hCA VII Research Articles

Synthesis, carbonic anhydrase inhibition studies and modelling investigations of phthalimide–hydantoin hybrids

A novel series of hydantoins incorporating phthalimides has been synthesised by condensation of activated phthalimides with 1-aminohydantoin and investigated for their inhibitory activity against a panel of human (h) carbonic anhydrase (CA, EC 4.2.1.1): the cytosolic isoforms hCA I, hCA II, and hCA VII, secreted isoform hCA VI, and the transmembrane hCA IX, by a stopped-flow CO2 hydrase assay. Although all newly developed compounds were totally inactive on hCA I and mainly ineffective towards hCA II, they generally exhibited moderate repressing effects on hCA VI, VII, and IX with K Is values in the submicromolar to micromolar ranges. The salts 3a and 3b, followed by derivative 5, displayed the best inhibitory activity of all the evaluated compounds and their binding mode was proposed in silico. These compounds can also be considered interesting starting points for the development of novel pharmacophores for this class of enzyme inhibitors.

Sulfonamide-incorporated bis(α-aminophosphonates) as promising carbonic anhydrase inhibitors: Design, synthesis, biological evaluation, and X-ray crystallographic studies.

A novel series of sulfonamide-incorporated bis(α-aminophosphonates) acting as effective carbonic anhydrase (CA, EC 4.2.1.1) inhibitors is reported. The synthesized bivalent ligands were tested against five human (h) isoforms, hCA I, hCA II, hCA VII, hCA IX, and hCA XIII. Such derivatives showed high activity and selectivity against the cancer-related, membrane-bound isoform hCA IX, and among them, compound 5h, tetraisopropyl (1,3-phenylenebis{[(4-sulfamoylphenyl)amino]methylene})bis(phosphonate)showed a KI of 15.1 nM, being highly selective against this isoform over all other investigated ones (hCA I/IX = 42; hCA II/IX = 6, hCA VII/IX = 3, hCA XIII/IX = 5). Therefore, compound 5h could be a potential lead for the development of selective anticancer agents. The newly developed sulfonamides were also found effective inhibitors against the cytosolic hCA XIII isoform. Compound 5i displayed the best inhibition against this isoform with a KI of 17.2 nM, equal to that of the well-known inhibitor acetazolamide (AAZ), but significantly more selective over all other tested isoforms (hCA I/XIII = 239; hCA II/XIII = 23, hCA VII/XIII = 2, hCA IX/XIII = 3) compared to AAZ.

4-Cyanamido-substituted benzenesulfonamides act as dual carbonic anhydrase and cathepsin inhibitors

A set of novel N-cyano-N-substituted 4-aminobenzenesulfonamide derivatives were synthesized and investigated for their inhibitory activity against four cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isoforms (hCA I, II, VII and XIII) and two cathepsins (S and B). N-alkyl/benzyl-substituted derivatives were revealed to be very potent inhibitors against brain-associated hCA VII, but inactive against both cathepsins. On the other hand, N-acyl-substituted derivatives displayed significant inhibitory activities against cathepsin S, but only moderate to poor inhibitory potency against hCA VII. Both hCA VII and cathepsin S have recently been validated as therapeutic targets in neuropathic pain. This study provided an excellent starting point for further structural optimization of this class of bifunctional compounds to enhance their inhibitory activity and selectivity against hCA VII and cathepsin S and to achieve new compounds with an attractive dual mechanism of action as anti-neuropathic agents.

6-Substituted Triazolyl Benzoxaboroles as Selective Carbonic Anhydrase Inhibitors: In Silico Design, Synthesis, and X-ray Crystallography.

Benzoxaborole is currently a scaffold of great relevance in medicinal chemistry. In 2016, it was reported to be a new and valuable chemotype for designing carbonic anhydrase (CA) inhibitors. Herein, using an in silico design, we report the synthesis and characterization of substituted 6-(1H-1,2,3-triazol-1-yl)benzoxaboroles. 6-Azidobenzoxaborole was described for the first time as a molecular platform to prepare libraries of inhibitors by a copper(I)-catalyzed azide-alkyne cycloaddition via a click chemistry strategy. With inhibition constants below 30 nM, some derivatives, such as compound 20, showed efficacy as selective hCA VII and IX inhibitors. The design hypothesis was validated by crystallographic investigation on the hCA II/20 adduct, which provided explanations over the different inhibition behavior observed against the five evaluated hCA isoforms. Overall, this study identified 20 as a new promising lead compound to develop novel anticancer agents targeting the tumor-associated hCA IX but also potent neuropathic pain relievers targeting hCA VII.

