Carbonic Anhydrase Isoforms Research Articles

Design, synthesis, and carbonic anhydrase inhibition activity of benzenesulfonamide-linked novel pyrazoline derivatives

Carbonic anhydrases (CA, EC 4.2.1.1) are Zinc metalloenzymes and are present throughout most living organisms. Among the catalytically active isoforms are the cytosolic CA I and II, and tumor-associated CA IX and CA XII. The carbonic anhydrase (CA) inhibitory activities of newly synthesized pyrazoline-linked benzenesulfonamides 18–33 against human CA (hCA) isoforms I, II, IX, and XII were measured and compared with that of acetazolamide (AAZ), a standard inhibitor. Potent inhibitory activity against hCA I was exerted by compounds 18–25, with inhibition constant (KI) values of 87.8–244.1 nM, which were greater than that of AAZ (KI, 250.0 nM). Compounds 19, 21, 22, 29, 30, and 32 were proven to have inhibitory activities against hCA IX with KI values (5.5–37.0 nM) that were more effective than or nearly equal to that of AAZ (KI, 25.0 nM). Compounds 20–22, and 30 exerted potent inhibitory activities (KIs, 7.1–10.1 nM) against hCA XII, in comparison with AAZ (KI, 5.7 nM).

Novel 8-Substituted Coumarins That Selectively Inhibit Human Carbonic Anhydrase IX and XII.

A novel series of 8-substituted coumarin-based compounds, characterized by the presence of alkylpiperazine and arylpiperazine chains, were synthesized and tested for their inhibitory activity against four human carbonic anhydrase (hCA) isoforms. All compounds displayed nanomolar potency against the cancer-related hCA IX and hCA XII; moreover, they were shown to be devoid of any inhibitory activity toward the cytosolic hCA I and hCA II up to 10 µM concentration in the assay system. Therefore, the synthesized coumarin ligands demonstrated to be potent and selective hCA IX/XII inhibitors, and were shown to be as potent as the reference inhibitor acetazolamide against hCA XII, with single-digit nanomolar Ki values. Molecular modeling studies provided a rationale for explaining the selectivity profile of these non-classic hCA inhibitors and their interactions with the enzymes, according to their specific mechanism of action, thus paving the way for future structure-based lead optimization studies.

Synthesis, biological activity and multiscale molecular modeling studies of bis-coumarins as selective carbonic anhydrase IX and XII inhibitors with effective cytotoxicity against hepatocellular carcinoma

A series of novel bis-coumarin derivatives containing triazole moiety as a linker between the alkyl chains was synthesized and their inhibitory activity against the human carbonic anhydrase (hCA) isoforms I, II, IX and XII were evaluated. In addition, cytotoxic effects of the synthesized compounds on renal adenocarcinoma (769P), hepatocellular carcinoma (HepG2) and breast adeno carcinoma (MDA-MB-231) cell lines were examined. While the hCA I and II isoforms were inhibited in the micromolar range, the tumor-associated isoform hCA IX and XII were inhibited in the high nanomolar range. 4-methyl-7-((1-(12-((2-oxo-2H-chromen-7-yl)oxy)dodecyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (5p) showed the strongest inhibitory activity against hCA IX with the Ki of 144.6 nM and 4-methyl-7-((1-(10-((2-oxo-2H-chromen-7-yl)oxy)decyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (5n) exhibited the highest hCA XII inhibition with the Ki of 71.5 nM. In order to better understand the inhibitory profiles of studied molecules, multiscale molecular modelling approaches were applied. Low energy docking poses of studied molecules at the binding sites of targets have been predicted. In addition, electrostatic potential surfaces (ESP) for binding sites were also generated to understand interactions between proteins and active ligands.

Synthesis of benzensulfonamides linked to quinazoline scaffolds as novel carbonic anhydrase inhibitors

Carbonic anhydrase (CA) inhibitory activities of newly synthesized quinazoline-linked benzensulfonamides 10–29, 31, 32, 35, 36, and 45–51 against human CA (hCA) isoforms I, II, IX, and XII were measured and compared to that of acetazolamide (AAZ) as a standard inhibitor. Potent selective inhibitory activity against hCA I was exerted by compounds 14, 15, 17, 19, 20, 21, 24, 25, 28, 29, 31, 35, 45, 47, 49, and 51 with inhibition constant (KIs) values of 39.4–354.7 nM that were nearly equivalent or even greater than that of AAZ (KI, 250.0 nM). Compounds 15, 20, 24, 28, 29, 45 and 47 proved to have inhibitory activities against hCA II with (KIs, 0.73–16.5 nM) that were similar or improved to that of AAZ (KI, 12.0 nM). Compounds 13–29, 31–32, and 45–51 displayed potent hCA IX inhibitory activities (KIs, 1.6–32.2 nM) that were more effective than or nearly equal to AAZ (KI, 25.0 nM). Compounds 14, 15, 20, 21, 26, 45, and 47 exerted potent hCA XII inhibitory activities (KIs, 5.2–9.2 nM), indicating similar CAI activities as compared to that of AAZ (KI, 5.7 nM).

