Human Carbonic Anhydrase Research Articles

Identification of New Carbonic Anhydrase VII Inhibitors by Structure-Based Virtual Screening

Human carbonic anhydrase VII (hCA VII) constitutes a promising molecular target for the treatment of epileptic seizures and other central nervous system disorders due to its almost exclusive expression in neurons. Achieving isoform selectivity is one of the main challenges for the discovery of new hCA inhibitors, since nonspecific inhibition may lead to tolerance and side effects. In the present work, we report the development of a molecular docking protocol based on AutoDock4Zn for the search of new hCA VII inhibitors by virtual screening. The docking protocol was applied to the screening of two sets of compounds: a ZINC15 subset of sulfur-containing structures and an in-house library consisting of synthetic and commercial candidates (including approved drugs). Five compounds were selected from the first screening campaign and three from the second one, and they were tested in vitro against the enzyme. Among the eight selected structures, four showed Ki values in the low nanomolar range. These confirmed hits include three approved drugs: meloxicam, piroxicam, and nitrofurantoin, which also showed good selectivity for hCA VII versus hCA II.

Synthesis and biological evaluation of coumarin-thiazole hybrids as selective carbonic anhydrase IX and XII inhibitors.

A series of coumarin-linked thiazoles (6a-p) was synthesized and the synthesized compounds were evaluated against human carbonic anhydrases (hCAs) IX and XII, which have been implicated in cancer. All the compounds exhibited selective inhibition of both isoforms. The designed compounds inhibited hCA IX in a moderate nanomolar to submicromolar range. The hCA XII was inhibited in a low to moderate nanomolar range. Compound 6o exhibited the best inhibition of hCA XII with a Ki value of 91.1 nM. The hydrolyzed form of compound 6o also exhibited favorable interactions as well as good docking scores with both the isoforms. Hence, this compound can be taken as a template for the design of selective and potent hCA XII inhibitors.

Simultaneous carbon dioxide sequestration and utilization for cadaverine production using dual promoters in engineered Escherichia coli strains

Human carbonic anhydrase II (hCAII) is a rapid-acting zinc-metalloenzyme that catalyzes CO2 hydration reversibly, with encouraging applications in carbon capture, sequestration, and utilization (CCSU). However, biocatalyst durability is a major challenge. Herein, hCAII is emphasized in 4 different Escherichia coli strains and designated under dual promoters from sigma factor 70 (σ70) and heat shock protein (HSP70A) to suppress the usage of inducer and stimulate activity in heat environments. As a result, hCAII under high-efficient dual promoters regulation retained high residual activity in CO2 biomineralization of 68.8 % after 4 cycles at 40 °C. Moreover, co-expression of CAC9 with lysine decarboxylase (CadA) simultaneously sequestered CO2 release up to 95.7 % and increased cadaverine titer from 18.0 to 36.7 g/L by using E. coli MG1655. The remnant biomass from cadaverine synthesis sustained converting CO2 to 57.9 mg-CaCO3. Thus, the dual promoters design demonstrated the promising potential for CCSU through simultaneous CO2 utilization and cadaverine synthesis.

Development of Hydrogen Sulfide-Releasing Carbonic Anhydrases IX- and XII-Selective Inhibitors with Enhanced Antihyperalgesic Action in a Rat Model of Arthritis.

An effective therapeutic approach based on the anti-inflammatory action of hydrogen sulfide (H2S) and inhibition of carbonic anhydrases (CAs) IX and XII is proposed here for the management of arthritis. H2S is a human gasotransmitter that modulates inflammatory response at low concentrations. Inhibition of CAs IX and XII can repristinate normal pH in the acidic inflamed synovial fluid, alleviating arthritis symptoms. We report here the design of H2S donor—CA inhibitor (CAI) hybrid derivatives. The latter were tested in vitro as inhibitors of human CAs I, II, IV, IX, and XII, showing a markedly increased inhibition potency/isoform selectivity compared to the CAI synthetic precursors. The best compounds demonstrated the ability to consistently release H2S and produce a potent pain-relieving effect in a rat model of arthritis. Compound 26 completely reverted the pain state 45 min after administration with enhanced antihyperalgesic effect in vivo compared to the single H2S donor, CAI fragment, or their co-administration.