Inhibition Studies on Human and Mycobacterial Carbonic Anhydrases with N-((4-Sulfamoylphenyl)carbamothioyl) Amides.

A library of structurally diverse N-((4-sulfamoylphenyl)carbamothioyl) amides was synthesized by selective acylation of easily accessible 4-thioureidobenzenesulfonamide with various aliphatic, benzylic, vinylic and aromatic acyl chlorides under mild conditions. Inhibition of three α-class cytosolic human (h) carbonic anhydrases (CAs) (EC 4.2.1.1); that is, hCA I, hCA II and hCA VII and three bacterial β-CAs from Mycobacterium tuberculosis (MtCA1-MtCA3) with these sulfonamides was thereafter investigated in vitro and in silico. Many of the evaluated compounds displayed better inhibition against hCA I (KI = 13.3-87.6 nM), hCA II (KI = 5.3-384.3 nM), and hCA VII (KI = 1.1-13.5 nM) compared with acetazolamide (AAZ) as the control drug (KI values of 250, 12.5 and 2.5 nM, respectively, against hCA I, hCA II and hCA VII). The mycobacterial enzymes MtCA1 and MtCA2 were also effectively inhibited by these compounds. MtCA3 was, on the other hand, poorly inhibited by the sulfonamides reported here. The most sensitive mycobacterial enzyme to these inhibitors was MtCA2 in which 10 of the 12 evaluated compounds showed KIs (KI, the inhibitor constant) in the low nanomolar range.

Antihistamines, phenothiazine-based antipsychotics, and tricyclic antidepressants potently activate pharmacologically relevant human carbonic anhydrase isoforms II and VII

Carbonic anhydrases (CAs) are important regulators of pH homeostasis and participate in many physiological and pathological processes. CA activators (CAAs) are becoming increasingly important in the biomedical field since enhancing CA activity may have beneficial effects at neurological level. Here, we investigate selected antihistamines, phenothiazine-based antipsychotics, and tricyclic antidepressants (TCAs) as potential activators of human CAs I, II, IV, and VII. Our findings indicate that these compounds are more effective at activating hCA II and VII compared to hCA I and IV. Overall, hCA VII was the most efficiently activated isoform, particularly by phenothiazines and TCAs. This is especially relevant since hCA VII is the most abundant isoform in the central nervous system (CNS) and is implicated in neuronal signalling and bicarbonate balance regulation. This study offers additional insights into the pharmacological profiles of clinically employed drugs and sets the ground for the development of novel optimised CAAs.

2-((1H-Benzo[d]imidazol-2-yl)amino)benzo[d]thiazole-6-sulphonamides: a class of carbonic anhydrase II and VII-selective inhibitors

A small library of substituted cyclic guanidine incorporated benzothiazole-6-sulphonamides was synthesized. All obtained compounds were investigated for their inhibitory activity against the key brain-associated human carbonic anhydrase isoform hCA VII (a promising target for the treatment of neuropathic pain) and three isoforms expressed in brain and other tissues, hCA I, II, and IV. Sulphaguanidine derivatives 9a–d were inactive on the all investigated isoforms while the primary sulphonamide containing guanidines 6a–c and 7a–c were inactive towards hCA IV but displayed inhibiting properties on hCA I, II, and VII with KIs values in the low nanomolar to micromolar ranges. The results indicated that isoforms hCA II and VII were potently and selectively inhibited by these compounds, whereas the cytosolic hCA I was less sensitive to inhibition. The derivatives reported in this study might be useful for design of more potent and selective inhibitors of hCA II and VII.

