Current Pharmaceutical Design - Volume 14, Issue 7, 2008

Carbonic anhydrases (CAs), the metalloenzymes that catalyze the conversion between carbon dioxide and bicarbonate, continue to be surprising targets, as many exciting new discoveries related to them emerge constantly. This is indeed unprecedented as these are quite “old” enzymes, which were discovered in 1933, and thoroughly investigated since then as drug targets. Furthermore, their inhibitors are in clinical use since the 50s. However, in the last years, a host of interesting reports were made regarding the catalytic/inhibition mechanism as well as isolation/characterization of new isozymes belonging to this family, as well as of CAs of non-vertebrate origin. The first paper [1] in this issue of the Journal dedicated to these enzymes and their inhibitors, represents an overview of CAs as drug targets. In fact, among the 16 isoforms known up to now in mammals, 12 catalytically active ones seem to be appropriate for designing inhibitors with various therapeutic applications (only CA III seems to remain an orphan target). In addition, many carbonic anhydrases isolated from other organisms were recently shown to be possible targets for the drug design, such as the α-CAs from Plasmodium falciparum and Helicobacter pylori, the β-CAs from Mycobacterium tuberculosis, Candida albicans, Cryptococcus neoformans, etc. Work is in progress in many laboratories for developing specific inhibitors targeting these enzymes, that would lead to conceptually novel therapies. An exhaustive review regarding the design of such inhibitors possessing different metal-binding functions than the classical sulfonamide one is then presented by Winum et al. [2]. The last years saw many relevant developments in this field with the report of several interesting classes of such derivatives, among which the sulfamates, sulfamides, substituted sulfonamides/sulfamides, etc., as well as a detailed X-ray crystallographic dscription of their interactions with various pharmacologically relevant isoforms. The next contribution deals with the carbonic anhydrases belonging to the α- and β-classes recently cloned and characterized from the widespread pathogen Helicobacter pylori, producing a wide range of diseases. In the last years, Nishimori's group [3] demonstrated that both these enzymes are druggable targets. Furthermore, in one of their papers it has been demonstrated for the first time that a non-α-CA, i.e., just the beta-CA from H. pylori, can be a druggable target. At the same level of importance is the report of Krungkrai et al. [4] regarding the presence of several α-CAs in the protozoa causing malaria, belonging to the genus Plasmodium. In several seminal papers, this group reported the cloning, characterization and inhibition studies of one of these enzymes, showing it to be a druggable target. The paper in this issue is just an excellent review of this work, potentially leading to novel antimalarial drugs. A reinvestigation of the sulfonamide diuretics belonging to the thiadiazine and high-ceiling diuretic type provided interesting clues regarding possible new applications of sulfonamide CAIs. Indeed, most of these drugs were discovered in a period when only isoform CA II was wellknown. Retesting these compounds on all the mammalian isozymes, Supuran's group [5] showed that many of these clinically used sulfonamide diuretics act as nanomolar inhibitors against many pharmacologically relevant “new” CA isoforms. Historically, in addition to their well-known role for the development of diuretics, the CA inhibitors were mainly used as antiglaucoma agents. The review by Mincione et al. [6] in this issue presents up-to-date data regarding the ophthalmologic use of systemically- and topically-acting CA inhibitors, as well as some drug design studies reported ultimately, which substantially extended the current knowledge in obtaining water-soluble such derivatives, potentially useful not only in the treatment of glaucoma but also for the management of macular degeneration. In the last years, there are also encouraging reports linking CA inhibitors to novel antiobesity therapies, field reviewed in a nice paper by De Simone et al. [7]. In fact two mitochondrial CA isoforms, CA VA and CA VB are involved in lipogenesis and their inhibition leads to diminished fatty acid biosynthesis. De Simone's group resolved the X-ray crystallographic structures of many important, clinically used inhibitors with various isozymes, and performed modelling studies regarding their binding to targets which have not been crystallized yet (such as the human CA VA/VB or the human CA IX). Such data are extremely useful for the drug design of inhibitors with various applications, not only as antiobesity agents, but also as antitumor or antiglaucoma drugs.

Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread metalloenzymes all over the phylogenetic tree, with at least 4 distinct gene families encoding for them. At least 16 different α- CA isoforms were isolated in mammals, where these enzymes play crucial physiological roles. Representatives of the β - δ-CA family are highly abundant in plants, diatoms, eubacteria and archaea. These enzymes are efficient catalysts for the reversible hydration of carbon dioxide to bicarbonate, but at least the α-CAs possess a high versatility, being able to catalyze different other hydrolytic processes The catalytic mechanism of the α-CAs is understood in detail: the active site consists of a Zn(II) ion co-ordinated by three histidine residues and a water molecule/hydroxide ion. The latter is the active species, acting as a potent nucleophile. For β- and γ-CAs, the zinc hydroxide mechanism is valid too, although at least some β-class enzymes do not have water directly coordinated to the metal ion. CAs are inhibited by two classes of compounds: the metal complexing anions and the sulfonamides and their isosteres (sulfamates, sulfamides etc.) possessing the general formula RXSO2NH2 (R = aryl; hetaryl; perhaloalkyl; X = nothing, O or NH). At least 25 clinically used drugs/agents in clinical development show applications as diuretics and antiglaucoma drugs, anticonvulsants, with some compounds being developed as anticancer agents/diagnostic tools for tumors, antiobesity agents, and antimicrobials/antifungals (inhibitors targeting CAs from pathogenic organisms such as Helicobacter pylori, Mycobacterium tuberculosis, Plasmodium falciparum, Candida albicans, etc). Several important physiological and physio-pathological functions are played by CA isozymes present in organisms all over the phylogenetic tree, related to respiration and transport of CO2/bicarbonate between metabolizing tissues and the lungs, pH and CO2 homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions, such as the gluconeogenesis and ureagenesis among others (in animals), CO2 fixation (in plants and algae), etc. The presence of these ubiquitous enzymes in so many tissues and in so different isoforms, represents an attractive goal for the design of inhibitors or activators with biomedical applications.

Zinc ion plays a crucial role in the protein's functions and is linked to a variety of physiological processes. It constitutes an essential component of numerous enzymes especially carbonic anhydrase (CAs, EC 4.2.1.1), a pharmaceutically-important metalloprotein which catalyses efficiently the reversible hydration of carbon dioxide to bicarbonate with discharge of a proton. The potential therapeutic applications of selective carbonic anhydrase inhibitors has become an important challenge over the last few years, as some isoforms of this enzyme on the 16 described in higher vertebrates have been found to be involved in important pathologies such as cancer, obesity and ophthalmologic diseases. Coordination of the inhibitor with the zinc ion present in the active site is an important determinant which has to be taken into consideration for the design of isozyme-specific and organ-selective inhibitors. Besides the well known sulfonamide function, others zinc binding groups have been described constituting a new platform for the development of novel pharmacological agents. In this review, recent studies on the discovery of new zinc binding function will be discussed.

Helicobacter pylori (H. pylori) successfully resides in the human stomach in highly acidic conditions, causing a variety of gastroduodenal lesions, including gastric ulcer, gastric cancer and MALT lymphoma. For acid acclimation of H. pylori, two types of enzymes, urease and carbonic anhydrase (CA), play a central role. They cooperatively function to maintain neutral pH in the bacterial cytoplasm and periplasm. The genome project of H. pylori identified two different classes of CA with different subcellular localization: a periplasmic α-class CA (hpαCA) and a cytoplasmic β-class CA (hpβCA). These two CAs are catalytically efficient with almost identical activity to that of the human isoform CA I for the CO2 hydration reaction, and highly inhibited by many sulfonamides/sulfamates, including acetazolamide, ethoxzolamide, topiramate and sulpiride, all clinically used drugs. Furthermore, certain CA inhibitors, such as acetazolamide and methazolamide, were shown to inhibit the bacterial growth in vitro. Since the efficacy of eradication therapies currently employed has been decreasing due to drug resistance and side effects of the commonly used drugs, the dual inhibition of α- and/or ß-CAs of H. pylori could be applied as an alternative therapy in patients with H. pylori infection or for the prevention of gastroduodenal diseases provoked by this widespread pathogen.

