Current Pharmaceutical Design - Volume 16, Issue 29, 2010

I use this title to introduce this issue of Curr Pharm Des. dedicated to the carbonic anhydrase (CA) conference held in September 2009 in Florence, Italy, as I found it in the not very happy comments of a reviewer of one of my manuscripts sent recently for publication, who was complaining that we publish too much in this field. Thus, I found this situation quite illustrative for this entire research field, in which important advancements have been achieved ultimately with a very high frequency. In fact, the prevalent view of many “important” scientists from both academia and industry was that CAs are “boring enzymes”, at least until recently. For several good reasons: (i) they catalyze a very simple reaction, CO2 hydration to bicarbonate and protons, which anyhow can occur without a catalyst [1]; (ii) their inhibition was not very successful in the past from the pharmaceutical viewpoint, especially considering the diuretic or antiepileptic activity of some old drugs, such as acetazolamide, ethoxzolamide, etc. [1]; (iii) these enzymes are widespread in many tissues/cells, and their inhibition by compounds originally designed for other applications, may only lead to side effects and complications with FDA or other regulatory agencies, when a new such drug has to be launched clinically. There are in fact examples of pharmaceutical companies which published very unrealistic inhibition data of their compounds (e.g., both topiramate and zonisamide were claimed to be weak or very weak CA inhibitors (CAIs) by their discoverers, whereas they are low nanomolar ones against many relevant isoforms [1]). However this distorted view started to change steadfastly in the last years. And again for multiple reasons. For example, when we proposed sulfonamide CAIs as anticancer agents more than 10 years ago, there was a great reluctance to accept this idea and the first paper presenting such data [2] had great difficulties to be published. Nowadays several CA isozymes are widely accepted as antitumor drug targets [1,3-5], the mechanims of action of such compounds started to be understood both at the molecular level and as pharmacology, and there are drug companies developing them both as imaging tools for hypoxic tumors or as anticancer drugs [3-5]. This aspect is presented in several reviews of this issue [3-5], both from the biochemical, medicinal chemistry and pharmacological points of view. It has been in fact possible to understand in detail these aspects due to the report of the X-ray crystal structure of isoform CA IX, the main anticancer target in this family of enzymes, which is reviewed in the second paper of the issue from De Simone's group [4]. The latest developments regarding the biochemistry, molecular biology, genetic regulation and pharmacology of CA IX are then detailed in the next paper, by Pastorekova's group [5], which are in fact the discoverers of this highly interesting enzyme. Their contributions in understanding many aspects related to CA IX were seminal in all the years that passed since their initial report of this first tumor-associated isoform. The next paper, from Parkkila's group [6], deals with a rather neglected topic in the CA research, and more precisely the acatalytic isoform CA VIII. In this excellent review a thorough analysis of the genetics, phylogeny, possible physiologic and pathological roles and distribution of CA VIII are provided, allowing thus a much better understanding of this and possibly of the related acatalytic isoforms, CA X and XI [6]. Many novel chemotypes of CAIs have been discovered ultimately (coumarins, polyamines, etc), as shown in the introductory paper [3], but sulfonamides still remain the main class of such derivatives. One of the highly versatile and innovative techniques which has been used to generate large libraries of such compounds is the “click chemistry”, which is reviewed for the first time here by Poulsen's group [7]. The next paper, by Said's group [8], deals just with such a group of sulfonamides which seem to be useful in the treatment of brain cancers, again by inhibiting CA IX, the isoform overexpressed in many hypoxic tumors. The next two papers deal with CAs isolated, characterized and studied from the inhibition point of view from pathogenic bacteria, such as Mycobacterium tuberculosis [9] and Brucella suis [10]. Indeed, many pathogenic bacteria contain one or more CAs, generally belonging to the β-CA class. Only recently, several groups showed that these enzymes may be drug targets for developing antibacterials with a novel mechanism of action [9,10], although this fact is difficult to accept for many scientists (who generally make quite bad reviews to such papers in which this innovative approach is being proposed). However, this was exactly the same situation with the anticancer effects of CAIs (as I stressed above), and probably sooner or later this idea will start to be widely accepted, but we still have to fight a lot to impose it. And these two reviews [9,10] on the inhibition of important human pathogens (but Brucella spp. also provokes zoonotic disease in many species) are thus welcome in this emerging new field. It should be stressed that these are the first reviews on mycobacterial or Brucella CAs.The next paper, by Guzel et al. [11] deals with the drug design campains of a very particular scaffold, i.e., 3-phenyl-1H-indole-5- sulfonamides, which have led ultimately to a range of highly potent inhibitors directed both against mammalian, α-CA isoforms, but also some β-CAs from pathogenic bacteria (Mycobacterium tuberculosis) or fungi (Candida albicans, Cryptococcus neoformans, etc). This field has also not been reviewed up until now, and as this is the most interesting new scaffold which has emerged, this review presents an updated view of drug design of sulfonamide CAIs based on the ring approach [11]. β-CAs are also present in fungi and yeasts, and Saccharomyces cerevisiae is no exception. Thus, the last paper in this issue deals just with the enzyme cloned, characterized and studied in this important species by Kockar et al. [12]. In conclusion, CAs are widely spread enzymes, found all over the phylogenetic tree, in prokaryotes and eukaryotes, being present in humans under the form of multiple isozymes with various activity, susceptibility to inhibition and diverse physiological roles. Their inhibitors are clinically used as antiglaucoma agents with topical activity, anticonvulsants, antipain, antiobesity and probably soon, as antitumor agents/diagnostic tools for cancer therapy [13]. Furthermore, although it is still difficult to make this idea widely acceptable, there is a real potential to develop anti-infectives (antimalarials, antifungal and antibacterial agents) belonging to the CAIs, targeting enzymes from various pathogens. Last but not least, the recent years saw the discovery of many new chemotypes showing significant CA inhibitory activity and a novel mechanism of action, in addition to the classical sulfonamides and their bioisosteres. All these facts prove how dynamic this research field is, where novel drug targets and novel chemotypes are emerging constantly worldwide, and why there is a CA conference each three years.

Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes which catalyze CO2 hydration to bicarbonate and protons. Five genetically distinct classes are known, which represent an excellent example of convergent evolution. Inhibition of α-CAs from vertebrates, including humans, with sulfonamides was exploited clinically for decades for various classes of diuretics and systemically acting antiglaucoma agents, whereas newer inhibitors are used as topically acting antiglaucoma drugs, anticonvulsants, antiobesity, antipain and antitumor agents/diagnostic tools. Recently, novel interesting chemotypes, in addition to the sulfonamides and sulfamates were discovered, such as the phenols, coumarins/thiocoumarins/lacosamide, fullerenes, boronic acids and some protein tyrosine kinase inhibitors. Furthermore, their detailed mechanism of inhibition has been explained and can be used for the rational drug design of other agents. Such new classes of enzyme inhibitors show promise for designing interesting pharmacological agents and understanding in detail protein-drug interactions at molecular level. CAs belonging to the α-,β-,γ-,δ- and ξ-families found in many organisms all over the phylogenetic tree and their inhibition were studied ultimately in nematodes, corals, some pathogenic protozoa (Plasmodium falciparum), fungi/yeasts (Cryptococcus neoformans, Candida albicans, C. glabrata, Saccharomyces cerevisiae) and bacteria (Helicobacter pylori, Mycobacterium tuberculosis, Brucella suis, Streptococcus pneumoniae), being demonstrated that anti-infectives based on their inhibitors might be obtained. Possible applications for these new chemotypes are envisaged and discussed in detail, based on a chemo-geographical approach which took the author around the world and the chemical space.

The carbonic anhydrase (CA) family has recently become an important target for the drug design of inhibitors with potential use as diagnostic and therapeutic tools. However, given the high degree of sequence and structure similarity among the different CA isoforms, no CA-directed drug developed so far has displayed selectivity for a specific isozyme. Since X-Ray crystallography is a very useful tool for the rational drug design of selective enzyme inhibitors, in recent years extensive research efforts have been devoted to the structural studies of all catalytically active α-CA isoforms, with the consequent resolution of the crystallographic structures of nearly all such enzyme isoforms. In this paper we review the progress that has recently been made in this field. In particular, we summarize the main structural features of hCA XIII and hCA IX, the most recently characterized human CA isoforms, and recapitulate how 3D structures of these enzymes, together with kinetic experiments, have been used either to deepen our knowledge on the structural features responsible of the catalytic properties of this protein family or to obtain important information for the rational drug design of inhibitors with better selectivity properties.