Novel thiazolone-benzenesulphonamide inhibitors of human and bacterial carbonic anhydrases

A small library of novel thiazolone-benzenesulphonamides has been prepared and evaluated for their ability to inhibit three human cytosolic carbonic anhydrases (hCA I, hCA II, and hCA VII) and three bacterial carbonic anhydrases (MscCAβ, StCA1, and StCA2). All investigated hCAs were inhibited by the prepared compounds 4a–4j in the low nanomolar range. These compounds were effective hCA I inhibitors (KIs of 31.5–637.3 nM) and excellent hCA II (KIs in the range of 1.3–13.7 nM) and hCA VII inhibitors (KIs in the range of 0.9–14.6 nM). The most active analog in the series, 4-((4-oxo-5-propyl-4,5-dihydrothiazol-2-yl)amino)benzenesulphonamide 4d, strongly inhibited bacterial MscCAβ, with KI of 73.6 nM, considerably better than AAZ (KI of 625 nM). The tested compounds displayed medium inhibitory potency against StCA1 (KIs of 69.2–163.3 nM) when compared to the standard drug (KI of 59 nM). However, StCA2 was poorly inhibited by the sulphonamides reported here, with KIs in the micromolar range between 275.2 and 4875.0 nM.

Resveratrol Analogues as Dual Inhibitors of Monoamine Oxidase B and Carbonic Anhydrase VII: A New Multi-Target Combination for Neurodegenerative Diseases?

Neurodegenerative diseases (NDs) are described as multifactorial and progressive syndromes with compromised cognitive and behavioral functions. The multi-target-directed ligand (MTDL) strategy is a promising paradigm in drug discovery, potentially leading to new opportunities to manage such complex diseases. Here, we studied the dual ability of a set of resveratrol (RSV) analogs to inhibit two important targets involved in neurodegeneration. The stilbenols 1-9 were tested as inhibitors of the human monoamine oxidases (MAOs) and carbonic anhydrases (CAs). The studied compounds displayed moderate to excellent in vitro enzyme inhibitory activity against both enzymes at micromolar/nanomolar concentrations. Among them, the best compound 4 displayed potent and selective inhibition against the MAO-B isoform (IC50 MAO-A 0.43 µM vs. IC50 MAO-B 0.01 µM) with respect to the parent compound resveratrol (IC50 MAO-A 13.5 µM vs. IC50 MAO-B > 100 µM). It also demonstrated a selective inhibition activity against hCA VII (KI 0.7 µM vs. KI 4.3 µM for RSV). To evaluate the plausible binding mode of 1-9 within the two enzymes, molecular docking and dynamics studies were performed, revealing specific and significant interactions in the active sites of both targets. The new compounds are of pharmacological interest in view of their considerably reduced toxicity previously observed, their physicochemical and pharmacokinetic profiles, and their dual inhibitory ability. Compound 4 is noteworthy as a promising lead in the development of MAO and CA inhibitors with therapeutic potential in neuroprotection.

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.

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.

Recombinant human carbonic anhydrase VII: Purification, characterization, inhibition, and molecular docking studies.

Human carbonic anhydrase VII (hCA VII), a cytosolic enzyme, defends against oxidative stress by preventing reactive oxygen species from forming. In our study, first, hCA VII was cloned into Escherichia coli (One Shot Mach1-T1R) strain by using cDNA of the human brain and successfully expressed. The integrity of the plasmid generated by colony PCR was checked, and after, for protein expression, the plasmid was transformed into E. coli BL21 (DE-3) strain. hCA VII expression was observed after 6h of isopropyl-D-1-thiogalactopyranoside (IPTG) induction. The fusion protein containing hexahistidine (6xHis) was purified with 7.02EU/mg of specific activity, had 48.07% of purification yield, and approximately 21-folds using a ProbondTM nickel chelating resin affinity column. Then, both molecular mass determination and purity control of the purified recombinant enzyme was done by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The mass of the SUMO-hCA VII fusion protein was calculated as 46.77kDa. As a result of Western blot analysis using anti-His G-HRP antibody, the fusion protein was detected as approximately 45kDa. Furthermore, the characterization assays and in vitro inhibition studies were done for the recombinant enzyme. KI values of these agents were found between 0.29μM and 157.6mM. Finally, molecular docking investigations of these antibiotics were undertaken to understand further the binding interactions on the active site of this recombinant enzyme.

