Current Topics in Medicinal Chemistry - Volume 7, Issue 9, 2007

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 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. 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. 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. 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. CA inhibitors were also used as antiepileptic drugs, but with less fortune. Indeed, the classical derivatives, acetazolamide and methazolamide, showed reduced utility in the treatment of seizure, as presented thoroughly in the review of Thiry et al., regarding the anticonvulsants belonging to this class of pharmacological agents. However, the last years saw the discovery of many CA isoforms present in the brain and a somehow better understanding of their role in this organ. Furthermore, some newer antiepileptics, such as topiramate and zonisamide also show substantial CA inhibitory activity, although it is unclear to what extent this activity is essential for their anticonvulsant effects, since these drugs possess a complex mechanism of action. Probably the most unexpected applications of the CA inhibitors are those regarding the diagnosis and treatment of tumors. This very important and dynamic research field is reviewed in the excellent paper of Pastorekova et al., the discoverer of the first tumor-associated CA isozyme, CA IX. In several seminal papers from her group, it has recently been demonstrated that CA IX (and probably also CA XII, the other tumor-associated isozyme) is overexpressed in hypoxic tumors being involved in tumor acidification processes which lead to metastatic spread and non-responsiveness to chemotherapeutic agents/radiation treatment. Furthermore, the same group demonstrated that sulfonamide CA IX-selective inhibitors may revert these processes, opening the way to conceptually novel anticancer therapies and diagnostic tools based on CA IX inhibitors. 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.

At least 15 different α-carbonic anhydrase (CA, EC 4.2.1.1) isoforms were isolated in mammals, where these zinc enzymes play crucial physiological roles. Some of these isozymes are cytosolic (CA I, CA II, CA III, CA VII, CA XIII), others are membranebound (CA IV, CA IX, CA XII, CA XIV and CA XV), CA VA and CA VB are mitochondrial, and CA VI is secreted in saliva and milk. Three acatalytic forms are also known, the CA related proteins (CARP), CARP VIII, CARP X and CARP XI. Representatives of the β - δ-CA family are highly abundant in plants, diatoms, eubacteria and archaea. These enzymes are very efficient catalysts for the reversible hydration of carbon dioxide to bicarbonate, but at least the β-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 primarily by two classes of compounds: the metal complexing anions (such as cyanide, cyanate, thiocyanate, azide, hydrogensulfide, etc) and the sulfonamides/sulfamates/sulfamides possessing the general formula RXSO2NH2 (R = aryl; hetaryl; perhaloalkyl; X = nothing, O or NH). Several important physiological and physio-pathological functions are played by the 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 with biomedical applications. Indeed, CA inhibitors are clinically used as antiglaucoma drugs, some other compounds being developed as antitumor agents/diagnostic tools for tumors, antiobesity agents, anticonvulsants, and antimicrobials/antifungals (inhibitors targeting CAs from pathogenic organisms such as Helicobacter pylori, Mycobacterium tuberculosis, Plasmodium falciparum, Candida albicans, etc).

The carbonic anhydrases (CAs, EC 4.2.1.1) are zinc containing metalloenzymes which catalyse efficiently the reversible hydration of carbon dioxide to bicarbonate with discharge of a proton, playing important physiological and physiopathological functions. To date, 16 different carbonic anhydrase isoforms have been described in higher vertebrates, including humans, and some of them have been considered as important targets for inhibitors with therapeutic applications. The catalytic and structural role of zinc in these enzyme are understood in great detail, and this provided molecular basis for the design of potent inhibitors, some of which possessing important clinical applications mainly as topically acting anti-glaucoma drugs, anticancer or antiobesity agents. The metal binding function is a critically important factor in the development of isozyme-specific and organ-selective inhibitors. Discovery of compounds that possess zinc binding function different from that of the classical one (sulfonamide type) is in constant progress and can offer opportunities for developing novel pharmacological agents. In the present review we will discuss the different zinc binding function reported in the literature up to now in the design of carbonic anhydrase inhibitors.

Carbonic anhydrase inhibitors (CAIs) such as acetazolamide, methazolamide, ethoxzolamide and dichlorophenamide were and still are widely used systemic antiglaucoma drugs. Their mechanism of action consists in inhibition of CA isozymes present in ciliary processes of the eye (such as CA II, IV and XII), with the consequent reduction of bicarbonate and aqueous humour secretion, and of elevated intraocular pressure (IOP) characteristic of this disease. Since 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. In order to avoid these undesired side effects, recently, topically effective CAIs have been developed. Two drugs are available clinically: dorzolamide and brinzolamide. Both these drugs are applied topically as water solutions/suspensions, alone or in combination with other agents (such as 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 oedema and other macular degeneration diseases, for which pharmacological treatment was unavailable up to now. Much research is in act in the search of even more effective topically acting CAIs, free of the inconveniences and side effects of the presently available drugs. For achieving this goal, a recently reported strategy, the tail approach, was extensively applied for the synthesis of large numbers of derivatives possessing various physico-chemical properties. Many such new sulfonamides showed promising antiglaucoma activity in animal models of the disease.