Carbonic anhydrase IX (CA IX) is a suitable target for various anticancer strategies. It is a cell surface protein that is present in human tumors, but not in the corresponding normal tissues. Expression of CA IX is induced by hypoxia and correlates with cancer prognosis in many tumor types. Moreover, CA IX is functionally implicated in cancer progression as a pro-survival factor protecting cancer cells against hypoxia and acidosis via its capability to regulate pH and cell adhesion. Cancer-related distribution of CA IX allows for targeting cancer cells by antibodies binding to its extracellular domain, whereas functional involvement of CA IX opens the possibility to hit cancer cells by blocking their adaptation to physiologic stresses via inhibition of CA IX enzyme activity. The latter strategy is recently receiving considerable attention and great efforts are made to produce CA IX-selective inhibitor derivatives with anticancer effects. On the other hand, targeting CA IX-expressing cells by immunotherapy has reached clinical trials and is close to application in treatment of renal cell carcinoma patients. Nevertheless, development and characterization of new CA IX-specific antibodies is still ongoing. Here we describe a mouse monoclonal antibody VII/20 directed to catalytic domain of CA IX. We show that upon binding to CA IX, the VII/20 MAb undergoes efficient receptor-mediated internalization, which is a process regulating abundance and signaling of cell surface proteins and has a considerable impact on immunotherapy. We evaluated biological properties of the MAb and demonstrated its capacity to elicit anti-cancer effect in mouse xenograft model of colorectal carcinoma. Thus, the VII/20 MAb might serve as a tool for preclinical studies of immunotherapeutic strategies against non-RCC tumors. These have not been explored so far and include broad spectrum of cancer types, treatment of which might benefit from CA IX-mediated targeting.

Mammalian carbonic anhydrase (α-CA) gene family comprises sixteen isoforms, thirteen of which are active isozymes and three isoforms lack classical CA activity of reversible hydration of CO2 due to absence of one or more histidine residues required for CA catalytic activity. The inactive isoforms are known as carbonic anhydrase related proteins (CARPs) VIII, X and XI. Among these three, CARP VIII was reported first in 1990 from a mouse brain cDNA library and is well studied structurally as well as functionally compared to CARP X and XI. CARP VIII is an intriguing protein and is widely distributed and evolutionarily well-conserved across the species. It is mainly expressed in the Purkinje cells of cerebellum and in wide variety of other tissues both in mouse and human. CARP VIII has been linked to development of colorectal and lung cancers in humans, and overexpression of CARP VIII has been observed in several other cancers. A mutation in the CA8 gene has been associated with ataxia, mild mental retardation and quadrupedal gait in humans and with lifelong gait disorder in mice, suggesting an important role for CARP VIII in the brain. However, the precise function of CARP VIII is still an enigma. The present review article describes the previous data on CARP VIII, including its structure, role in neurodegeneration and cancer; and bioinformatic and expression analyses recently performed in our laboratory.

In recent years there has been renewed activity in the literature concerning the 1,3-dipolar cycloaddition reaction (1,3-DCR) of organic azides (R-N3) with alkynes (R´ C≡CH)to form 1,2,3-triazoles, i.e. the Huisgen synthesis. The use of catalytic Cu(I) leads to a dramatic rate enhancement (up to 107-fold) and exclusive synthesis of the 1,4-disubstituted 1,2,3-triazole product. The reaction, now referred to as the copper-catalyzed azide-alkyne cycloaddition (CuAAC), meets the stringent criteria of a click-reaction in that it is modular, wide in scope, high yielding, has no byproducts, operates in water at ambient temperature, product purification is simple and the starting materials are readily available. The 1,3-DCR reaction has rapidly become the premier click chemistry reaction with applications spanning modern chemistry disciplines, including medicinal chemistry. Recently the ‘tail’ approach initiative for the development of carbonic anhydrase inhibitors (CAIs) has been combined with the synthetic versatility of click chemistry. This has proven a powerful combination leading to the synthesis of CAIs with useful biopharmaceutical properties and activities. This review will discuss complementary and contrasting applications that have utilized ‘click tailing’ development of CAIs. Applications encompass i) medicinal chemistry and drug discovery; ii) radiopharmaceutical development of positron emission topography (PET) chemical probes; and iii) in situ click chemistry.