Sultam based Carbonic Anhydrase VII inhibitors for the management of neuropathic pain

We report a series of compounds 1–17 derived from the antiepileptic drug Sulthiame (SLT) from which both the benzenesulfonamide and the sultam moiety were retained. All compounds were tested in vitro for their inhibition activity against the human (h) Carbonic Anhydrase (CA; EC 4.2.1.1) I, II, VII, IX and XII isoforms. Among the series, derivatives 1 and 11 showed great enhancement of both inhibition potency and selectivity towards the hCA VII isoform, when compared to the reference SLT drug. The binding mode of 11 within the hCA VII active site was deciphered by means of X-ray crystallography and revealed the sultam moiety being exposed to the rim of the active site. In vivo experiments on a model of neuropathic pain induced by oxaliplatin clearly showed 11 being an effective pain relieving agent and therefore worth of further exploitation towards the validation of the hCA VII as new target for the management of neuropathies.

Quantum mechanical study on the activation mechanism of human carbonic anhydrase VII cluster model with bis-histamine schiff bases and bis-spinaceamine derivatives

The activation mechanism of human carbonic anhydrase (hCA) isoform VII, hCA VII, with histamine, histamine bis-Schiff bases and bis-spinaceamine derivatives has been investigated using quantum mechanical calculations. The DFT-D3 method has been employed to calculate in detail the electronic structure and electronic energy of different compounds and complexes throughout the reaction pathway. The model system of hCA VII included the core catalytic center, the Zn2+ ion, its three histidine ligands and a hydroxide ion or water molecule coordinated to it. Furthermore, Thr199, Glu106 and the deep water molecule were considered in the model. Five activators of this enzyme, including histamine as standard, in complex with the cluster model of hCA VII were investigated. Thermodynamic functions for the overall reaction and for the complexation between activators and hCA VII were evaluated. Our results demonstrate that the protonatable moiety of these activators participates in proton transfer reactions from the zinc-bound water molecule to the reaction medium, promoting the formation of the catalytically active zinc hydroxide species of the enzyme. The QM analysis revealed that the electrostatic interactions between activators and hCA VII are the driving force of the enzyme-activator complex formation.

Design, synthesis and biochemical evaluation of novel carbonic anhydrase inhibitors triggered by structural knowledge on hCA VII

To tackle the challenge of isoform selectivity, we explored the entrance of the cavity for selected druggable human Carbonic Anhydrases (hCAs). Based on X-ray crystallographic studies on the 4-(4-(2-chlorobenzoyl)piperazine-1-carbonyl)benzenesulfonamide in complex with the brain expressed hCA VII (PDB code: 7NC4), a series of 4-(4(hetero)aroylpiperazine-1-carbonyl)benzene-1-sulfonamides has been developed. To evaluate their capability to fit the hCA VII catalytic cavity, the newer benzenesulfonamides were preliminary investigated by means of docking simulations. Then, this series of thirteen benzenesulfonamides was synthesized and tested against selected druggable hCAs. Among them, the 4-(4-(furan-2-carbonyl)piperazine-1-carbonyl)benzenesulfonamide showed remarkable affinity towards hCA VII (Ki: 4.3 nM) and good selectivity over the physiologically widespread hCA I when compared to Topiramate (TPM).

Discovery of Potent Carbonic Anhydrase Inhibitors as Effective Anticonvulsant Agents: Drug Design, Synthesis, and In Vitro and In Vivo Investigations.

Two sets of benzenesulfonamide-based effective human carbonic anhydrase (hCA) inhibitors have been developed using the tail approach. The inhibitory action of these novel molecules was examined against four isoforms: hCA I, hCA II, hCA VII, and hCA XII. Most of the molecules disclosed low to medium nanomolar range inhibition against all tested isoforms. Some of the synthesized derivatives selectively inhibited the epilepsy-involved isoforms hCA II and hCA VII, showing low nanomolar affinity. The anticonvulsant activity of selected sulfonamides was assessed using the maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (sc-PTZ) in vivo models of epilepsy. These potent CA inhibitors effectively inhibited seizures in both epilepsy models. The most effective compounds showed long duration of action and abolished MES-induced seizures up to 6 h after drug administration. These sulfonamides were found to be orally active anticonvulsants, being nontoxic in neuronal cell lines and in animal models.