Discovery of new ureido benzenesulfonamides incorporating 1,3,5-triazine moieties as carbonic anhydrase I, II, IX and XII inhibitors.

A series of twenty novel ureido benzenesulfonamides incorporating 1,3,5-triazine moieties substituted on one side with aromatic amines and on the other side with dimethylamine, morpholine and piperidine is reported. The compounds were synthesized from the 4-(3-(4,6-dichloro-1,3,5-triazin-2-yl)ureido)benzensulfonamide (1) by using stepwise nucleophilic substitution of the chlorine atoms of cyanuric chloride. The intermediates 2(a-e) and final compounds 3(a-o) were tested for their efficiency as carbonic anhydrase (CA) inhibitors against four selected physiologically relevant human carbonic anhydrase (CA, EC 4.2.1.1) isoforms, namely, the cytosolic ones hCA I and II, and the transmembrane, tumor associated ones hCA IX, and XII. The compounds 2a, 2e and 3m showed the highest activity for hCA IX with Kis in the range of 11.8-14.6 nM. Most of the compounds showed high hCA IX selectivity over the abundant off-target isoforms hCA I and II. Since hCA IX is a validated drug target for anticancer/antimetastatic agents, these isoform-selective and potent inhibitors may be considered of interest for further medicinal/pharmacologic studies.

Isothermal Analysis of ThermoFluor Data can readily provide Quantitative Binding Affinities

Differential scanning fluorimetry (DSF), also known as ThermoFluor or Thermal Shift Assay, has become a commonly-used approach for detecting protein-ligand interactions, particularly in the context of fragment screening. Upon binding to a folded protein, most ligands stabilize the protein; thus, observing an increase in the temperature at which the protein unfolds as a function of ligand concentration can serve as evidence of a direct interaction. While experimental protocols for this assay are well-developed, it is not straightforward to extract binding constants from the resulting data. Because of this, DSF is often used to probe for an interaction, but not to quantify the corresponding binding constant (Kd). Here, we propose a new approach for analyzing DSF data. Using unfolding curves at varying ligand concentrations, our “isothermal” approach collects from these the fraction of protein that is folded at a single temperature (chosen to be temperature near the unfolding transition). This greatly simplifies the subsequent analysis, because it circumvents the complicating temperature dependence of the binding constant; the resulting constant-temperature system can then be described as a pair of coupled equilibria (protein folding/unfolding and ligand binding/unbinding). The temperature at which the binding constants are determined can also be tuned, by adding chemical denaturants that shift the protein unfolding temperature. We demonstrate the application of this isothermal analysis using experimental data for maltose binding protein binding to maltose, and for two carbonic anhydrase isoforms binding to each of four inhibitors. To facilitate adoption of this new approach, we provide a free and easy-to-use Python program that analyzes thermal unfolding data and implements the isothermal approach described herein (https://sourceforge.net/projects/dsf-fitting).

Carbonic anhydrases are involved in mitochondrial biogenesis and control the production of lactate by human Sertoli cells

The process that allows cells to control their pH and bicarbonate levels is essential for ionic and metabolic equilibrium. Carbonic anhydrases (CAs) catalyse the conversion of CO2 to and H+ and are thus essential for this process. Herein, we inhibited CAs with acetazolamide - ACT and SLC-0111 - to study their involvement in the metabolism, mitochondrial potential, mitochondrial biogenesis and lipid metabolism of human Sertoli cells (hSCs), obtained from biopsies from men with conserved spermatogenesis. We were able to identify three isoforms of CAs, one mitochondrial isoform (CA VB) and two cell membrane-bound isoforms (CA IX and CA XII) in hSCs. When assessing the expression of markers for mitochondrial biogenesis, we observed a decrease in HIF-1α, SIRT1, PGC1α and NRF-1 mRNAs after all CAs were inhibited, resulting in decreased mitochondrial DNA copy numbers. This was followed by an increased production of lactate and alanine in the same conditions. In addition, consumption of glucose was maintained after inhibition of all CAs in hSCs. These results indicate a reduced conversion of pyruvate to acetyl-coA, possibly due to decreased mitochondrial function, caused by CA inhibition in hSCs. Inhibition of CAs also caused alterations in lipid metabolism, since we detected an increased expression of hormone-sensitive lipase (HSL) in hSCs. Our results suggest that CAs are essential for mitochondrial biogenesis, glucose and lipid metabolism in hSCs. This is the first report showing that CAs play an essential role in hSC metabolic dynamics, being involved in mitochondrial biogenesis and controlling lactate production.

