BACKGROUND AND PURPOSE:Carbonic anhydrase (CA) IX expression is increased upon hypoxia and has been proposed as a therapeutic target since it has been associated with poor prognosis, tumor progression and pH regulation. The aim of this study was to evaluate the antitumor activity of a high CAIX-affinity indanesulfonamide (11c) combined with irradiation, compared with the general CA inhibitor acetazolamide (AZA).MATERIAL AND METHODS:HT-29 carcinoma cells with or without (genetic knockdown, KD) CAIX expression were incubated with 11c/AZA under different oxygen levels and proliferation, apoptosis and radiosensitivity were evaluated. 11c/AZA was administered intravenously (1×/day; 5 days) to tumor-bearing mice and tumor irradiation (10 Gy) was performed at day 3 of the injection period. Tumor growth and potential treatment toxicity were monitored (3×/week).RESULTS:Treatment with 11c/AZA alone resulted in tumor regression, which was further increased in CAIX expressing cells by combining 11c with irradiation. AZA demonstrated also an additional effect in the KD tumors when combined with irradiation. CAIX inhibition in vitro significantly reduced proliferation and increased apoptosis upon hypoxia exposure without affecting intrinsic radiosensitivity.CONCLUSIONS:Specific inhibition of CAIX activity enhanced the effect of tumor irradiation and might, therefore, be an attractive strategy to improve overall cancer treatment.

Background and purpose Carbonic anhydrase (CA) IX is suggested to be an endogenous marker of hypoxia. Fluorescent sulfonamides with a high affinity for CA IX (CAI) have been developed and shown to bind to cells only when CA IX protein was expressed and while cells were hypoxic. The aim of this study was to investigate the in vivo CAI binding properties in a xenograft tumour model using fluorescent imaging. Materials and methods NMRI-nu mice subcutaneously transplanted with HT-29 colorectal tumours were treated with 7% oxygen or with nicotinamide and carbogen and were compared with control animals. CAI accumulation was monitored by non-invasive fluorescent imaging. Results Specific CAI accumulation could be observed in delineated tumour areas as compared with a non-sulfonamide analogue (P<0.01). Administration of nicotinamide and carbogen, decreasing acute and chronic hypoxia, respectively, prevented CAI accumulation (P<0.05). When treated with 7% oxygen breathing, a 3-fold higher CAI accumulation (P<0.01) was observed. Furthermore, the bound CAI fraction was rapidly reduced upon tumour reoxygenation (P<0.01). Conclusions Our in vivo imaging results confirm previous in vitro data demonstrating that CAI binding and retention require exposure to hypoxia. Fluorescent labelled sulfonamides provide a powerful tool to visualize hypoxia response. An important step is made towards clinical applicability, indicating the potential of patient selection for CA IX-directed therapies.

Seizures are one of the most common neurological disorders in clinical medicine. Triggering mechanisms by which seizures form remain unclear, but are related to a rapid change in ionic composition, including an increase of intracellular potassium concentration and pH shifts within the brain. pH buffering of extra- and intracellular spaces is mainly carried out by the CO(2)/ HCO(3)(-) buffer, the equilibration of the two species being assured by the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). Some carbonic anhydrase inhibitors (CAIs) are used as anticonvulsants in the treatment of epilepsy. In this review, we will describe the link between CA and seizures on the basis of several putative mechanisms. Several CA isozymes have been pointed out for their contribution to epileptiform activity. An overview of the CA isozyme expression in the brain and of their specifics roles is also discussed. This article reviews the research achievements published on CA inhibitors, clinically used as anticonvulsant and those under development.

We report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors prepared regio- and stereoselectively by reacting sulfanilamide with ethyl trans-phenylglycidate in the presence of cobalt(II) chloride. Various derivatizations of the ester moiety in the parent compound led to a small library of derivatives (2R,3R and 2S,3S) which displayed interesting inhibitory activities towards the human tumor-associated isoform CA IX. One of the new compounds shows high selectivity in inhibiting hCA IX compared to the two physiologically relevant, cytosolic isozymes hCA I and hCA II. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active sites of hCA IX and hCA II.

A three-dimensional pharmacophore model of CA IX inhibitors was generated and used to screen the ZINC database of commercially available compounds. The hits were docked in a CA IX homology model. By visualizing the binding mode and score of these compounds, six derivatives were selected and evaluated for their inhibitory potency against CA IX. A highly active CA IX inhibitor was identified which may be used as a lead to design novel such derivatives.

The carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous zinc enzymes present in prokaryotes and eukaryotes, being encoded by four distinct, evolutionarily unrelated gene families: the R-CAs (present in vertebrates, bacteria, algae, and cytoplasm of green plants), the -CAs (predominantly in bacteria, algae, and chloroplasts of both monoand dicotyledonds), the γ-CAs (mainly in archaea and some bacteria), and the δ-CAs, present in some marine diatoms. In higher vertebrates, including humans, 14 R-CAs isozymes have been discovered and numbered from I to XIV. Recently, an additional CA isozyme (CA XV) has been highlighted in several animal species except in humans and chimpanzees. CA isozymes are distinguished, on one hand, by different subcellular and tissue distribution and, on the other hand, by a modulation of the catalytic activity (CO2 hydration) due to structural differences present in their active site. CAs are cytosolic (isozymes I, II, III, VII, and XIII), mitochondrial (isozymes VA and VB), membrane-bound (isozymes IV, IX, XII, XIV, and XV) or secreted in the saliva (isozyme VI). Other isozymes, called CARPs (carbonic anhydrase related proteins) are acatalytic and are present in the cytosol. The loss of classical CA catalytic activity is the consequence of a lack of histidine residues required to bind the zinc ion. CAs catalyze a very simple reaction, the interconversion between carbon dioxide and bicarbonate ion (CO2 + H2O T HCO3 + H). They are thus involved in crucial physiological processes connected with respiration and transport of CO2/bicarbonate between metabolizing tissues and lungs, pH and CO2 homeostasis, electrolytes secretion in a variety of tissues/organs, biosynthetic reactions (such as gluconeogenesis, lipogenesis, and ureagenesis), bone resorption, and calcification. Moreover, some recent evidence suggests the role of CAs in pathological processes (i.e., tumorigenicity, obesity, epilepsy). Many of these isozymes are targets of inhibitors with different therapeutic applications. Clinical CA inhibitors include acetazolamide, methazolamide, and ethoxzolamide. Recently, it has been shown that two CA isozymes are prominently associated with cancer, where they are involved in pH regulation and tumor progression. Recent advances in understanding their exact role led to the development of new carbonic anhydrase inhibitors as therapeutic and/or diagnostic agents.

Nonsteroidal anti-inflammatory drugs (NSAIDs) represent the most commonly used medications for the treatment of pain and inflammation, but numerous well-described adverse drug reactions (ADRs) limit their use. These drugs act via the inhibition of cyclooxygenase (COX) enzyme of which at least two isoforms were described: COX-1 which plays important roles in homeostatic processes such as thrombogenesis and homeostasis of the gastrointestinal tract and kidneys and COX-2 expressed in pathological conditions such as inflammation or cancer proliferation. Selective COX-2 inhibitors or "coxibs" were initially developed as a therapeutic strategy to avoid not only the gastrointestinal but also the renal and cardiovascular side effects of non specific NSAIDs. However, this class of drug did not fulfill all their promises. Indeed, numerous unexpected side effects have limited their use and some of them have been withdrawn or suspended from the market for different safety reasons including cardiovascular, hepatic and skin adverse reactions. For instance, cardiovascular warnings have been applied to the whole class of coxibs and more recently for all classical NSAIDs as well. However, differences in the chemical structures should be taken into consideration in order to discriminate between coxibs and the development of some ADRs of which renal events and hypertension. The aim of this paper is to focus on the differences in chemical structures of all marketed COX-2 inhibitors and their unexpected effects on carbonic anhydrase in order to provide non-COX-2 mechanistic insights into some of the differences observed between coxibs.

The marketed antiepileptic drugs can not solve entirely the problem of seizure in patients suffering from refractory epilepsies. Therefore, new anticonvulsant compounds structurally and pharmacologically different of the currently prescribed drugs are needed. Carbonic anhydrase (CA) inhibitors are known to act as anticonvulsant since several decades while the link between CA and seizure is not straightforward. However, the recent discovery that several CA isozymes are expressed in brain and the better knowledge of their physiological/pathological role, lead to the emergence of new CA inhibitors with anticonvulsant effect including: analogues of acetazolamide, analogues of topiramate, aromatic or heterocyclic sulfonamides incorporating valproyl or adamantyl moieties. Different strategies are developed for the design of new more selective CA inhibitors with anticonvulsant properties.