Sulfonamide-phosphonate hybrids as new carbonic anhydrase inhibitors: In vitro enzymatic inhibition, molecular modeling, and ADMET prediction

In the presented work, we report the synthesis of a new series of sulfonamide-phosphonate hybrids 4a-m. These newly synthesized compounds were assessed for their inhibitory effects toward two human carbonic anhydrase isoforms I and II (hCA I and II). These examined isoforms were as well inhibited by the most of prepared sulfonamide-phosphonates in comparison to standard inhibitor acetazolamide. Obtained data exhibited that compounds 4b-m with Ki values in the range of 8.11–48.08 nM were more potent than standard drug acetazolamide with Ki value of 64.52 Nm against hCA I. Moreover, all the synthesized compounds (Ki values = 7.08–64.24 nM), with the exception of compound 4b, were more potent than acetazolamide (Ki value = 75.36) against hCA II. In particular, sulfonamide-phosphonates 4l and 4j, respectively, with substituents 5‑chloro-2-nitro and 2,3-dichloro emerged as the most potent hCA inhibitors. Thereafter, the molecular docking of compounds 4l and 4j at hCA I and II active sites was performed and the obtained results revealed that these compounds interacted whit the important amino acids of the active site. Finally, the predicted parameters of Lipinski's rule of five, ADME, and toxicity analysis showed that compounds 4l and 4j had good drug-likeness and acceptable physicochemical properties. Therefore, the hybridization of the sulfonamide and phosphonate moieties could be promising strategy for achieve to potent lead compounds for inhibition of hCA.

Antioxidant, Antidiabetic, Anticholinergic, and Antiglaucoma Effects of Magnofluorine.

Magnofluorine, a secondary metabolite commonly found in various plants, has pharmacological potential; however, its antioxidant and enzyme inhibition effects have not been investigated. We investigated the antioxidant potential of Magnofluorine using bioanalytical assays with 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+), N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD•+), and 1,1-diphenyl-2-picrylhydrazyl (DPPH•) scavenging abilities and K3[Fe(CN)6] and Cu2+ reduction abilities. Further, we compared the effects of Magnofluorine and butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), α-Tocopherol, and Trolox as positive antioxidant controls. According to the analysis results, Magnofluorine removed 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals with an IC50 value of 10.58 μg/mL. The IC50 values of BHA, BHT, Trolox, and α-Tocopherol were 10.10 μg/mL, 25.95 μg/mL, 7.059 μg/mL, and 11.31 μg/mL, respectively. Our results indicated that the DPPH· scavenging effect of Magnofluorine was similar to that of BHA, close to that of Trolox, and better than that of BHT and α-tocopherol. The inhibition effect of Magnofluorine was examined against enzymes, such as acetylcholinesterase (AChE), α-glycosidase, butyrylcholinesterase (BChE), and human carbonic anhydrase II (hCA II), which are linked to global disorders, such as diabetes, Alzheimer’s disease (AD), and glaucoma. Magnofluorine inhibited these metabolic enzymes with Ki values of 10.251.94, 5.991.79, 25.411.10, and 30.563.36 nM, respectively. Thus, Magnofluorine, which has been proven to be an antioxidant, antidiabetic, and anticholinergic in our study, can treat glaucoma. In addition, molecular docking was performed to understand the interactions between Magnofluorine and target enzymes BChE (D: 6T9P), hCA II (A:3HS4), AChE (B:4EY7), and α-glycosidase (C:5NN8). The results suggest that Magnofluorine may be an important compound in the transition from natural sources to industrial applications, especially new drugs.

New Sulfamethoxazole Derivatives as Selective Carbonic Anhydrase IX and XII Inhibitors: Design, Synthesis, Cytotoxic Activity and Molecular Modeling