Pyridazinone-substituted benzenesulfonamides display potent inhibition of membrane-bound human carbonic anhydrase IX and promising antiproliferative activity against cancer cell lines

An expanded set of pyridazine-containing benzene sulfonamides was investigated for inhibition of four human carbonic anhydrase isoforms, which revealed a pronounced inhibition trend toward hCA IX, a cancer-related, membrane-bound isoform of the enzyme. Comparison of antiproliferative effects of these compounds against cancer (PANC-1) and normal (ARPE-19) cells at 50 μM concentration narrowed the selection of compounds to the eight which displayed selective growth inhibition toward the cancer cells. More detailed investigation in concentration-dependent mode against normal (ARPE-19) and two cancer cell lines (PANC-1 and SK-MEL-2) identified two lead compounds one of which displayed a notable cytotoxicity toward pancreatic cancer cells while the other targeted the melanoma cells. These findings significantly expand the knowledge base concerning the hCA IX inhibitors whose inhibitory potency against a recombinant enzyme translates into selective anticancer activity under hypoxic conditions which are aimed to model the environment of a growing tumor.

Design, synthesis and biological evaluation of coumarin-3-carboxamides as selective carbonic anhydrase IX and XII inhibitors

A series of novel 7-hydroxycoumarin-3-carboxamides was synthesized by the reaction of 7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid with various substituted aromatic amines. The newly synthesized compounds were evaluated for their inhibitory activity against the four physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms CA I, CA II, CA IX and CA XII. The CA inhibition results show that the newly synthesized 7-hydroxycoumarin-3-carboxamides (4a-n) exhibited selective inhibition of the tumor associated isoforms, CA IX and CA XII over CA I and II isoforms. The inhibition constants ranged from sub micromolar to low micromolar. Amongst all the compounds tested, compound 4m was the most effective inhibitor exhibiting sub micromolar potency against both hCA IX and hCA XII, with a Ki of 0.2 µM. Therefore, it can be anticipated that compound 4m can serve as a lead for development of anticancer therapy by exhibiting a novel mechanism of action. The binding modes of the most potent compounds within hCA IX and XII catalytic clefts were investigated by docking studies.

- THE IN VITRO IMPACTS OF SOME PLANT EXTRACTS ON CARBONIC ANHYDRASE I, II AND PARAOXONASE-1

The presented article focuses on the in vitro inhibition of plant extracts on the human carbonic anhydrase isoforms (hCA I and hCAII), and paraoxonase-1 (PON1) activities. Five different plants (Alcea rosea, Foeniculum vulgare, Elettaria cardamomum, Laurus azorica and Lavandula stoechas) were selected in this study. Methanol, ethanol, and water extracts of plants were prepared and the concentration-dependent inhibition degrees were found for hCA I and hCA II isozymes and hPON1 spectrophotometrically. Thus, IC50 (mg/mL) values were obtained for each extract. Methanolic extract of Elettaria cardamomum has the highest inhibitory effects (0.032 mg/mL). The water extracts of plants showed lower inhibitory impacts compared to the methanol and ethanol extracts.

Carbonic Anhydrase Inhibitors of Different Structures Dilate Pre-Contracted Porcine Retinal Arteries.

Carbonic anhydrase inhibitors (CAIs), such as dorzolamide (DZA), are used as anti-glaucoma drugs to lower intraocular pressure, but it has been found that some of these drugs act as vasodilators of retinal arteries. The exact mechanism behind the vasodilatory effect is not yet clear. Here we have addressed the issue by using small vessel myography to examine the effect of CAIs of the sulfonamide and coumarin type on the wall tension in isolated segments of porcine retinal arteries. Vessels were pre-contracted by the prostaglandin analog U-46619, and CAIs with varying affinity for five different carbonic anhydrase (CA) isoenzymes found in human tissue tested. We found that all compounds tested cause a vasodilation of pre-contracted retinal arteries, but with varying efficacy, as indicated by the calculated mean EC50 of each compound, ranging from 4.12 µM to 0.86 mM. All compounds had a lower mean EC50 compared to DZA. The dilation induced by benzolamide (BZA) and DZA was additive, suggesting that they may act on separate mechanisms. No clear pattern in efficacy and affinity for CA isoenzymes could be discerned from the results, although Compound 5, with a low affinity for all isoenzymes except the human (h) CA isoform IV, had the greatest potency, with the lowest EC50 and inducing the most rapid and profound dilation of the vessels. The results suggest that more than one isozyme of CA is involved in mediating its role in controlling vascular tone in retinal arteries, with a probable crucial role played by the membrane-bound isoform CA IV.