A small library of indanesulfonamides was screened for the inhibition of the human carbonic anhydrase (CA, EC 4.2.1.1) isoforms involved in neuronal excitation, that is, isoforms VII, XII and XIV. These CA isoforms are becoming interesting target for the design of agents useful for the treatment of epilepsy. The inhibition pattern of these indanesulfonamide compounds towards these three isoforms was excellent, with many nanomolar inhibitors detected (KIs in the range of 0.78–10nM against hCA VII; 0.32–56nM against hCA XII, and 0.47–1030nM against hCA XIV, respectively). The maximal electroshock seizure (MES) test performed on mice showed a good anticonvulsant activity for some compounds which protected the mice against convulsions in the 50–62.5% range at a dose of 50mg/kg. In parallel, the blood–brain barrier passive permeation of these sulfonamides was also estimated by using a computational approach.

The marketed antiepileptic drugs can not solve entirely the problem of seizure in patients suffering from refractory epilepsies. Therefore, new anticonvulsant compounds structurally and pharmacologically different of the currently prescribed drugs are needed. Carbonic anhydrase (CA) inhibitors are known to act as anticonvulsant since several decades while the link between CA and seizure is not straightforward. However, the recent discovery that several CA isozymes are expressed in brain and the better knowledge of their physiological/pathological role, lead to the emergence of new CA inhibitors with anticonvulsant effect including: analogues of acetazolamide, analogues of topiramate, aromatic or heterocyclic sulfonamides incorporating valproyl or adamantyl moieties. Different strategies are developed for the design of new more selective CA inhibitors with anticonvulsant properties.

Cyclooxygenase is a key enzyme responsible for metabolisation of arachidonic acid into prostaglandins and thromboxane. This enzyme is the target of non steroidal anti-inflammatory drugs (NSAIDs), used against inflammation and pain. The inducible COX-2 was associated with inflammatory conditions, whereas the constitutive form (COX-1) was responsible for the beneficial effects of the PGs. This observation led to the development of COX-2 inhibitors or "coxibs" of which rofecoxib (Vioxx) characterized by a methylsulfone moiety and the sulfonamides celecoxib (Celebrex) and valdecoxib (Bextra). Initially described as COX-2 "selective" inhibitors, recent reports revealed a nanomolar inhibition activity of the sulfonamide COX-2 inhibitors for several carbonic anhydrase (CA) isoforms, confirmed by X-ray crystal structures for the adducts of celecoxib and valdecoxib with isozyme CA II. This dual activity may help to explain differences in clinical observation between sulfonamide and methylsulfone COX-2 inhibitors. Moreover, the inhibition of CA isozymes, critical for the development and invasion of cancer cells, such as CA II, IX and XII, may constitute an important mechanism of antitumor action of such sulfonamide compounds.

Indanesulfonamides are interesting lead compounds for designing selective inhibitors of the different isoforms of the zinc enzyme Carbonic Anhydrase (CA). Herein, we report for the first time the X-ray crystal structure of two such derivatives, namely indane-5-sulfonamide and indane-2-valproylamido-5-sulfonamide, in complex with the physiologically dominant human isoform II. The structural analysis reveals that, although these two inhibitors have quite similar chemical structures, the arrangement of their indane ring within the enzyme active site is significantly diverse. Thus, our findings suggest that the introduction of bulky substituents on the indane-sulfonamide ring may alter the binding mode of this potent class of CA inhibitors, although retaining good inhibitory properties. Accordingly, the introduction of bulky tail moieties on the indane-sulfonamide scaffold may represent a powerful strategy to induce a desired physicochemical property to an aromatic sulfonamide or to obtain inhibitors with diverse inhibition profiles and selectivity for various mammalian CAs.

