In this paper, we reviewed the purification and characterization methods of the a-carbonic anhydrase (CA, EC 4.2.1.1) class. Six genetic families (a-, b-, g-, d-, z-and Z-CAs) all know to date, all encoding such enzymes in organisms widely distributed over the phylogenetic tree. Starting from the manuscripts published in the 1930s on the isolation and purification of a-CAs from blood and other tissues, and ending with the recent discovery of the last genetic family in protozoa, the Z-CAs, considered for long time an a-CA, we present historically the numerous and different procedures which were employed for obtaining these catalysts in pure form. a-CAs possess important application in medicine (as many human a-CA isoforms are drug targets) as well as biotechnological processes, in which the enzymes are ultimately used for CO 2 capture in order to mitigate the global warming effects due to greenhouse gases. Recently, it was discovered an involvement of CAs in cancerogenesis as well as infection caused by pathogenic agents such as bacteria, fungi and protozoa. Inhibition studies of CAs identified in the genome of the aforementioned organisms might lead to the discovery of innovative drugs with a novel mechanism of action.

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The carbonic anhydrase (CA, EC 4.2.1.1) isozymes IX and XII are predominantly found in tumor cells and show a restricted expression in normal tissues. By efficiently hydrating carbon dioxide to protons and bicarbonate, these CAs contribute significantly to the extracellular acidification of solid tumors. CA IX and XII are overexpressed in many such tumors in response to the hypoxia inducible factor (HIF) pathway, and research on the involvement of these isozymes in cancer has progressed in recent years. The report of the X-ray crystal structure of CA IX, which is a dimeric protein with a quaternary structure not evidenced earlier for this family of enzymes, allows for structure-based drug design campaigns of inhibitors against this novel antitumor target. Indeed, it has been known for some time that aromatic/ heterocyclic sulfonamides and sulfamates have good affinity for this isoform, but generally they do not show specificity for the inhibition of the tumor-associated isoform versus the remaining CA isozymes (CA I-VII, and XII-XV) found in mammals. Recently, we reported several classes of compounds with good selectivity for the tumor-associated CAs, being shown that CA IX/XII inhibition reverses the effect of tumor acidification, leading to inhibition of the cancer cells growth. CA IX/XII are now proposed as novel therapeutic antitumor targets. Furthermore, as some types of CA inhibitors (CAIs), such as the fluorescent sulfonamides accumulate only in hypoxic tumor cells overexpressing these enzymes, CAIs may be also used as diagnostic tools for imaging of hypoxic cancer cells. Work from several laboratories recently reported the proof-of-concept studies for the use of CA IX/XII inhibitors as well as antibodies both in the therapy and imaging of hypoxic tumors.

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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 CO(2) hydration to bicarbonate and protons, with a k(cat) of 9.4 x 10(5) s(-1), and k(cat)/K(M) of 9.8 x 10(7) 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 K(I)s of 8.7 - 10.8 µM. Benzenesulfonamides substituted in 2-, 4- and 3,4-positions with amino, alkyl, halogeno and hydroxyalkyl moieties had K(I)s in the range of 0.976 - 18.45 µM. Better inhibition (K(I)s 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 K(I)s in the range of 82.6 - 133 nM. The best inhibitor (K(I) of 15.1 nM) was 4-(2-amino-pyrimidin-4-yl)-benzenesulfonamide. L-adrenaline 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.

The ongoing Covid-19 is a contagious disease, and it is characterised by different symptoms such as fever, cough, and shortness of breath. Rising concerns about Covid-19 have severely affected the healthcare system in all countries as the Covid-19 outbreak has developed at a rapid rate all around the globe. Intriguing, a clinically used drug, acetazolamide (a specific inhibitor of carbonic anhydrase, CA, EC 4.2.1.1), is used to treat high-altitude pulmonary oedema (HAPE), showing a high degree of clinical similarities with the pulmonary disease caused by Covid-19. In this context, this preliminary study aims to provide insights into some factors affecting the Covid-19 patients, such as hypoxaemia, hypoxia as well as the blood CA activity. We hypothesise that patients with Covid-19 problems could show a dysregulated acidbase status influenced by CA activity. These preliminary results suggest that the use of CA inhibitors as a pharmacological treatment for Covid-19 may be beneficial.