Plasmodium falciparum is the protozoan parasite responsible for the majority of life-threatening cases of human malaria, causing more than one million deaths a year. The global emergence of drug-resistant malarial parasites necessitates identification and characterization of novel drug targets. At present, α-carbonic anhydrase (CA) genes are identified in limited numbers of parasites in both protozoa and helminthes, however, the malarial genes are found in four species of Plasmodium. The CA gene of P. falciparum encodes an α- carbonic anhydrase enzyme possessing catalytic properties distinct of that of the human host CA I and II isozymes. P. falciparum native and recombinant enzymes have been prepared. A library of aromatic sulfonamides, most of which were Schiff's bases derived from sulfanilamide/ homosulfanilamide/4-aminoethyl-benzenesulfonamide and substituted-aromatic aldehydes, or ureido-substituted sulfonamides are very good inhibitors for P. falciparum enzyme with Ki values in the range of 80 nM-0.50 μM. The 4-(3,4-dichlorophenylureidoethyl)- benzenesulfonamide is the most effective antimalarial activity against growth of P. falciparum in vitro with an IC50 of 2 μM. The structure of the groups substituting the aromatic-ureido- or aromatic-azomethine fragment of the molecule and the length of the parent sulfonamide (i.e., from sulfanilamide to 4-aminoethylbenzenesulfonamide) from which the Schiff's base obtained, are the critical parameters for the enzyme inhibitory activities of these aromatic sulfonamide derivatives, both against the malarial as well as human enzymes. This review provides further support that the CA may have essential roles in the parasite metabolism. Thus, the aromatic sulfonamide CA inhibitors may have potential for development of novel antimalarial drugs.

The widely clinically used benzothiadiazines and high ceiling diuretics, such as hydrochlorothiazide, hydroflumethiazide, quinethazone, metolazone, chlorthalidone, indapamide, furosemide and bumetanide, contain SO2NH2 moieties acting as an effective zincbinding function in carbonic anhydrases (CAs, EC 4.2.1.1) inhibitors. These drugs were launched in a period when only isoform CA II was known and considered physiologically/pharmacologically relevant. Although acting as moderate-weak inhibitors of CA II, all these drugs considerably inhibit other CA isozymes known nowadays to be involved in critical physiologic processes, among the 16 CAs present in vertebrates. Some low nanomolar (or even subnanomolar) inhibitors against such isoforms were recently detected, such as metholazone against CA VII, XII and XIII, chlorthalidone against CA VB, VII, IX, XII and XIII, indapamide against CA VII, IX, XII and XIII, furosemide against CA I, II and XIV, and bumethanide against CA IX and XII. The X-ray crystal structure of the CA II - indapamide adduct was also reported recently, revealing interesting aspects useful for the drug design of CA inhibitors. It has also been proposed that the recently observed beneficial effect of indapamide for the treatment of patients with hypertension and type 2 diabetes might be due to its potent inhibition of CA isoforms present in kidneys and blood vessels, which would thus explain both the blood pressure lowering effects as well as organ-protective activity of the drug. Thus, these old drugs may be useful as leads for new applications.