A series of aromatic/heterocyclic sulfonamides incorporating 2,3:4,5-bis-O-(isopropylidene)-β-d-fructopyranosyl-thioureido moieties has been synthesized and assayed for the inhibition of seven human isoforms of the zinc enzyme carbonic anhydrase (hCA, EC 4.2.1.1). The new derivatives behaved as weak hCA I inhibitors (KIs of 9.4 −13.3μM), were efficient hCA II inhibitors (KIs of 6–750nM), and slightly inhibited isoforms hCA IV and hCA VA. Only the sulfanilamide derivative showed efficient and selective inhibition of hCA IV (KI of 10nM). These derivatives also showed excellent hCA VII inhibitory activity (KIs of 10–79nM), being less efficient as inhibitors of the transmembrane isoforms hCA IX (KIs of 10–4500nM) and hCA XIV (KIs of 21–3500nM). Two of the new compounds showed anticonvulsant action in a maximal electroshock seizure test in mice, with the fluorosulfanilamide derivative being a more efficient anticonvulsant than the antiepileptic drug topiramate.

Convulsions are common neurological disorders in clinical medicine and are triggered by several mechanisms. The enhancement of neuronal excitability can be related, among other factors, to GABAergic depolarization. Carbonic anhydrase (CA) VII contributes to this electrophysiological behavior by providing bicarbon ate anion, which can mediate current through channels coupled to GABA(A) receptors. Among the cytosolic CAs, the mechanism of action and inhibition of CA VII is less understood. We present herein the pharmacological evaluation of both enantiomers of an indanesulfonamide compound substituted by a pentafluorophenyl moiety against CA VII and five other human CA isoforms to evaluate their selectivity. The investigated compounds are powerful inhibitors of hCA VII, with K, values in the range of 1.7-3.3 nM, but their selectivity needs to be improved. A molecular modeling study was conducted to rationalize the structure-activity relationships and provide useful insight into the future design of selective hCA VII inhibitors.

Carbonic anhydrases are ubiquitous metalloenzymes which are involved in fundamental processes (i.e., acid-base regulation, respiration, calcification, etc.). The carbonic anhydrase isozyme IX becomes an interesting pharmacological target due to its overexpression in cancer and its absence in normal tissue. Therefore, several indanesulfonamides were synthesized and tested for their inhibition both against the human CA IX and against two other biologically relevant isozymes (CA I and II). Structure-activity relationships are discussed and point out different compounds for its selectivity and activity against CA IX. To establish preliminary hypothesis for the design of new isozyme-selective CA IX inhibitors, we conducted molecular modeling. We describe here the first human CA IX model built by homology with another CA isozyme already crystallized. Docking studies were performed to explore the binding mode of our indanesulfonamide derivatives.

Carbonic anhydrase isoform IX (CA IX) is highly overexpressed in many types of cancer. Its expression, which is regulated by the HIF-1 transcription factor, is strongly induced by hypoxia and correlates with a poor response to classical chemo- and radiotherapies. CA IX contributes to acidification of the tumor environment by efficiently catalyzing the hydration of carbon dioxide to bicarbonate and protons, thereby leading to acquisition of metastasic phenotypes and chemoresistance to weakly basic anticancer drugs. Inhibition of this enzymatic activity by specific inhibitors, such as the sulfonamide indisulam, reverts these processes, establishing a clear-cut role for CA IX in tumorigenesis. Thus, selective CA IX inhibitors could prove useful for elucidating the role of CA IX in hypoxic cancers, for controlling the pH imbalance in tumor cells and for developing diagnostic or therapeutic applications for tumor management. Indeed, fluorescent inhibitors and membrane-impermeant sulfonamides have recently been used as proof-of-concept tools, demonstrating that CA IX is an interesting target for anticancer drug development.

A series of aromatic sulfonamides incorporating indane moieties were prepared starting from commercially available 1- and 2-indanamine, and their activity as inhibitors of two carbonic anhydrase (CA, EC 4.2.1.1) isozymes, hCA I and II was studied. The new sulfonamides incorporating acetamido, 4-chloro-benzoyl, valproyl, tetra-, and pentafluorobenzoyl moieties acted as very potent inhibitors of the slow red blood cell isozyme hCA I (Kis in the range of 1.6–8.5 nM), which usually has a lower affinity for such inhibitors, as compared to isozyme II. Some derivatives also showed excellent hCA II inhibitory properties (Kis in the range of 2.3–12 nM), but the anticonvulsant activity of these sulfonamides was rather low as compared to that of other sulfonamide/sulfamate CA inhibitors, such as methazolamide. Furthermore, the 2-amino/acetamido-indane-5-sulfonic acids prepared during this work also showed interesting CA inhibitory properties, with inhibition constants in the range of 43–89 nM against the two isozymes, being among the most potent sulfonic acid CA inhibitors reported so far.