Carbonic anhydrase activation profile of indole-based derivatives

Carbonic Anhydrase Activators (CAAs) could represent a novel approach for the treatment of Alzheimer’s disease, ageing, and other conditions that require remedial achievement of spatial learning and memory therapy. Within a research project aimed at developing novel CAAs selective for certain isoforms, three series of indole-based derivatives were investigated. Enzyme activation assay on human CA I, II, VA, and VII isoforms revealed several effective micromolar activators, with promising selectivity profiles towards the brain-associated cytosolic isoform hCA VII. Molecular modelling studies suggested a theoretical model of the complex between hCA VII and the new activators and provide a possible explanation for their modulating as well as selectivity properties. Preliminary biological evaluations demonstrated that one of the most potent CAA 7 is not cytotoxic and is able to increase the release of the brain-derived neurotrophic factor (BDNF) from human microglial cells, highlighting its possible application in the treatment of CNS-related disorders.

Biochemical profiling of anti-HIV prodrug Elsulfavirine (Elpida®) and its active form VM1500A against a panel of twelve human carbonic anhydrase isoforms

The non-nucleoside reverse transcriptase inhibitor VM1500A is approved for the treatment of HIV/AIDS in its N-acyl sulphonamide prodrug form elsulfavirine (Elpida®). Biochemical profiling against twelve human carbonic anhydrase (CA, EC 4.2.1.1) isoforms showed that while elsulfavirine was a weak inhibitor of all isoforms, VM1500A potently and selectively inhibited human (h) hCA VII isoform, a proven target for the therapy of neuropathic pain. The latter is a common neurologic complication of HIV infection and we hypothesise that by using Elpida® in patients may help alleviate this debilitating symptom.

Computer-aided identification of human carbonic anhydrase isoenzyme VII inhibitors as potential antiepileptic agents

Human carbonic anhydrase (hCA) belongs to a superfamily of metalloenzymes that reversibly catalyse the hydration of carbon dioxide to give bicarbonate (HCO3 −) and proton (H+). As HCO3 − ions play an important role in neuronal signalling hence, hCA enzymes are an attractive target for antiepileptic drugs. Out of all the isoforms, hCA VII is predominantly expressed in the brain cortex and hippocampus region, which are the most affected area during seizure activity. Hence, we have identified some hCA VII inhibitors employing computational tools like atom-based 3D quantitative structure–activity relationship (QSAR), auto-QSAR, pharmacophore-based virtual screening, molecular docking, and molecular dynamics (MD) simulations. Atom-based 3D QSAR modelling outperformed auto-QSAR with an R 2 and Q 2 value of 0.9634 and 0.9646, respectively. A four-feature pharmacophore model (AADR_1) was developed and a focussed library of around 3,00,000 compounds was screened. Compounds with a phase screen score >2.40 were selected for docking studies. The activity of the selected hits was predicted employing the developed 3D QSAR model. Finally, three compounds were taken up for the MD simulation studies which also suggest that the identified hits might form a stable complex with hCA VII enzyme. A comparative docking study was also done with other hCA isoforms like I, II, IV, IX, and XII to examine the selectivity of the identified hits towards hCA VII. Based on these studies, three hits have been identified as potential hCA VII inhibitor which is drug-like molecules. Further, in vitro studies are required to develop leads from these identified hits. Communicated by Ramaswamy H. Sarma

Activation of carbonic anhydrases from human brain by amino alcohol oxime ethers: towards human carbonic anhydrase VII selective activators

The synthesis and carbonic anhydrase (CA; EC 4.2.1.1) activating effects of a series of oxime ether-based amino alcohols towards four human (h) CA isoforms expressed in human brain, hCA I, II, IV and VII, are described. Most investigated amino alcohol derivatives induced a consistent activation of the tested CAs, with KAs spanning from a low micromolar to a medium nanomolar range. Specifically, hCA II and VII, putative main CA targets when central nervous system (CNS) diseases are concerned, were most efficiently activated by these oxime ether derivatives. Furthermore, a multitude of selective hCA VII activators were identified. As hCA VII is one of the key isoforms involved in brain metabolism and other brain functions, the identified potent and selective hCA VII activators may be considered of interest for investigations of various therapeutic applications or as lead compounds in search of even more potent and selective CA activators.