Inhibition of carbonic anhydrase (CA, EC 4.2.1.1) isoforms present in the eyes (CA I, II, IV and XII), with sulfonamides such as acetazolamide, methazolamide, ethoxzolamide and dichlorophenamide, is still widely used for the systemic treatment of glaucoma. The mechanism of action of these drugs consists in inhibition of CA isozymes present in ciliary processes of the eye, with the consequent reduction of bicarbonate and aqueous humour secretion, and of elevated intraocular pressure (IOP) characteristic of this disease. As isoforms CA II/IV/XII are present in many other tissues/organs, generally, systemic CAIs possess undesired side effects such as numbness and tingling of extremities; metallic taste; depression; fatigue; malaise; weight loss; decreased libido; gastrointestinal irritation; metabolic acidosis; renal calculi and transient myopia. For avoiding these side effects, recently, topically effective CAIs have been developed in the last 10 years, with two drugs available clinically: dorzolamide and brinzolamide. Both these drugs are applied topically as water solutions/ suspensions, alone or in combination with other agents (β-blockers, prostaglandin derivatives, etc) and produce a consistent and prolonged reduction of IOP. Furthermore, recent reports show both the systemically as well as topically acting sulfonamide CAIs to be effective in the treatment of macular edema, macular degeneration disease, or diabetic retinopathy, for which pharmacological treatment is unavailable up to now. Much research is in act in the search of more effective topically acting CAIs, free of the inconveniences and side effects of the presently available drugs. For achieving this goal, two recently reported strategy, the tail approach and its variant, the sugar-tail approach, were extensively applied for the synthesis of large numbers of derivatives possessing desired physico-chemical properties. Many such new sulfonamides showed promising antiglaucoma activity in animal models of the disease.

Obesity is widespread disease both in the developed and developing world, which currently affects over 300 million individuals worldwide and is associated with premature mortality and chronic morbidity. Although diet, physical activity and behavioral modifications should theoretically help in controlling this condition, very often these strategies are insufficient to normalize the multiple risks associated with this condition. Thus, pharmacological interventions for the treatment of this disease are essential. Paradoxically, the currently available drugs for the treatment of obesity are very few, their mechanism of action is hardly understood and their side effects are generally quite serious. Therefore, novel effective anti-obesity drugs possessing different mechanisms of action are needed. In this review we describe in detail a possible new approach for the treatment and prophylaxis of this disease based on the inhibition of Carbonic Anhydrases (CAs, EC 4..2.1.1), enzymes involved in several steps of de novo lipogenesis. In particular, we summarize here a series of kinetic and structural studies recently reported on Topiramate (TPM) and Zonisamide (ZNS), two antiepileptic drugs showing strong CA inhibitory properties, that were shown to induce persistent weight loss in obese patients. On the basis of the reviewed studies we suggest that the use of TPM and ZNS as lead molecules for the design of CA inhibitors targeting isozymes involved in lipogenesis could represent the beginning of a very promising approach for the treatment of obesity.

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.

The carbonic anhydrase (CA) enzyme family consists of thirteen active isozymes in mammals. The most recently characterized members of this family are cytosolic CA XIII and membrane-bound CA XV. This article describes recent advances in the CA family, especially CA XIII and XV. We have also included catalytic activity data on human CA XIII and mouse CA XV. Additionally, the inhibition constants of acetazolamide toward these isozymes were determined to be kcat = 1.5 x 105 s-1, kcat/KM = 1.1 x 107 M-1s-1 and KI = 16 nM for human CA XIII and kcat= 4.7 x 105 s-1, kcat/KM = 3.3 x 107 M-1s-1 and KI = 72 nM for mouse CA XV. Although the activity of CA XIII is the second lowest reported thus far for any of the human CAs, it may have a role in maintaining the acid-base balance in the kidney and the gastrointestinal and reproductive tracts. CA XV is an exceptional enzyme, as it seems to be active in numerous species, such as rodents, birds and fish, but is absent from humans and chimpanzees. Mouse CA XV is a moderately active enzyme, suggesting that it may play a physiological role at least in the kidney. It is likely that other isozymes have substituted for this protein in humans. In addition to the novel data on CA XIII and XV, we present the catalytic activities as well as inhibition constants of acetazolamide for all mammalian CA isozymes in this review.