In this study new sulphamethoxazole derivatives (S1–S4, S6–S12, and S14–S22) were designed and synthesized and their structures were fully characterized and validated using NMR, mass, and IR spectroscopy, as well as elemental analyses. All new derivatives (S1–S22) were assayed against human carbonic anhydrase (hCAs IX and XII) for their inhibitory activities. hCAs IX and XII were chosen due to the fact that CAIX expression is recognized as a hypoxia marker with a poor prognosis in breast cancer. When compared to Dorzolamide HCl as a standard reference, derivatives S2, S3, S8, S9, and S15 had the most effective inhibition with low IC50 values. The active compounds were further evaluated against hCAs I and II inhibitory activity and compounds S8, S9 and S15 showed the least inhibitory effect compared to the reference standard, acetazolamide, indicating that their effect in normal cells is the lowest. Cell viability tests for the selected compounds were carried out on MCF7 (normoxia and hypoxia) and on the normal breast cell line (MCF10a) with Staurosporine as a standard. The results showed that compound S15 had a highly potent cytotoxic effect. Furthermore, cell cycle analysis results showed that compound S15 triggered cell cycle arrest and apoptosis in G1/S of MCF7 cancer cells. Finally, molecular docking was performed to point out the possible explanation for the vital structural features and key-interactions exerted by our ligands with hCAs IX and XII that might share additional designs and highlight possible leads for a hopeful anticancer agent. Consequently, sulphamethoxazole Derivative S15 could be the potential lead for emerging selective cytotoxic compounds directing h CAs IX and XII.

Synthesis and In Vitro Human Carbonic Anhydrase I and II Inhibition Properties of Some Novel Hydrazone Compounds Containing an Aryl Sulfonate Moiety

The aim of this research was to evaluate the enzyme inhibitory potential of some new hydrazone derivatives bearing an aryl sulfonate moiety against the human carbonic anhydrase isoenzymes I and II (hCA I and II), which were attained from commercial suppliers. In the current study, the structures of targeted molecules (5-8) were characterized by some spectroscopic techniques. The inhibition capacities of the compounds on hCA I and II activities were examined by employing the esterase activity method under in vitro conditions. The IC50 values of the tested molecules were determined in the range of 15.7-135.2 µM against hCA I and in the range of 13.5-76.3 µM against hCA II. Among them, compound 7 exhibited the highest activity against both hCA I and II. The inhibitory activity results of all tested compounds were also compared to the standard drug Acetazolamide (AAZ). It was established that some of the tested molecules showed the inhibitory activities close to AAZ.

Structure and Dynamics of the Isozymes II and IX of Human Carbonic Anhydrase.

Human carbonic anhydrases (HCAs) are responsible for the pH control and sensing in our body and constitute key components in the central pH paradigm connected to cancer therapeutics. However, little or no molecular level studies are available on the pH-dependent stability and functional dynamics of the known isozymes of HCA. The main objective of this Article is to report the first bench-marking study on the structure and dynamics of the two most efficient isozymes, HCA II and IX, at neutral pH using classical molecular dynamics (MD) and constant pH MD (CpHMD) simulations combined with umbrella sampling, transition path sampling, and Markov state models. Starting from the known crystal structures of HCA II and the monomeric catalytic domain of HCA IX (labeled as HCA IX-c), we have generated classical MD and CpHMD trajectories (of length 1 μs each). In all cases, the overall stability, RMSD, and secondary structure segments of the two isozymes are found to be quite similar. Functionally important dynamics of these two enzymes have been probed in terms of active site hydration, coordination of the Zn(II) ion to a transient excess water, and the formation of putative proton transfer paths. The most important difference between the two isozymes is observed for the side-chain fluctuations of His-64 that is expected to shuttle an excess proton out of the active site as a part of the rate-determining intramolecular proton transfer reaction. The relative stability of the stable inward and outward conformations of the His-64 side-chain and the underlying free energy surfaces are found to depend strongly on the isozyme. In each case, a lower free energy barrier is detected between predominantly inward conformations from predominantly outward ones when simulated under constant pH conditions. The kinetic rate constants of interconversion between different free energy basins are found to span 107–108 s–1 with faster conformational transitions predicted at constant pH condition. The estimated rate constants and free energies are expected to validate if the fluctuation of the His-64 side-chain in HCA IX may have a significance similar to that known in the multistep catalytic cycle of HCA II.

Structure-assisted design of carborane inhibitors of human carbonic anhydrase IX

Structure-assisted design of carborane inhibitors of human carbonic anhydrase IX

Synthesis of Sulfonamides Incorporating Piperidinyl-Hydrazidoureido and Piperidinyl-Hydrazidothioureido Moieties and Their Carbonic Anhydrase I, II, IX and XII Inhibitory Activity.

Here we report a small library of hydrazinocarbonyl-ureido and thioureido benzenesulfonamide derivatives, designed and synthesized as potent and selective human carbonic anhydrase inhibitors (hCAIs). The synthesized compounds were evaluated against isoforms hCA I, II, IX and XII using acetazolamide (AAZ) as standard inhibitor. Several urea and thiourea derivatives showed inhibitory activity at low nanomolar levels with selectivity against the cytosolic hCA II isoform, as well as the transmembrane, tumor-associated enzymes hCA IX and XII. The thiourea derivatives showed enhanced potency as compared to urea analogues. Additionally, eight compounds 5g, 5m, 5o, 5q, 6l, 6j, 6o and 6u were selected for docking analysis on isoform I, II, IX, XII to illustrate the potential interaction with the enzyme to better understand the activity against the different isoforms.

New PEPPSI-Pd-NHC complexes bearing 4-hydroxyphenylethyl group: Synthesis, characterization, molecular docking, and bioactivity properties.

Five 4-hydroxyphenylethyl substituted pyridine enhanced, precatalyst, preparation, stabilization, and initiation-Pd-N-heterocyclic carbene(PEPPSI-Pd-NHC) complexes are synthesized in a straightforward way. All PEPPSI-Pd-NHC complexes were prepared by mixing 4-hydroxyphenylethyl substituted NHC precursors, palladium chloride, potassium carbonate, and potassium bromide in pyridine. All complexes were screened for human carbonic anhydrase I (hCA I)and hCA II, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glucosidase (α-Glu) inhibitory activities. The ChE inhibitory activities of the new PEPPSI-Pd-NHC complexes bearing the 4-hydroxyphenylethyl group (1a-e) against α-Glu, AChE, and BChE were determined by the Tao and Ellman methods. The results indicated that all the synthetic complexes exhibited potent inhibitory activities against all targets as compared to the standard inhibitors, revealed by IC50 values. The Ki values of the new PEPPSI-Pd-NHC complexes 1a-e for hCA I, hCA II, AChE, BChE, and α-Glu were obtained in the ranges of 18.98-32.65, 22.95-38.13, 3.67-11.65, 4.09-9.36, 186.92-287.45 µM, respectively. Among the synthesized complexes, the most potent complexes were 1c toward hCA I and II with Ki values 18.98 and 22.95 µM, and 1d toward AChE and BChE with Ki = 3.67 and 4.09 µM, respectively.

New Pyridinium Salt Derivatives of 2-(Hydrazinocarbonyl)-3-phenyl-1H-indole-5- sulfonamide as Selective Inhibitors of Tumour-Related Human Carbonic Anhydrase Isoforms IX and XII.

The positively charged membrane impermeant sulfonamides were evaluated as a remarkable class of carbonic anhydrase inhibitors (CAIs) previously. Without affecting the human carbonic anhydrase (hCA), cytosolic isoforms hCA I and II, inhibition of two membrane-associated isoforms hCA IX and XII especially overexpressed in hypoxic tumour cells, makes the pyridinium salt derivatives potent promising therapeutic agents. A novel series of tri, tetra, and cyclo-substituted pyridinium salt derivatives of the lead compound 2- (hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide has been prepared by using sixteen different pyrylium salts, for the search of selective inhibitors of transmembrane tumour-associated human carbonic anhydrase hCA IX and XII. Molecular modeling studies were carried out to understand and rationalize the in vitro enzyme inhibition data. Six of the new compounds showed good inhibitory profiles with low nanomolar range (< 100 nM) against hCA IX/XII, and compound 5 showed excellent potency with Ki values lower than 10 nM. In addition, molecular modelling studies have presented the possible binding modes of the ligands. Most of the compounds displayed potent inhibitory activity against the tumor-associated hCA IX and XII in the low nanomolar range and selectivity over the off-targeted isoforms hCA I and II. Due to their cationic structure and membrane-impermeant behavior, it is also expected to maximize the selectivity over cytosolic isoforms hCA I/II while inhibiting tumor overexpressed isoforms hCA XI/XII of new compounds in in vivo conditions.

Exploring enzyme inhibition profiles of novel halogenated chalcone derivatives on some metabolic enzymes: Synthesis, characterization and molecular modeling studies

Enzyme inhibition is a very active area of research in drug design and development. Chalcone derivatives have a broad enzyme inhibitory activity and function as potential molecules in the development of new drugs. In this study, the synthesized novel halogenated chalcones with bromobenzyl and methoxyphenyl moieties were evaluated toward the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes and human erythrocyte carbonic anhydrase I (hCA I), and II (hCA II) isoenzymes. They showed highly potent inhibition ability toward AChE with Ki values of 1.83 ± 0.21–11.19 ± 0.96 nM and BChE with Ki values of 3.35 ± 0.91–26.70 ± 4.26 nM; hCA I with Ki values of 29.41 ± 3.14–57.63 ± 4.95 nM, and hCA II with Ki values of 24.00 ± 5.39–54.74 ± 1.65 nM. Among the tested enzyme inhibitions, compounds 14 and 13 were the most active compounds against AChE and BChE. Docking studies were performed to the most active compounds against AChE, BChE, hCA I and hCA II to propose a binding mode in the active site and molecular dynamics simulations were studied to check the molecular interactions and the stability of the ligands in the active site. The results may contribute to the development of new drugs particularly to treat some global disorders including Alzheimer’s disease (AD), glaucoma, and diabetes.

Novel Insights on Human Carbonic Anhydrase Inhibitors Based on Coumalic Acid: Design, Synthesis, Molecular Modeling Investigation, and Biological Studies.

Human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms IX and XII are overexpressed in solid hypoxic tumors, and they are considered as prognostic tools and therapeutic targets for cancer. Based on a molecular simplification of the well-known coumarin scaffold, we developed a new series of derivatives of the pyran-2-one core. The new compounds are endowed with potent and selective inhibitory activity against the tumor-related hCA isoforms IX and XII, in the low nanomolar range, whereas they are inactive against the two cytosolic off-targets hCA I and II. The compounds exhibiting the best hCA inhibition were further investigated against the breast adenocarcinoma cell line (MCF7) in hypoxic conditions, evaluating their ability to eventually synergize with doxorubicin. The compounds’ biocompatibility on healthy cells was also tested and confirmed on Human Gingival Fibroblasts (HGFs). Furthermore, the possible binding mode of all compounds to the active site of the tumor-associated human CA IX was investigated by computational techniques which predicted the binding conformations and the persistency of binding poses within the active site of the enzyme, furnishing relevant data for the design of tight binding inhibitors.

Thiosemicarbazide-Substituted Coumarins as Selective Inhibitors of the Tumor Associated Human Carbonic Anhydrases IX and XII.

A novel series of thiosemicarbazide-substituted coumarins was synthesized and the inhibitory effects against four physiologically relevant carbonic anhydrase isoforms I, II, IX and XII showed selective activities on the tumor-associated IX and XII isozymes. Molecular modeling studies on selected compounds 14a and 22a were performed. The binding modes of such compounds were determined assuming their enzymatically active structures (i.e., cinnamic acid) in the thermodynamically favored, and not previously explored, E geometry. Molecular modelling suggests multiple interactions within the enzymatic cavity and may explain the high potency and selectivity reported for the hCAs IX and XII.

Physical–chemical studies of new, versatile carbazole derivatives and zinc complexes: Their synthesis, investigation of in vitro inhibitory effects on α‐glucosidase and human erythrocyte carbonic anhydrase I and II isoenzymes

