Carbonic anhydrase inhibitors: an editorial

Abstract

This issue of Expert Opinion on Therapeutic Patents is dedicated to carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs), a highly dynamic research topic in the last years. Several review articles and patent analyses on diuretics and antiglaucoma, antiepileptic, anti-obesity and anticancer agents belonging to the pharmacological class of the CAIs are presented, together with a review on bacterial, fungal and protozoan CAs and their inhibition as a novel means of designing anti-infectives.

Keywords::

• anticancer
• antiglaucoma
• anti-obesity
• carbonic anhydrase
• coumarin
• dithiocarbamate
• diuretic
• inhibitor
• polyamine
• sulfonamide

The carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes present in all life kingdoms, as they equilibrate the reaction among three simple but essential chemical species: CO₂, bicarbonate and protons [1-5]. Discovered exactly 80 years ago, in 1933, these enzymes were extensively investigated due to the biomedical applications of their inhibitors, and also because they are an extraordinary example of convergent evolution, with five genetically distinct CA families evolved independently in Bacteria, Archaea, and Eukarya, that is, the α-, β-, γ-, δ- and ζ-CA [1-5]. CAs are also among the most efficient enzymes known in nature [6,7], probably due to the fact that the uncatalyzed CO₂ hydration is a very slow process, and the physiological demands for its conversion to ionic, soluble species are very high [1-3].

These enzymes are also characterized by the presence of a large number of isoforms in most organisms investigated so far [1-20]. CAs are involved in many crucial physiological and pathological processes connected with pH regulation, secretion of electrolytes, biosynthetic processes, photosynthesis, tumorigenesis, etc. [1-10]. Drugs interfering with CA activity are clinically used for almost 60 years, most of them belonging to the sulfonamide class [1-3,11,12].

Inhibition of the CAs has pharmacological applications in many fields, such as antiglaucoma [1], anticonvulsant [1-3], anti-obesity [11], and anticancer agents/diagnostic tools [5]. Also, it is an emerging paradigm for designing anti-infectives, that is, antifungal, antibacterial and antiprotozoan agents with a novel mechanism of action [13-20]. As a consequence, the drug design of CA inhibitors (CAIs) is a highly dynamic field, with a large number of novel derivatives reported constantly in the scientific and patent literature [1-4]. Indeed, this special issue of the journal is publishing a series of comprehensive review articles on CAI research in all these main fields: antiglaucoma drugs, antiepileptics, anti-obesity agents, antitumor CAIs, and anti-infectives. CA inhibition is an emerging field with promising results for designing antibacterial, antifungal and antiprotozoan agents with a new mechanism of action (compared with such clinically used agents for which extensive drug resistance has been reported in the last years). In addition, a review dedicated to the diuretics possessing CA inhibitory action is presented in this issue too, together with several patent analyses of recent, interesting applications claiming sulfonamides, coumarins or monoclonal antibodies with CA inhibitory properties and therapeutic use.

The sulfonamides and their isosteres (sulfamates/sulfamides) constitute the main class of CAIs which bind to the metal ion from the enzyme active site, and are in clinical use for decades as diuretics and antiglaucoma, antiepileptic and altitude-sickness treatment agents [1,4]. However, many other families of CAIs were ultimately reported, mainly considering the natural products as a ‘gold mine' for their design [1-3]. Many of them possess a distinct mechanism of action compared with the sulfonamides; the phenols, polyamines, some carboxylates, and the sulfocoumarins were shown to anchor to the zinc-coordinated water molecule from the enzyme active site, not directly binding to the metal center [1-3,16]. Coumarins and some lactones were shown to be prodrug inhibitors [1,16], binding in a hydrolyzed form at the entrance of the active site cavity.