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.

Cells of the growing tumor tissue are exposed to physiological stresses connected with insufficient delivery of oxygen (hypoxia) and accumulation of acidic products of the glycolytic metabolism (acidosis). Adaptation to these microenvironmental stresses involves remodeling of the cellular expression program mediated by hypoxia-inducible factor (HIF), which activates broad array of genes functionally involved in angiogenesis, anaerobic glycolysis, de-adhesion, invasion etc. This leads to increased aggressiveness of tumors, metastatic spread and poor response to therapy. Genes coding for transmembrane carbonic anhydrase (CA) isoforms IX and XII are induced in response to low oxygen as a part of the hypoxic transcriptome. Moreover, CA IX is a direct target of HIF and serves as a surrogate marker of hypoxia and prognostic indicator. Its expression is strongly linked to different types of tumors with the HIF pathway activated due to genetic defect or physiological hypoxia. CA IX (and possibly also CA XII) is participates in pH regulation, which is important for survival of hypoxic cells. Both enzymes are therefore promising therapeutic molecules targetable by inhibitors of CA activity. Some of these sulfonamide compounds and their derivatives are capable to block CA-mediated pH regulation in hypoxia. This review summarizes research data related to distribution, regulation and functional aspects of CA IX and CA XII, and describes emerging possibilities for clinical exploitation of CA inhibitors as imaging tools and anticancer drugs.

Carbonic anhydrase IX (hCA IX) is a membrane-associated glycoprotein that is observed in many tumor tissues and is strongly overexpressed by hypoxia conditions. Hypoxia is a clinically important tumor parameter and this enzyme can play an important role as a potential marker of hypoxic tumor and as a therapeutic target too. In the last years, Carbonic Anhydrase IX Inhibitors which possess fluorescent probe were largely used for visualize hypoxic tumor cell lines and for understanding the biological roles of hCA IX in acidification of the external matrix. Here we resume the developement pathways of such compounds from the design to the final biological evaluation. Furthermore, spin-labeled CAIs were included to have a complete overview of the potenciality of this enzyme as marker of hypoxic tumors.

The activation mechanism of Carbonic Anhydrase was recently explained using kinetic, spectroscopic and X-ray techniques. It has been demonstrated that the activators molecules (CAAs) bind at the entrance of the enzyme active-site facilitating the ratedetermining step of CA catalitic cycle. Drug design studies have been performed in order to obtain strong CAAs belonging to several chemical classes: amino acids, azoles, amine and their derivatives, etc. Structure-activity correlations of different activators are discussed for the most studied Carbonic Anhydrase isozymes: isoform I and II. The physiological relevance of CA activation and the possible application of CAAs in Alzheimer's desease and for other memory therapies are also treated.

There are currently five (α,β,γ,δ,Ezgr;) classes of carbonic anhydrases (CA's) of which the α-class from mammalian sources has been studied to a much greater extent compared to the other four classes. Yet, CA's other than the α-class are widely distributed in Nature and play important roles in human health, the global carbon cycle, and industrial applications. In aerobic prokaryotes, β-class CA's are implicated in maintaining internal pH and CO2/bicarbonate balances required for biosynthetic reactions. In anaerobic prokaryotes, β- class CA's are implicated in the transport of CO2 and bicarbonate across the cytoplasmic membrane that regulates pH and facilitates acquisition of substrates and product removal required for growth. In phototrophic organisms, β-class CA's are particularly important for transport and concentration of CO2 and bicarbonate for photosynthesis. The δ- and ζ-classes are proposed to function in marine diatoms to concentrate CO2 for photosynthesis. Physiological roles for the γ-class are not as well documented; however, the active site architecture and catalytic mechanism is well understood as are patterns of inhibition by sulfonamides and anions.