α-Carbonic anhydrases are strongly activated by spinaceamine derivatives

A series of 4-substituted-spinaceamine (4,5,6,7-tetrahydro-imidazolo[4,5-c]pyridine) were prepared from histamine and aromatic aldehydes Schiff bases, and investigated as activators of four human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic hCA I, II and VII, and the membrane-associated hCA IV. All isoforms were effectively activated by the new derivatives, and the nature of the moiety in position 4 of the bicyclic system was the factor influencing activation properties against all isoforms. For hCA I, these compounds showed KAs in the range of 2.52-21.5 µM, the most effective activator being 4-(2-hydroxyphenyl)-spinaceamine. For hCA II the activation constants ranged between 0.60 and 17.2 µM, with 4-(2,3,5,6-tetrafluorophenyl)- spinaceamine the best activator. Affinity for hCA IV was in the range of 0.52-63.8 µM, and the same compound as for hCA II was the most effective activator. The most sensitive isoform for activation was the brain-associated hCA VII, for which KAs in the range of 82 nM-4.26 µM were observed. Effective hCA VII activators were the (2-bromophenyl)-, 2,3,5,6-tetrafluorophenyl- and furyl-substituted spineaceamines (KAs of 82-95 nM). As CA activators may have pharmacologic applications in various fields, this work provides interesting derivatives for further studies.

Synthesis of novel benzenesulfonamide bearing 1,2,3-triazole linked hydroxy-trifluoromethylpyrazolines and hydrazones as selective carbonic anhydrase isoforms IX and XII inhibitors

A series of twenty four hydroxy-trifluoromethylpyrazoline-carbonyl-1,2,3-triazoles and four hydrazones bearing benzenesulfonamide moieties was obtained by condensation of carboxyhydrazides with substituted 1,3-diketones. All the newly synthesized compounds were investigated as inhibitors of physiologically and pharmacologically relevant human (h) carbonic anhydrsae (CA, EC 4.2.1.1) cytosolic isoforms hCA I and II, as well as transmembrane tumor-assosciated isoforms hCA IX and XII. These compounds exhibited excellent CA inhibitory potency against the four CA isoenzymes as compared to clinically used reference drug acetazolamide (AAZ). Some compounds bearing bulkier group at C-5′ position of 1,2,3-triazoles ring were weaker inhibitors of hCA I. Inhibition assay against hCA II indicates, that several derivatives exhibited upto 27-fold more effective inhibitory activity compared to AAZ. Five of the assayed compounds displayed low nanomolar potency (Ki ≤ 10 nM) against hCA IX, whereas five compounds were found to be endowed with excellent inhibitory potencies (Ki ≤ 5 nM) against hCA XII. The biological activity profile presented herein will be useful for designing new leads and provide candidates for preclinical investigations.

Synthesis and biological evaluation of novel 3-(quinolin-4-ylamino)benzenesulfonamides as carbonic anhydrase isoforms I and II inhibitors

Carbonic anhydrases (CAs, EC 4.2.1.1) are crucial metalloenzymes that are involved in diverse bioprocesses. We report the synthesis and biological evaluation of novel series of benzenesulfonamides incorporating un/substituted ethyl quinoline-3-carboxylate moieties. The newly synthesised compounds were in vitro evaluated as inhibitors of the cytosolic human (h) isoforms hCA I and II. Both isoforms hCA I and II were inhibited by the quinolines reported here in variable degrees: hCA I was inhibited with KIs in the range of 0.966–9.091 μM, whereas hCA II in the range of 0.083–3.594 μM. The primary 7-chloro-6-flouro substituted sulphfonamide derivative 6e (KI = 0.083 μM) proved to be the most active quinoline in inhibiting hCA II, whereas, its secondary sulfonamide analog failed to inhibit the hCA II up to 10 μM, confirming the crucial role of the primary sulphfonamide group, as a zinc-binding group for CA inhibitory activity.