Hypoxia is a crucial factor in tumour aggressiveness and its treatment resistance, particularly in human brain cancer. Tumour resistance against radiation- and chemo- therapy is facilitated by oxygenation reduction at tumour areas. HIF-1α regulated genes are mostly responsible for this type of resistance. Among these genes, carbonic anhydrase isoform 9 (CA9) is highly overexpressed in many types of cancer especially in high grade brain cancer like GBM. CA IX contributes to tumour environment acidification by catalyzing the carbon dioxide hydration to bicarbonate and protons, leading to the acquisition of metastasic phenotypes and chemoresistance to weakly basic anticancer drugs and therefore to inadequate application of radio-therapeutic or chemotherapeutic anti-cancer treatment strategies. Inhibition of this enzymatic activity by application of specific chemical CA9 inhibitors (sulphonamide derivative compounds) or indirect inhibitors like HIF-1α inhibitors (chetomin) or molecular inhibitors like CA9-siRNA leads to reversion of these processes, leading to the CA9 functional role inhibition during tumourigenesis. Hypoxia significantly influences the tumour microenvironment behaviour via activation of genes involved in the adaptation to the hypoxic stress. It also represents an important cancer prognosis indicator and is associated with aggressive growth, malignant progression, metastasis and poor treatment response. The main objective in malignant GBM therapy is either to eradicate the tumour or to convert it into a controlled, quiescent chronic disease. Sulfonamide derivative compounds with CA9 inhibitory characteristics represent one of the optimal treatment options beside other CA9 inhibitory agents or chemical inhibitory compounds against its main regulating transcription factor which is the hypoxia induced HIF-1α when applied against human cancers with hypoxic regions like GBM, bearing potential for an effective role in human brain tumour therapeutic strategies. Glycolytic inhibitors, when added in controlled doses under hypoxia, lead to a reduced accumulation of HIF-1α and can function as indirect hypoxia regulated genes inhibitors like CA9. These may be used as alternative or in conjunction with other direct inhibitors like the sulphonamide derivate compounds, chetomin or specific siRNAs, or other different chemical compounds possessing similar functionality making them as optimal tools for optimized therapy development in cancer treatment, especially against human brain cancer. Further experimental analysis towards the tumour stage specific inhibitory CA9 characteristics determination are necessary to find the optimal therapeutic solutions among the different available modalities; whether they are direct or indirect chemical, molecular or natural inhibitors to be able to set up successful treatment approaches against the different human tumour diseases.

Three β-carbonic anhydrases (CAs, EC 4.2.1.1), encoded by the gene Rv1284 (mtCA 1) Rv3588c (mtCA 2) and Rv3273 (mtCA 3) are present in the human pathogen Mycobacterium tuberculosis. These enzymes were cloned and they showed appreciable catalytic activity for CO2 hydration, with kcat of 3.9 x 105 s-1, and kcat/Km of 3.7 x 107 M-1.s-1 for mtCA 1, of 9.8 x 105 s-1, and kcat/Km of 9.3 x 107 M-1.s-1 for mtCA 2 and kcat of 4.3 x 105 s-1, and a kcat/Km of 4.0 x 107 M-1.s-1 for mtCA 3, respectively. The Rv3273 gene product is predicted to be a 764 amino acid residues polypeptide, consisting of a sulfate transporter domain (amino acids 121-414) in addition to the β-CA mentioned above (which is encoded by residues 571-741). All these enzymes were inhibited appreciably by many sulfonamides and sulfamates, in the nanomolar - micromolar range, whereas some subnanomolar inhibitors were also reported for two of them (mtCA 1 and mtCA 3). As sulfonamides also efficiently inhibit dehydropteroate synthetase (DHPS), the contribution of mtCAs and DHPS inhibition to a possible antimycobacterial action of these drugs must be better understood. It has been however proven that mtCAs are druggable targets, with a real potential for developing antimycobacterial agents with a diverse mechanism of action compared to the clinically used drugs for which many strains exhibit multi-drug resistance and extensive multi-drug resistance, although for the moment no in vivo inhibition of the bacteria could be evidenced with the presently avilable drugs due to lack of penetrability through the mycolic acid cell wall of M. tuberculosis.