Seizures is 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 CO2 / HCO3 - 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.

Recent progress in understanding the role of catalytically active carbonic anhydrases in tumors has opened new possibilities for diagnostic and/or therapeutic applications of carbonic anhydrase inhibitors selectively blocking the enzyme activity of cancer-related isoforms, namely CA IX and CA XII. Different classes of inhibitors have been investigated in order to evaluate their usefulness as in vivo imaging tools, as modulators of intratumoral pH that influences uptake of conventional chemotherapeutics, or as drugs impeding survival of tumor cells exposed to physiological stresses including hypoxia and acidosis. Here we summarize the most important data related to expression, regulation and functional aspects of cancer-related carbonic anhydrases and discuss advances in synthesis and preclinical studies of isozyme-selective and highly efficient carbonic anhydrase inhibitors.

Few pharmacological approaches for the treatment of obesity exist at this time, and most of them are unsatisfactory, whereas this disease is widespread both in the developed and developing world. Novel effective approaches are needed for the development of antiobesity agents possessing different mechanisms of action. A possible new approach for the treatment and prophylaxis of obesity is based on the inhibition of carbonic anhydrases (CAs, EC 4.2.1.1), enzymes involved in several steps of de novo lipogenesis, both in the mitochondria and the cytosol of cells. Topiramate and zonisamide are two antiepileptic drugs that were shown to induce persistent weight loss in obese patients, but their mechanism of action is largely unknown. We demonstrated strong CA inhibitory properties for these two drugs, by means of kinetic studies in solution and X-ray crystallography, against several physiologically relevant isoforms, such as CA II, VA and VB. It has been proved that topiramate also inhibits lipogenesis in adipocytes, similarly to other sulfonamide CA inhibitors investigated earlier. A large number of new sulfonamides have been synthesized and assayed as possible inhibitors of CA isoforms involved in lipogenesis. This is the beginning of a very new and promising approach for the treatment of obesity, with the hope that new compounds showing this property will be soon developed and available for clinical use.

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.

The carbonic anhydrase (CA) protein family consists of twelve active isozymes in humans and thirteen in most other mammals. The most recently discovered members of this family include cytosolic CA XIII and membrane-bound CAs XIV and XV. In this article we will review the characterization and inhibition profiles of these isozymes. CA XIII is expressed in the kidney as well as in the gastrointestinal and reproductive tracts, and therefore it may have a role in various physiological processes. The inhibition studies with CA XIII have shown that this isozyme can be inhibited efficiently with some sulfonamide inhibitors, while it is resistant to inhibition with chloride and bicarbonate ions. CA XIV is a membrane-bound enzyme that is expressed in numerous organs such as the brain, kidney, eye, liver and epididymis, where it has a role in the regulation of acid-base balance. The inhibitory properties of CA XIV have been studied, but no specific inhibitors have been found for this isozyme. The membrane-bound CA XV is an exceptional member of this protein family, because it is expressed in numerous species but absent in humans and chimpanzees. A detailed biochemical characterization of this isozyme is under way in our laboratories.

Five independently evolved classes (α-, β-, γ-, δ-, ζ-) of carbonic anhydrases facilitate the reversible hydration of carbon dioxide to bicarbonate of which the α-class is the most extensively studied. Detailed inhibition studies of the α-class with the two main classes of inhibitors, sulfonamides and metal-complexing anions, revealed many inhibitors that are used as therapeutic agents to prevent and treat many diseases. Recent inhibitor studies of the archaeal β-class (Cab) and the γ-class (Cam) carbonic anhydrases show differences in inhibition response to sulfonamides and metal-complexing anions, when compared to the α-class carbonic anhydrases. In addition, inhibition between Cab and Cam differ. These inhibition patterns are consistent with the idea that although, α-, β-, and γ-class carbonic anhydrases participate in the same two-step isomechanism, diverse active site architecture among these classes predicts variations on the catalytic mechanism. These inhibitor studies of the archaeal β- and γ-class carbonic anhydrases give insight to new applications of current day carbonic anhydrase inhibitors, as well as direct research to develop new compounds that may be specific inhibitors of prokaryotic carbonic anhydrases.

Plasmodium falciparum is responsible for the majority of life-threatening cases of human malaria. The global emergence of drug-resistant malarial parasites necessitates identification and characterization of novel drug targets. At present, carbonic anhydrase (CA) genes are identified in limited numbers of protozoa and helminthes parasites, however, they are demonstrated in at least 4 Plasmodium species. The CA gene of P. falciparum encodes an α-carbonic anhydrase enzyme possessing catalytic properties distinct of that of the human host enzymes. A small library of aromatic sulfonamides, most of which were Schiff's bases derived from sulfanilamide/homosulfanilamide/4-aminoethylbenzenesulfonamide and substituted-aromatic aldehydes, or ureido-substituted sulfonamides are good inhibitors of P. falciparum enzyme. The 4-(3,4-dichlorophenylureido-ethyl)-benzenesulfonamide is the most effective antimalarial activity against growth of P. falciparum in vitro. The nature 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 CA inhibitory activities of these aromatic sulfonamide derivatives, both against the malarial as well as human enzymes. Thus, the sulfonamide CA inhibitors may have the potential for the development of novel antimalarial drugs.