Organoruthenium(II) complexes of acetazolamide potently inhibit human carbonic anhydrase isoforms I, II, IX and XII

Two acetazolamide (AAZ) complexes with ruthenium(II) η6-p-cymene chloride were synthesised, characterised and tested for their inhibitory effects on several carbonic anhydrase (CA, EC 4.2.1.1) isoforms with pharmacological applications. Against human (h) isoform hCA I, the two complexes showed inhibition constants in the range of 8.5–23.4 nM (AAZ has a KI of 250 nM), against hCA II of 0.48–4.2 nM, whereas against hCA IX of 0.63–3.8 nM and against hCA XII of 0.04–0.52 nM, respectively. These highly effective ruthenium acetazolamide derivatives against the tumour-associated CA isoforms IX and XII warrant further in vivo studies, in hypoxic tumours overexpressing these enzymes.

Design, synthesis and biological activity of selective hCAs inhibitors based on 2-(benzylsulfinyl)benzoic acid scaffold

A large library of derivatives based on the scaffold of 2-(benzylsulfinyl)benzoic acid were synthesised and tested as atypical inhibitors against four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). The exploration of the chemical space around the main functional groups led to the discovery of selective hCA IX inhibitors in the micromolar/nanomolar range, thus establishing robust structure-activity relationships within this versatile scaffold. HPLC separation of some selected chiral compounds and biological evaluation of the corresponding enantiomers was performed along with molecular modelling studies on the most active derivatives.

Activation of human α-carbonic anhydrase isoforms I, II, IV and VII with bis-histamine schiff bases and bis-spinaceamine substituted derivatives

A series of histamine bis-Schiff bases and bis-spinaceamine derivatives were synthesised and investigated as activators of four human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic hCA I, II and VII, and the membrane-associated hCA IV. All isoforms were effectively activated by the new derivatives, with activation constants in the range of 4.73–10.2 µM for hCA I, 6.15–42.1 µM for hCA II, 2.37–32.7 µM for hCA IV and 32 nM–18.7 µM for hCA VII, respectively. The nature of the spacer between the two histamine/spinaceamine units of these molecules was the main contributor to the diverse activating efficacy, with a very different fine tuning for the diverse isoforms. As CA activators recently emerged as interesting agents for enhancing cognition, in the management of CA deficiencies, or for therapy memory and artificial tissues engineering, our compounds may be considered as candidates for such applications.

Synthesis of a new series of 3-functionalised-1-phenyl-1,2,3-triazole sulfamoylbenzamides as carbonic anhydrase I, II, IV and IX inhibitors

The synthesis of a novel series of 3-functionalised benzenesulfonamides incorporating phenyl-1,2,3-triazole with an amide linker was achieved by using the “click-tail” approach. The new compounds, including the intermediates, were assayed as inhibitors of human carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I and II (cytosolic isoforms) and also for hCA IV and IX (transmembrane isoforms) taking acetazolamide as standard drug. Most of these compounds exhibited excellent activity against all these isoforms. hCA I was inhibited with Kis in the range of 50.8–966.8 nM, while the glaucoma associated hCA II was inhibited with Kis in the range of 6.5–760.0 nM. Isoform hCA IV was inhibited with Kis in the range of 65.3–957.5 nM, whereas the tumor associated hypoxia induced hCA IX was inhibited with Kis in the range of 30.8–815.9 nM. The structure activity relationship study for the 3-functionalised-1-phenyl-1,2,3-triazole sulfamoylbenzamides against these isoforms was also inferred from the results.

(Hetero)aryl substituted thiazol-2,4-yl scaffold as human carbonic anhydrase I, II, VII and XIV activators

Using histamine as lead molecule, a library of (hetero)aryl substituted thiazol-2,4-yl derivatives incorporating pyridine as proton shuttling moiety were obtained and investigated as activators of human carbonic anhydrase (CA, EC 4.2.1.1) isoforms I, II, VII and XIV. Some derivatives displayed good activating and selectivity profiles. This study provides an interesting opportunity to study the thiazole scaffold for the design of CA activators (CAAs), possibly acting on the central nervous system and targeting pathologies involving memory and learning impairments.

Exploring new structural features of the 4-[(3-methyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzenesulphonamide scaffold for the inhibition of human carbonic anhydrases

A library of 4-[(3-methyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulphonamides (EMAC8002a–m) was designed and synthesised to evaluate the effect of substituents in the positions 3 and 4 of the dihydrothiazole ring on the inhibitory potency and selectivity toward human carbonic anhydrase isoforms I, II, IX, and XII. Most of the new compounds preferentially inhibit the isoforms II and XII. Both electronic and steric features on the aryl substituent in the position 4 of the dihydrothiazole ring concur to determine the overall biological activity of these new derivatives.