The facultative intracellular pathogen and zoonotic agent Brucella sp. possesses two carbonic anhydrases (CAs, EC 4.2.1.1), termed bsCA I and bsCA II (in Brucella suis), belonging to the β-class of these metalloproteins. These zinc enzymes, present in many other pathogenic bacteria, have been considered recently as potential antibacterial targets. The catalytic activity of bsCA II is higher than that of bsCA I (for the conversion of CO2 to bicarbonate). Both enzymes were inhibited by the well-studied inhibitor acetazolamide, a sulfonamide drug. A library of 41 sulfonamides and one sulfamate, among which were 13 clinically tested drugs, was used for inhibition studies with bsCA I and II. These compounds were generally much more potent inhibitors of bsCA I (KIs of 17-75 nM) than of bsCA II (KIs of 84-923nM). However, certain glycosidic sulfonamide derivatives exhibited the same strong inhibitory activity on both bsCA I and bsCA II (KIs of 8.9-20 nM). Furthermore, at least one of these glycosylsulfonamides showed a significant inhibition of B. suis growth after 8-11 days of culture in minimal medium. In conclusion, as β-CAs of Brucella are susceptible to inhibition by a wide range of aromatic and heteroaromatic sulfonamides, they may represent novel targets for the development of clinically useful antibacterial agents.

A series of 2-(hydrazinocarbonyl)-3-substituted-phenyl-1H-indole-5-sulfonamides possessing various 2-, 3- or 4- substituted phenyl groups with methyl-, halogeno- and methoxy- functionalities, as well as the perfluorophenyl moiety have been synthesized and evaluated as inhibitors of both α- and β-class carbonic anhydrases (CAs, EC 4.2.1.1). All human isoforms with medicinal chemistry applications were included in such studies, among which CA I, II, VA, VB, VII, IX and XII. Several low nanomolar, sometimes isoformselective compounds were thus detected. Two β-CAs from the pathogenic bacterium Mycobacterium tuberculosis encoded by the genes Rv1284 Rv3588c were also highly inhibited (sometimes in the sub-nanomolar range) by some pyridinium derivatives incoprorating this scaffold, obtained from the corresponding 2-(hydrazinocarbonyl)-3-substituted-phenyl-1H-indole-5-sulfonamides by reaction with pyrylium salts. The fungal β-CAs from Candida albicans (Nce103) and Cryptococcus neoformans (Can2) were also investigated for their inhibition with this family of sulfonamides and some highly effective inhibitors detected. As the X-ray crystal structure of one such sulfonamide with the human isoform CA II is also known, the 3-substituted-phenyl-1H-indole-5-sulfonamides represent a totally new class of inhibitors obtained by structure-based drug design, which show efficiency in inhibiting both α- and β-CAs from several species.

The β-carbonic anhydrase from Saccharomyces cerevisiae (CA, EC 4.2.1.1), scCA, which is encoded by the Nce103 gene, is an effective catalyst for CO2 hydration to bicarbonate and protons, with a kcat of 9.4 x 105 s-1, and kcat/KM of 9.8 x 107 M-1.s-1. Its inhibition with anions and sulfonamides has been investigated, as well as its activation with amines and amino acids. Bromide, iodide and sulfamide, were the best anion inhibitors, with KIs of 8.7 - 10.8 μM. Benzenesulfonamides substituted in 2-, 4- and 3,4-positions with amino, alkyl, halogeno and hydroxyalkyl moieties had KIs in the range of 0.976 - 18.45 μM. Better inhibition (KIs in the range of 154 - 654 nM) was observed for benzenesulfonamides incorporating aminoalkyl/carboxyalkyl moieties or halogenosulfanilamides; benzene- 1,3-disulfonamides; simple heterocyclic sulfonamides and sulfanilyl-sulfonamides. The clinically used sulfonamides/sulfamate (acetazolamide, ethoxzolamide, methazolamide, dorzolamide, topiramate, celecoxib, etc.) generally showed effective scCA inhibitory activity, with KIs in the range of 82.6 - 133 nM. The best inhibitor (KI of 15.1 nM) was 4-(2-amino-pyrimidin-4-yl)-benzenesulfonamide. Ladrenaline and some piperazines incorporating aminoethyl moieties were the most effective scCA activators. These studies may lead to a better understanding of the role of this enzyme in yeasts/fungi, and since the Nce103 gene is also present in many pathogenic organisms (Candida spp., Cryptococcus neoformans, etc) they may be useful to develop antifungal drugs.