Bad news for cancer. The Bcl-2 (B-cell lymphoma 2) family of proteins is comprised of both pro-apoptotic and anti-apoptotic members that operate through a complex series of protein-protein interactions [1-3]. The pro-apoptotic proteins are categorized into two groups based on the number of Bcl homology (BH) domains: 1) those with 3 BH domains (BH1-BH3 such as Bax and Bak) and 2) those with a single BH domain (BH-3 only such as Bad, Bik, Puma, etc...). The prosurvival (anti-apoptotic) members contain four BH domains (BH1-BH4 such as Bcl-2, Bcl-xL, etc...). Overexpression of Bcl-2 and its closely related counterpart Bcl-xL affords a mechanism by which cancer cells suppress apoptotic signaling, confer a survival advantage and provide resistance to chemotherapy [1-3]. Recognizing that inhibition of Bcl-2 family members should specifically target the abnormal cell death pathway in the overexpressing cancer cells led Elmore and co-workers at Abbott labs to launch a drug discovery campaign aimed at developing small molecule protein-protein inhibitors of the Bcl-2 family [4-6]. Initial work centered on a class of potent biarylacylsulfonamide antagonists - outside the usual α-helical mimetics - and a lead compound which bound Bcl-xL with a Ki of 800 pM [4]. Despite effective cytotoxic activity with multiple cytotoxic agents and UV irradiation, this Bcl-xL antagonist had no single agent efficacy across a large panel of human tumor cell lines. Elmore and co-workers then reasoned that since this inhibitor was developed by structure-based design aimed at Bcl-xL, it was not surprising that it exhibited much lower affinity for Bcl-2 [5,6]. Since Bcl-2 overexpression is a major hallmark of many human cancers, the team sought to broaden their inhibitor profile to target both Bcl-xL and Bcl-2; interestingly, despite only 49% homology, the three-dimensional structures of Bcl-xL and Bcl-2 are similar. The researchers focused in on the largest structural distinction between the two - a different helical fold of the α3 helix [5]. The α3 helix borders one side of the hydrophobic binding groove, and there exists a wider groove for Bcl-2 than Bcl-xL and a deeper hydrophobic pocket within the groove for Bcl-2 than Bcl-xL. Based on this key structural difference, the team designed modified inhibitors to access this deep hydrophobic pocket in the floor of the groove and thereby increasing affinity for Bcl-2. Their strategy worked! A chemical lead optimization campaign along with structure-guided design resulted in the identification of a clincal candidate, ABT-737, the first dual, subnanomolar inhibitors of Bcl- 2 and Bcl-xL (Ki <1 nM and <0.5 nM, respectively) and with EC50s of 8 nM and 30 nM, respectively [5]. ABT-737 showed single agent efficacy against human follicular lymphoma cell lines that overexpress Bcl-2 and efficacy in a murine tumor xenograft model of lymphoma when given as either a single agent or in combination with etoposide. ABT-737 was also efficacious in two mice small cell lung carcinoma xenograft models known to be resistant to most cytotoxic agents. For example, ABT-737 administered i.p. at 75 mg/kg/day for 21 days in a H146 model caused complete regression of established xenografts and 58 days after cessation of therapy, the tumors did not grow back in 77% of the mice [5,6]. ABT-737 represents a significant advance in the discovery and development of small molecule protein-protein inhibitors and clinical data in human patients is eagerly awaited. REFERENCES [1] Cory, S.; Adams, J.M. The Bcl2 family: regulators of the cellular life-ordeath switch. Nat. Rev. Cancer 2002, 2, 647-656. [2] Borner, C. The Bcl2 protein family: sensor and checkpoints for life-or-death decisions. Mol. Immunol. 2003, 39, 615-647. [3] van Delft, M.F.; Huang, D.S.C. How the Bcl-2 family of proteins interact to regulate apoptosis. Cell. Res. 2006, 16, 203-213. [4] Wendt, M.D.; Shen, W.; Kunzer, A.; McClellan, W.J.; Bruncko, M.; Oost, T.K.; Ding, H.; Joseph, M. K.; Zhang, H.; Nimmer, P.M.; Ng, S.-C.; Shoemaker, A.R.; Petros, A.M.; Oleksijew, A.; Marsh, K.; Bauch, J.; Oltersdorf, T.; Belli, B.A.; Martineau, D.; Fesik, S.W.; Rosenberg, S. H.; Elmore, S.W. Discovery and structure-activity relationship of antagonists of B-Cell Lymphoma 2 Family Proteins with chemopotentiation activity in vitro and in vivo. J. Med. Chem. 2006, 49, 1165-1181.