, yet unexplored scenarios for different mechanisms of inhibition that could explain the high inhibitory selectivity. A computational approach has been carried out to further rationalize the biological data and to characterize the binding mode of some of these inhibitors.     

Synthesis of 4-(thiazol-2-ylamino)-benzenesulfonamides with carbonic anhydrase I,II and IX inhibitory activity and cytotoxic effects against breast cancer cell lines

A series of 4-(thiazol-2-ylamino)-benzenesulfonamides was synthesized and screened for their carbonic anhydrase (CA, EC 4.2.1.1) inhibitory and cytotoxic activity on human breast cancer cell line MCF-7. Human (h) CA isoforms I, II and IX were included in the study. The new sulfonamides showed excellent inhibition of all three isoforms, with K_Is in the range of 0.84–702 nM against hCA I, of 0.41–288 nM against hCA II and of 5.6–29.2 against the tumor-associated hCA IX, a validated anti-tumor target, with a sulfonamide (SLC-0111) in Phase I clinical trials for the treatment of hypoxic, metastatic solid tumors overexpressing CA IX. The new compounds showed micromolar inhibition of growth efficacy against breast cancer MCF-7 cell lines.     

Carbonic anhydrase inhibitors: Two-prong versus mono-prong inhibitors of isoforms I,II, IX,and XII exemplified by photochromic cis-1,2-α-dithienylethene derivatives

We investigated the inhibition of five physiologically relevant CA isoforms with photochromic cis-1,2-α-dithienylethene-based compounds incorporating either a benzenesulfonamide and Cu(II)-iminodiacetic acid (IDA)-, bis-benzenesulfonamide-, bis-Cu(II)-IDA-, and bis-ethyleneglycol-methyl ether moieties, in both their open- and closed-ring forms. For hCA I the best inhibitors were the mono-prong bis-sulfonamide and the bis-Cu-IDA complexes (K_Is of 2–3 nM) in their open form. For hCA II, best inhibitors were the open and closed forms of the mono-prong bis-sulfonamide (K_Is of 13–18 nM). hCA IX was moderately inhibited by these compounds (K_Is of 9–376 nM) whereas hCA XII and XIV were less susceptible to inhibition (K_Is of 1.12–16.7 μM).     

Fluorescent sulfonamide carbonic anhydrase inhibitors incorporating 1,2,3-triazole moieties: Kinetic and X-ray crystallographic studies

Fluorescent sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) were essential for demonstrating the role played by the tumor-associated isoform CA IX in acidification of tumors, cancer progression towards metastasis and for the development of imaging and therapeutic strategies for the management of hypoxic tumors which overexpress CA IX. However, the presently available such compounds are poorly water soluble which limits their use. Here we report new fluorescent sulfonamides 7, 8 and 10 with increased water solubility. The new derivatives showed poor hCA I inhibitory properties, but were effective inhibitors against the hCA II (K_Is of 366–127 nM), CA IX (K_Is of 8.1–36.9 nM), CA XII (K_Is of 4.1–20.5 nM) and CA XIV (K_Is of 12.8–53.6 nM). A high resolution X-ray crystal structure of one of these compounds bound to hCA II revealed the factors associated with the good inhibitory properties. Furthermore, this compound showed a three-fold increase of water solubility compared to a similar derivative devoid of the triazole moiety, making it an interesting candidate for ex vivo/in vivo studies.     

Acetylcholinesterase and carbonic anhydrase inhibitory properties of novel urea and sulfamide derivatives incorporating dopaminergic 2-aminotetralin scaffolds

In the present study a series of urea and sulfamide compounds incorporating the tetralin scaffolds were synthesized and evaluated for their acetylcholinesterase (AChE), human carbonic anhydrase (CA, EC 4.2.1.1) isoenzyme I, and II (hCA I and hCA II) inhibitory properties. The urea and their sulfamide analogs were synthesized from the reactions of 2-aminotetralins with N,N-dimethylcarbamoyl chloride and N,N-dimethylsulfamoyl chloride, followed by conversion to the corresponding phenols via O-demethylation with BBr₃. The novel urea and sulfamide derivatives were tested for inhibition of hCA I, II and AChE enzymes. These derivatives exhibited excellent inhibitory effects, in the low nanomolar range, with K_i values of 2.61–3.69 nM against hCA I, 1.64–2.80 nM against hCA II, and in the range of 0.45–1.74 nM against AChE. In silico techniques such as, atomistic molecular dynamics (MD) and molecular docking simulations, were used to understand the scenario of the inhibition mechanism upon approaching of the ligands into the active site of the target enzymes. In light of the experimental and computational results, crucial amino acids playing a role in the stabilization of the enzyme–inhibitor adducts were identified.     

The extremo-α-carbonic anhydrase (CA) from Sulfurihydrogenibium azorense,the fastest CA known,is highly activated by amino acids and amines

The α-carbonic anhydrase (CA, EC 4.2.1.1) 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 3–23 nM. l- and d-His, l-Phe, l-Tyr, 2-pyridyl-methylamine and L-adrenaline were also effective activators (K_As in the range of 62–90 nM), 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 CO₂ capture processes.     

Carbonic anhydrase inhibitors: Synthesis and inhibition of the human carbonic anhydrase isoforms I,II, IX and XII with benzene sulfonamides incorporating 4- and 3-nitrophthalimide moieties

A series of 4 and 5 nitro-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1–8) was synthesized by reaction of benzenesulfonamide derivatives with 4 and 3-nitrophthalic anhydrides. These new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated hCA IX and XII. Most of the novel compounds were medium potency-weak hCA I inhibitors (K_is in the range of 295–10,000 nM), but were more effective hCA II inhibitors (K_is of 1.7–887 nM). The tumor-associated hCA IX was also inhibited, with K_is in the micromolar range, whereas against hCA XII the inhibition constants were in the range of 90–3746 nM. The structure–activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoforms being established. The high sequence hCA alignment homology and molecular docking studies was performed in order to rationalize the activities reported and binding mode to different hCA as inhibitors.