Novel drug design strategies have been reported principally based on the tail approach [17] for obtaining all these types of CAIs, which exploit more external binding regions within the enzyme active site (in addition to coordination to the metal ion), leading thus to a large number of isoform-selective compounds [4]. In fact, the first- and second-generation (sulfonamide) inhibitors showed a large number of side effects due to the indiscriminate inhibition of most CA isoforms (from mammals), which was detrimental to their wide clinical use [1-10]. Sugar-based tails as well as click chemistry were the most fruitful and latest developments of the tail approach [1]. Promising compounds that inhibit CAs from bacterial and fungal pathogens [19], of the dithiocarbamate [18], phenol and carboxylate types have also been reported [1] in the last period.

As mentioned earlier, most patents from the last several years deal with CAIs targeting the tumor-associated isoforms CA IX and XII [5], and several of them also with novel antiglaucoma therapies based on CAIs (e.g., combining in the same pharmacophore the sulfonamide and nitric oxide-donating moieties). Indeed, the publication of the proof-of-concept studies regarding the potent antitumor and antimetastasis effects of CA IX inhibition (in several animal tumor models) led to a lot of research and patenting in these fields [5].

It is, on the other hand, quite probable that the most important advances in this research field in future years will deal with anti-infective agents which can be obtained based on the CAIs. Indeed, enzymes from several classes are present in many pathogenic bacteria [8,9]. For the moment they have been investigated in some detail in a restricted number of them, among which are Neisseria spp., Helicobacter pylori, Vibrio cholerae (which all encode α-class enzymes) as well as the β-class enzymes from Escherichia coli, H. pylori, Mycobacterium tuberculosis, Brucella spp., Streptococcus pneumoniae, Salmonella enterica, and Haemophilus influenzae [8,9]. For some of these enzymes the X-ray crystal structures were determined at rather high resolution, allowing for a good understanding of the catalytic/inhibition mechanisms [4,8,9]. However no adducts with inhibitors of these enzymes have been characterized so far, although in vitro and in vivo inhibition studies with various classes of inhibitors, such as anions, sulfonamides, sulfamates, phenols and dithiocarbamates have been reported [4]. Efficient in vitro inhibitors have been reported for many such enzymes, but only for Neisseria spp., H. pylori, Brucella suis, S. pneumoniae, M. tuberculosis and CAs it has been possible to evidence inhibition of bacterial growth in vivo [4]. Thus, bacterial CAs represent at this moment very promising targets for obtaining antibacterials devoid of the resistance problems of such clinically used agents but further studies are needed to validate these and other less investigated enzymes as novel drug targets. In addition, only a very limited number of bacterial genomes have been examined for the presence of CAs. We predict that bacterial CAs may represent an active research frontline, for academia and industry, in the following years.

Fungal CAs have also been investigated in some detail in few organisms, such as the model one Saccharomyces cerevisiae as well as the pathogenic fungi Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia globosa [19]. Many interesting inhibitors of the β-CAs encoded by these fungi have been detected, belonging to the sulfonamide, sulfamate, carboxylate and dithiocarbamate classes, but in vivo inhibition of growth was evidenced so far only for the Malassezia enzyme, which has been proposed as a novel antidandruff target. Indeed, an interesting patent dealing with this discovery has received much attention.

More recently, protozoan genomes have also been investigated for the presence of CAs. In the malaria parasite Plasmodium falciparum and the parasite provoking Chagas disease Trypanosoma cruzi, interesting α-CAs have been cloned, characterized and investigated for their inhibition. Several classes of sulfonamides or thiols were found to be low nanomolar in vitro inhibitors, also showing in vivo inhibition of growth of the protozoan parasite [20].

All these important advances in the CAI field are highlighted in the present issue of the journal. The large number of good-quality patents and publications on all aspects of CA research testifies the crucial role that these enzymes may have for future biomedical as well as biotechnological applications.

Declaration of interest

The author declares conflict of interest being an author on many patents dealing with various classes of CA inhibitors (most of which were cited in the reviews published in this issue). He was not paid for writing this review.

Acknowledgments

Research from the author's laboratory was financed in part by several grants of the 6th and 7th Framework Programme of the European Union (DeZnIT, Metoxia, Gums and Joints, and Dynano projects).