CA Isoforms Research Articles

Probing the surface of human carbonic anhydrase for clues towards the design of isoform specific inhibitors.

The alpha carbonic anhydrases (α-CAs) are a group of structurally related zinc metalloenzymes that catalyze the reversible hydration of CO2 to HCO3 −. Humans have 15 different α-CAs with numerous physiological roles and expression patterns. Of these, 12 are catalytically active, and abnormal expression and activities are linked with various diseases, including glaucoma and cancer. Hence there is a need for CA isoform specific inhibitors to avoid off-target CA inhibition, but due to the high amino acid conservation of the active site and surrounding regions between each enzyme, this has proven difficult. However, residues towards the exit of the active site are variable and can be exploited to design isoform selective inhibitors. Here we discuss and characterize this region of “selective drug targetability” and how these observations can be utilized to develop isoform selective CA inhibitors.

Saccharin: A lead compound for structure-based drug design of carbonic anhydrase IX inhibitors

Carbonic anhydrase IX (CA IX) is a key modulator of aggressive tumor behavior and a prognostic marker and target for several cancers. Saccharin (SAC) based compounds may provide an avenue to overcome CA isoform specificity, as they display both nanomolar affinity and preferential binding, for CA IX compared to CA II (>50-fold for SAC and >1000-fold when SAC is conjugated to a carbohydrate moiety). The X-ray crystal structures of SAC and a SAC-carbohydrate conjugate bound to a CA IX-mimic are presented and compared to CA II. The structures provide substantial new insight into the mechanism of SAC selective CA isoform inhibition.

Inhibition studies of quinazoline-sulfonamide derivatives against the γ-CA (PgiCA) from the pathogenic bacterium, Porphyromonas gingivalis

Carbonic anhydrases (CAs, EC 4.2.1.1) began to be investigated in detail in pathogenic bacteria, in the search for antibiotics with a novel mechanism of action, since it has been demonstrated that in many bacteria CAs are essential for the life cycle of the organism. The presence of CAs in pathogenic bacteria allows the development of anti-infectives with a new mechanism of action, less explored to date. Here, novel quinazoline derivatives crowned with sulfonamide functionality at position-2 were tested for their ability to inhibit the bacterial γ-CA (PgiCA), identified in the genome of Porphyromonas gingivalis. Six compounds were highly effective, nanomolar inhibitors of the pathogenic enzyme γ-PgCA. Three of them were also highly effective sub-nanomolar inhibitors of the cytosolic human isoform II (hCAII). The best γ-PgCA inhibitor was compound 8c, with a KI of 3.53 nM and selectivity ratio of 24.5 and 24.8 against hCA I and hCA II, respectively. Many of these new compounds showed a high selectivity for bacterial enzyme respect to the mammalian CA isoforms (hCAI and hCAII). These results suggest that sulfonamides with quinazoline scaffold could be considered as suitable candidates for further derivatization to better understand the role of bacterial CAs in pathogenesis.

A class of 4-sulfamoylphenyl-ω-aminoalkyl ethers with effective carbonic anhydrase inhibitory action and antiglaucoma effects.

We report a series of 4-sulfamoylphenyl-ω-aminoalkyl ethers as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The structure–activity relationship was drawn for the inhibition of four physiologically relevant isoforms: hCA I, II, IX, and XII. Many of these compounds were highly effective, low nanomolar inhibitors of all CA isoforms, whereas several isoform-selective were also identified. X-ray crystal structures of two new sulfonamides bound to the physiologically dominant CA II isoform showed the tails of these derivatives bound within the hydrophobic half of the enzyme active site through van der Waals contacts with Val135, Leu198, Leu204, Trp209, Pro201, and Pro202 amino acids. One of the highly water-soluble compound (as trifluoroacetate salt) showed effective IOP lowering properties in an animal model of glaucoma. Several fluorescent sulfonamides incorporating either the fluorescein-thiourea (7a–c) or tetramethylrhodamine-thiourea (9a,b) moieties were also obtained and showed interesting CA inhibitory properties for the tumor-associated isoforms CA IX and XII.

Inhibition studies of new ureido-substituted sulfonamides incorporating a GABA moiety against human carbonic anhydrase isoforms I–XIV

Reaction of γ-Boc-GABA, prepared by protecting the γ-amino moiety of the amino butyric acid with the tert-butyloxycarbonyl (Boc) protecting group, with 4-methyl/ethyl benzenesulfonamide, followed by removal of the Boc protecting group in 3M HCl afforded the corresponding hydrochlorides, which were further derivatized by reaction with a varying of aryl isocyanates to give a new classes of ureido substituted benzenesulfonamide containing a GABA moiety. Inhibition studies of the human carbonic anhydrase (CA, EC 4.2.1.1) isoforms, CA I–XIV with these new compounds revealed that they possess moderate–weak inhibition potency against hCA III, IV, VA, VI and XIII, rather efficient inhibitory power against hCA I, VI, and IX, and excellent inhibition of the physiologically relevant hCA II and VII, as well as of the two tumor-associated isoforms CA IX and XII. The inhibition profile of the new ureido-substituted benzenesulfonamides reported here is thus very different from the corresponding ureido-substituted analogs incorporating sulfanilamide, which were previously investigated as inhibitors of some of these enzymes.

Structural Insights on Carbonic Anhydrase Inhibitory Action, