Insights towards sulfonamide drug specificity in α-carbonic anhydrases

Carbonic anhydrases (CAs, EC 4.2.1.1) are a group of metalloenzymes that play important roles in carbon metabolism, pH regulation, CO₂ fixation in plants, ion transport etc., and are found in all eukaryotic and many microbial organisms. This family of enzymes catalyzes the interconversion of CO₂ and HCO₃^?. There are at least 16 different CA isoforms in the alpha structural class (α-CAs) that have been isolated in higher vertebrates, with CA isoform II (CA II) being ubiquitously abundant in all human cell types. CA inhibition has been exploited clinically for decades for various classes of diuretics and anti-glaucoma treatment. The characterization of the overexpression of CA isoform IX (CA IX) in certain tumors has raised interest in CA IX as a diagnostic marker and drug target for aggressive cancers and therefore the development of CA IX specific inhibitors. An important goal in the field of CA is to identify, rationalize, and design potential compounds that will preferentially inhibit CA IX over all other isoforms of CA. The variations in the active sites between isoforms of CA are subtle and this causes non-specific CA inhibition which leads to various side effects. In the case of CA IX inhibition, CA II along with other isoforms of CA provide off-target binding sites which is undesirable for cancer treatment. The focus of this article is on CA IX inhibition and two different structural approaches to CA isoform specific drug designing: tail approach and fragment addition approach.     

Carbonic anhydrases as targets for medicinal chemistry

Carbonic anhydrases (CAs, EC 4.2.1.1) are zinc enzymes acting as efficient catalysts for the reversible hydration of carbon dioxide to bicarbonate. 16 different alpha-CA isoforms were isolated in mammals, where they play crucial physiological roles. Some of them are cytosolic (CA I, CA II, CA III, CA VII, CA XIII), others are membrane-bound (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 beta-delta-CA family are highly abundant in plants, diatoms, eubacteria and archaea. The catalytic mechanism of the alpha-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 beta- and gamma-CAs, the zinc hydroxide mechanism is valid too, although at least some beta-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 and the sulfonamides/sulfamates/sulfamides possessing the general formula RXSO(2)NH(2) (R=aryl; hetaryl; perhaloalkyl; X=nothing, O or NH). Several important physiological and physio-pathological functions are played by CAs present in organisms all over the phylogenetic tree, related to respiration and transport of CO(2)/bicarbonate between metabolizing tissues and the lungs, pH and CO(2) homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions, such as the gluconeogenesis and ureagenesis among others (in animals), CO(2) 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 antitumour agents/diagnostic tools for tumours, antiobesity agents, anticonvulsants and antimicrobials/antifungals (inhibitors targeting alpha- or beta-CAs from pathogenic organisms such as Helicobacter pylori, Mycobacterium tuberculosis, Plasmodium falciparum, Candida albicans, etc.).     

Structure-based drug discovery of carbonic anhydrase inhibitors

Inhibition of the metalloenzyme carbonic anhydrase (CA; EC 4.2.1.1) has pharmacologic applications in the field of anti-glaucoma, anti-convulsant and anti-cancer agents. But recently, it has also emerged that these enzymes have the potential for designing anti-infective drugs (anti-fungal and anti-bacterial agents) with a novel mechanism of action. Sulphonamides and their isosteres (sulphamates/sulphamides) constitute the main class of CA inhibitors (CAIs), which bind to the metal ion from the enzyme active site. Recently, the dithiocarbamates (DTCs), possessing a similar mechanism of action, were reported as a new class of inhibitors. These types of CAIs will be discussed in detail in this review. Novel drug design strategies have been reported ultimately based on the tail approach for obtaining sulphonamides/DTCs, which exploit more external binding regions within the enzyme active site (in addition to coordination to the metal ion), leading thus to isoform-selective compounds. Most of the promising data have been obtained by combining x-ray crystallography of enzyme-inhibitor adducts with novel synthetic approaches for generating chemical diversity. Whereas sulphonamide – NO donating hybrid drugs were reported as effective anti-glaucoma agents, most of the interesting new inhibitors were designed for inhibiting specifically the tumour-associated isoforms CA IX and XII, validated targets for imaging and treatment of hypoxic tumours. Promising compounds that inhibit CAs from bacterial and fungal pathogens, of the DTC and carboxylate types, will be also reviewed.     

Herbicide oryzalin inhibits human carbonic anhydrases in vitro

Herbicides of the dinitroaniline chemical class, widely used oryzalin and trifluralin, and also nitralin were tested as inhibitors of recombinant human carbonic anhydrases (CAs). Oryzalin bound and inhibited 11 out of 12 catalytically active CA isoforms present in the human body with the affinities in the same range as clinically used CA drugs, while no effect was detected for the other two compounds. Binding of all three herbicides was examined by fluorescence‐based thermal shift assay, isothermal titration calorimetry, and the inhibition of carbon dioxide hydratase activity. During the last decade, dinitroaniline compound‐based therapies against protozoan diseases are being developed. Therefore, it is important to investigate their potential off‐target effects, including human CAs.     

Structural study of interaction between brinzolamide and dorzolamide inhibition of human carbonic anhydrases

Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes that catalyze the reversible hydration of carbon dioxide and bicarbonate. Their pivotal role in metabolism, ubiquitous nature, and multiple isoforms (CA I–XIV) has made CAs an attractive drug target in clinical applications. The usefulness of CA inhibitors (CAIs) in the treatment of glaucoma and epilepsy are well documented. In addition several isoforms of CAs (namely, CA IX) also serve as biological markers for certain tumors, and therefore they have the potential for useful applications in the treatment of cancer. This is a structural study on the binding interactions of the widely used CA inhibitory drugs brinzolamide (marketed as Azopt®) and dorzolamide (marketed as Trusopt®) with CA II and a CA IX-mimic, which was created via site-directed mutagenesis of CA II cDNA such that the active site resembles that of CA IX. Also the inhibition of CA II and CA IX and molecular docking reveal brinzolamide to be a more potent inhibitor among the other catalytically active CA isoforms compared to dorzolamide. The structures show that the tail end of the sulfonamide inhibitor is critical in forming stabilizing interactions that influence tight binding; therefore, for future drug design it is the tail moiety that will ultimately determine isoform specificity.     

Discovering a new class of antifungal agents that selectively inhibits microbial carbonic anhydrases

Infections caused by pathogens resistant to the available antimicrobial treatments represent nowadays a threat to global public health. Recently, it has been demonstrated that carbonic anhydrases (CAs) are essential for the growth of many pathogens and their inhibition leads to growth defects. Principal drawbacks in using CA inhibitors (CAIs) as antimicrobial agents are the side effects due to the lack of selectivity toward human CA isoforms. Herein we report a new class of CAIs, which preferentially interacts with microbial CA active sites over the human ones. The mechanism of action of these inhibitors was investigated against an important fungal pathogen, Cryptococcus neoformans, revealing that they are also able to inhibit CA in microbial cells growing in vitro. At our best knowledge, this is the first report on newly designed synthetic compounds selectively targeting β-CAs and provides a proof of concept of microbial CAs suitability as an antimicrobial drug target.     

Carbonic anhydrases: current state of the art, therapeutic applications and future prospects

Carbonic anhydrases (CAs, EC 4.2.1.1) are wide-spread enzymes, present in mammals in at least 14 different isoforms. Some of these isozymes are cytosolic (CA I, CA II, CA III, CA VII, CA XIII), others are membrane-bound (CA IV, CA IX, CA XII and CA XIV), CA V is mitochondrial and CA VI is secreted in the saliva and milk. Three cytosolic acatalytic forms are also known (CARP VIII, CARP X and CARP XI). The catalytically active isoforms, which play important physiological and patho-physiological functions, are strongly inhibited by aromatic and heterocyclic sulfonamides. The catalytic and inhibition mechanisms of these enzymes are understood in great detail, and this greatly helped the design of potent inhibitors, some of which possess important clinical applications. The use of such CA inhibitors (CAIs) as antiglaucoma drugs are discussed in detail, together with the recent developments that led to isozyme-specific and organ-selective inhibitors. A recent discovery is connected with the involvement of CAs and their sulfonamide inhibitors in cancer: many potent CAIs were shown to inhibit the growth of several tumor cell lines in vitro and in vivo, thus constituting interesting leads for developing novel antitumor therapies. Future prospects for drug design of inhibitors of these ubiquitous enzymes are dealt with. Although activation of CAs has been a controversial issue for some time, recent kinetic, spectroscopic and X-ray crystallographic experiments offered an explanation of this phenomenon, based on the catalytic mechanism. It has been demonstrated recently, that molecules that act as carbonic anhydrase activators (CAAs) bind at the entrance of the enzyme active site participating in facilitated proton transfer processes between the active site and the reaction medium. In addition to CA II-activator adducts, X-ray crystallographic studies have been also reported for ternary complexes of this isozyme with activators and anion (azide) inhibitors. Structure-activity correlations for diverse classes of activators is discussed for the isozymes for which the phenomenon has been studied, i.e., CA I, II, III and IV. The possible physiological relevance of CA activation/inhibition is also addressed, together with recent pharmacological/ biomedical applications of such compounds in different fields of life sciences.     

可用于二氧化碳捕获过程的微生物碳酸酐酶的挖掘与改造

二氧化碳排放量的急剧上升引起全球温室效应加剧。碳酸酐酶是地球上反应速率最快的几种酶之一,可以大幅提高CO_2捕获和生物矿化的效率,从而降低大气中CO_2的排放量。但捕获过程在高温条件,而CO_2生物矿化形成CaCO_3的过程则需要碱性条件。因此,迫切需要筛选出既嗜热又耐碱的碳酸酐酶以用于CO_2捕获,极端微生物是这类酶的重要来源之一。文中系统、深入地介绍了目前从极端微生物或利用蛋白质工程技术获取嗜热、耐碱的碳酸酐酶的最新研究进展,同时简要介绍了一些新型固定化碳酸酐酶的方法。最后指出当前研究的重点应致力于拓宽寻找碳酸酐酶的范围,改良蛋白质工程改造技术,研发高效廉价、易于放大的固定化方法,为减轻温室效应、延缓全球变暖这一迫切需要解决的问题提供新思路。     

Gadonanotubes as new high-performance MRI contrast agents

Gadolinium-based carbon nanostructures are poised to make a significant impact as advanced contrast agents (CAs) for magnetic resonance imaging (MRI) in medicine. This paper reviews and forecasts gadonanotubes as synthons for the design of high-performance MRI CA probes with efficacies up to 100 times greater than current clinical CAs. This level of performance is vital for achieving the goal of cellular and molecular imaging with MRI. These new materials will be useful for in vivo MRI applications as circulating drug nanocapsules because of their low toxicities, extremely high relaxivities, and potential for cellular targeting and induced cell death by magnetic hyperthermia.     

Carbonic anhydrase IX: Biochemical and crystallographic characterization of a novel antitumor target

Isoform IX of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), CA IX, is a transmembrane protein involved in solid tumor acidification through the HIF-1α activation cascade. CA IX has a very high catalytic activity for the hydration of carbon dioxide to bicarbonate and protons, even at acidic pH values (of around 6.5), typical of solid, hypoxic tumors, which are largely unresponsive to classical chemo- and radiotherapy. Thus, CA IX is used as a marker of tumor hypoxia and as a prognostic factor for many human cancers. CA IX is involved in tumorigenesis through many pathways, such as pH regulation and cell adhesion control. The X-ray structure of the catalytic domain of CA IX has been recently reported, being shown that CA IX has a typical α-CA fold. However, the CA IX structure differs significantly from the other CA isozymes when the protein quaternary structure is considered. Thus, two catalytic domains of CA IX associate to form a dimer, which is stabilized by the formation of an intermolecular disulfide bond. The active site clefts and the proteoglycan (PG) domains are located on one face of the dimer, while the C-termini are located on the opposite face to facilitate protein anchoring to the cell membrane. As all mammalian CAs, CA IX is inhibited by several main classes of inhibitors, such as the inorganic anions, the sulfonamides and their bioisosteres (sulfamates, sulfamides, etc.), the phenols, and the coumarins. The mechanism of inhibition with all these classes of compounds is understood at the molecular level, but the sulfonamides and their congeners have important applications. It has been recently shown that both in vitro, in cell cultures, as well as in animals with transplanted tumors, CA IX inhibition with sulfonamides lead to a return of the extracellular pH to more normal values, which leads to a delay in tumor growth. As a consequence, CA IX represents a promising antitumor target for the development of anticancer agents with an alternative mechanism of action.     

Review Article

Carbonic anhydrases (CAs, EC 4.2.1.1) are wide-spread enzymes, present in mammals in at least 14 different isoforms. Some of these isozymes are cytosolic (CA I, CA II, CA III, CA VII, CA XIII), others are membrane-bound (CA IV, CA IX, CA XII and CA XIV), CA V is mitochondrial and CA VI is secreted in the saliva and milk. Three cytosolic acatalytic forms are also known (CARP VIII, CARP X and CARP XI). The catalytically active isoforms, which play important physiological and patho-physiological functions, are strongly inhibited by aromatic and heterocyclic sulfonamides. The catalytic and inhibition mechanisms of these enzymes are understood in great detail, and this greatly helped the design of potent inhibitors, some of which possess important clinical applications. The use of such CA inhibitors (CAIs) as antiglaucoma drugs are discussed in detail, together with the recent developments that led to isozyme-specific and organ-selective inhibitors. A recent discovery is connected with the involvement of CAs and their sulfonamide inhibitors in cancer: many potent CAIs were shown to inhibit the growth of several tumor cell lines in vitro and in vivo, thus constituting interesting leads for developing novel antitumor therapies. Future prospects for drug design of inhibitors of these ubiquitous enzymes are dealt with. Although activation of CAs has been a controversial issue for some time, recent kinetic, spectroscopic and X-ray crystallographic experiments offered an explanation of this phenomenon, based on the catalytic mechanism. It has been demonstrated recently, that molecules that act as carbonic anhydrase activators (CAAs) bind at the entrance of the enzyme active site participating in facilitated proton transfer processes between the active site and the reaction medium. In addition to CA II-activator adducts, X-ray crystallographic studies have been also reported for ternary complexes of this isozyme with activators and anion (azide) inhibitors. Structure-activity correlations for diverse classes of activators is discussed for the isozymes for which the phenomenon has been studied, i.e, CA I, II, III and IV. The possible physiological relevance of CA activation/inhibition is also addressed, together with recent pharmacological/biomedical applications of such compounds in different fields of life sciences.     

Insights into the role of reactive sulfhydryl groups of Carbonic Anhydrase III and VII during oxidative damage

Carbonic anhydrases (CAs) III and VII are two cytosolic isoforms of the α-CA family which catalyze the physiological reaction of carbon dioxide hydration to bicarbonate and proton. Despite these two enzymes share a 49% sequence identity and present a very similar three-dimensional structure, they show profound differences when comparing the specific activity for CO₂ hydration reaction, with CA VII being much more active than CA III. Recently, CA III and CA VII have been proposed to play a new role as scavenger enzymes in cells where oxidative damage occurs. Here, we will examine functional and structural features of these two isoforms giving insights into their newly proposed protective role against oxidative stress.     

A systematic quantification of carbonic anhydrase transcripts in the mouse digestive system

Background  

Carbonic anhydrases (CAs) are physiologically important enzymes which participate in many gastrointestinal processes such as acid and bicarbonate secretion and metabolic pathways including gluconeogenesis and ureagenesis. The genomic data suggests that there are thirteen enzymatically active members of the mammalian CA isozyme family. In the present study, we systematically examined the mRNA expression levels of all known CA isozymes by quantitative real-time PCR in eight tissues of the digestive system of male and female mice.     

Advances in understanding the physiological role and locations of carbonic anhydrases in C3 plant cells

The review describes the structures of plant carbonic anhydrases (CAs), enzymes catalyzing the interconversion of inorganic carbon forms and belonging to different families, as well as the interaction of inhibitors and activators of CA activity with the active sites of CAs in representatives of these families. We outline the data that shed light on the location of CAs in green cells of C3 plants, algae and angiosperms, with the emphasis on the recently obtained data. The proven and proposed functions of CAs in these organisms are listed. The possibility of the involvement of several chloroplast CAs in acceleration of the conversion of bicarbonate to CO₂ and in supply of CO₂ for fixation by Rubisco is particularly considered. Special attention is paid to CAs in various parts of thylakoids and to discussion about current knowledge of their possible physiological roles. The review states that, despite the significant progress in application of the mutants with suppressed CAs synthesis, the approach based on the use of the inhibitors of CA activity in some cases remains quite effective. Combination of these two approaches, namely determining the effect of CA activity inhibitors in plants with certain knocked-out CA genes, turns out to be very useful for understanding the functions of other CAs.

     

A novel carbonic anhydrase from the giant clam Tridacna gigas contains two carbonic anhydrase domains

This report describes the presence of a unique dual domain carbonic anhydrase (CA) in the giant clam, Tridacna gigas. CA plays an important role in the movement of inorganic carbon (Ci) from the surrounding seawater to the symbiotic algae that are found within the clam's tissue. One of these isoforms is a glycoprotein which is significantly larger (70 kDa) than any previously reported from animals (generally between 28 and 52 kDa). This alpha-family CA contains two complete carbonic anhydrase domains within the one protein, accounting for its large size; dual domain CAs have previously only been reported from two algal species. The protein contains a leader sequence, an N-terminal CA domain and a C-terminal CA domain. The two CA domains have relatively little identity at the amino acid level (29%). The genomic sequence spans in excess of 17 kb and contains at least 12 introns and 13 exons. A number of these introns are in positions that are only found in the membrane attached/secreted CAs. This fact, along with phylogenetic analysis, suggests that this protein represents the second example of a membrane attached invertebrate CA and it contains a dual domain structure unique amongst all animal CAs characterized to date.     

Carbonic anhydrase inhibitors

Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread enzymes in all organisms, catalyzing CO₂ hydration to bicarbonate and protons. Their inhibition is exploited clinically for decades for various classes of diuretics and systemically acting antiglaucoma agents. In the last years novel applications of CA inhibitors (CAIs) emerged, such as topically acting antiglaucoma, anticonvulsants, antiobesity, antipain, and antitumor agents/diagnostic tools. Such CAIs target diverse isozymes of the 13 catalytically active α-CA isoforms present in mammals. CAs belonging to the α-, β-, γ-, δ-, and ζ-families are found in many organisms all over the phylogenetic tree, and their inhibition was studied ultimately for some pathogenic protozoa (Plasmodium falciparum), fungi (Cryptococcus neoformans, Candida albicans, Candida glabrata, and Saccharomyces cerevisiae), and bacteria (Helicobacter pylori, Mycobacterium tuberculosis, and Brucella suis). Novel interesting chemotypes, in addition to the sulfonamide and sulfamate CAIs, such as coumarins, phenols, and fullerenes, were also reported recently, together with their mechanism of inhibition. This class of enzyme inhibitors shows promise for designing interesting pharmacological agents and understanding in detail protein–drug interactions at molecular level.     

Inhibition of the alpha- and beta-carbonic anhydrases from the gastric pathogen Helycobacter pylori with anions

The gastric pathogen Helicobacter pylori encodes two carbonic anhydrases (CAs, EC 4.2.1.1), an α- and a β-class one, hpαCA and hpβCA, crucial for its survival in the acidic environment from the stomach. Sulfonamides, strong inhibitors of these enzymes, block the growth of the pathogen, in vitro and in vivo. Here we report the inhibition of the two H. pylori CAs with inorganic and complex anions and other molecules interacting with zinc proteins. hpαCA was inhibited in the low micromolar range by diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic acid, and in the submillimolar one by cyanide, cyanate, hydrogen sulfide, divanadate, tellurate, perruthenate, selenocyanide, trithiocarbonate, iminodisulfonate. hpβCA generally showed a stronger inhibition with most of these anions, with several low micromolar and many submillimolar inhibitors detected. These inhibitors may be used as leads for developing anti-H. pylori agents with a diverse mechanism of action compared to clinically used antibiotics.     

Biochemical characterization of the δ-carbonic anhydrase from the marine diatom Thalassiosira weissflogii,TweCA

Abstract

Diatom genome sequences clearly reveal the presence of different systems for HCO₃^? uptake. Carbon-concentrating mechanisms (CCM) based on HCO₃^? transport and a plastid-localized carbonic anhydrase (CA, EC 4.2.1.1) appear to be more probable than the others because CAs have been identified in the genome of many diatoms. CAs are key enzymes involved in the acquisition of inorganic carbon for photosynthesis in phytoplankton, as they catalyze efficiently the interconversion between carbon dioxide and bicarbonate. Five genetically distinct classes of CAs exist, α-, β-, γ-, δ- and ζ and all of them are metalloenzymes. Recently we investigated for the first time the catalytic activity and inhibition of the δ-class CA from the marine diatom Thalassiosira weissflogii, named TweCA. This enzyme is an efficient catalyst for the CO₂ hydration and its inhibition profile with sulfonamide/sulfamate and anions have also been investigated. Here, we report the detailed biochemical characterization and chemico-physical properties of the δ-CA of T. weissflogii. The δ-CA encoding gene was cloned and expressed in Artic Express cells and the recombinant protein purified to homogeneity. Interesting to note that TweCA has no intrinsic esterase activity with 4-nitrophenyl acetate (pNpA) as substrate although the phylogenetic analysis showed that δ-CAs are closer to the α-CAs than to the other classes of such enzymes.