In this study, two novel zinc–phthalocyanine complexes (4a and 6a), containing carbazole rings, were investigated. The first phthalocyanine 4a has been synthesized from 4‐(2‐(3‐acetyl‐9H‐carbazol‐9‐yl)ethoxy at the peripheral position. Likewise, the second phthalocyanine 6a has been synthesized from 4‐(2‐(3‐cinnamoyl‐9H‐carbazol‐9‐yl)ethoxy at the same position. The new phthalocyanines (Pcs) and their ligands are soluble in organic solvents, such as dimethylformamide, dimethyl sulfoxide, dichloromethane, ethyl acetate, and methanol/ethanol. The Zn (II) complexes were characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance (NMR) (except 13C NMR for complexes), elemental analysis, mass spectrometry, and UV–Vis spectroscopy. Also, photophysical and photochemical properties and cyclic voltammogram for the N‐substituted carbazole zinc–Pcs have been reported. The evaluation of carbazole Zn (II) complexes (4a and 6a) and ligands (3, 4, 5, and 6) for in vitro α‐glucosidase inhibitory activities was performed compared to standard drugs acarbose, and kinetic studies were also carried out to determine their inhibition modes. The inhibitory effects of these new Pcs (4a and 6a) and their ligands (3, 4, 5, and 6) on human erythrocytes carbonic anhydrase I (hCA I) and II (hCA II) isoenzymes were investigated. Moreover, in silico studies were also carried out to better investigate the interactions of N‐substituted carbazole containing zinc Pcs with the active sites of hCA I and II isoforms.

Improvement of photochemical and enzyme inhibition properties of new BODIPY compound by conjugation with cisplatin

In this study, new BODIPY complexes were synthesized, characterized and investigated for their some metabolic enzyme inhibition and photophysicochemical properties. BODIPY central core unit was synthesized by using conventional method followed by Suzuki-Miyaura Cross-Coupling reaction. After converting the obtained compound into the diiodinated derivative in the presence of HIO3/I2, quaternization of BODIPY derivative was achieved with cisplatin [cis-PtCl(NO3)(NH3)2] over the pyridine segment. Characterizations of all intermediates and the target compound were determined by 1H-NMR, 13C-NMR and MS spectroscopic methods. The potentials of the synthesized compounds in photodynamic therapy applications were determined by calculating the fluorescence, singlet oxygen and photodegradation quantum yields. Also, all complexes were screened for human carbonic anhydrase I, II (hCAs I and II), acetylcholinesterase (AChE), butyryl cholinesterase (BChE), and α-glucosidase inhibitory activities. Results indicated that all the synthesized complexes exhibited potent inhibitory activities against standard inhibitors, revealed by IC50 values.

Isolation of Some Phenolic Compounds from Plantago subulata L. and Determination of Their Antidiabetic, Anticholinesterase, Antiepileptic and Antioxidant Activity.

In the current study, some phenolic compounds, including acteoside, isoacteoside, echinacoside, and arenarioside purified and characterized from Plantago subulata. These compounds were tested for its antioxidant potential, including Fe3+ and Cu2+ reductive ability and Fe2+ chelating effects. The inhibitory effects of isolated phenolic compounds were tested towards human carbonic anhydrase I and II isoenzymes (hCA I and hCA II), butyrylcholinesterase (BChE) acetylcholinesterase (AChE), aldose reductase (AR) and α-glycosidase (α-gly). Ki values were found these compounds in range of 0.24±0.05-1.38±0.34 μM against hCA I, 0.194±0.018-1.03±0.06 μM against hCA II, 0.043±0.01-0.154±0.02 μM against AChE, 3.92±1.08-11.93±4.45 μM against BChE, 0.082±0.0008-1.68±0.42 μM against AR, and 6.93±2.74-17.17±6.70 μM against α-glycosidase. As a result, isolated compounds displayed inhibition effects against studied all metabolic enzymes. They are promising candidates for treating disorders like Alzheimer's disease, diabetes mellitus, glaucoma, leukemia, and epilepsy.

Sulfonamide diuretic azosemide as an efficient carbonic anhydrase inhibitor

Azosemide, a sulfonamide loop diuretic, has been investigated in vitro as inhibitor of human Carbonic Anhydrases (hCAs, EC 4.2.1.1). The inhibition profile shows the same high potency as the structural analog loop diuretic furosemide towards the most abundant isoforms hCA I and hCA II. On the other hand, replacement of the carboxylic group of furosemide with the tetrazole bioisostere in azosemide leads to important differences in activity for other isoforms such as hCA XII and in particular against hCA IV and XIV, thus explaining possible differences of potency in vivo. The higher inhibitory potency of azosemide compared to furosemide and bumetamide against hCA VII might also explain its efficacy in the management of epilepsy. By means of X-ray crystallographic studies we elucidated the binding mode of azosemide to hCA I and hCA II, which is rather different from that of furosemide when considering the thienylmethylamino and furanylmethylamino fragments present in the two diuretics.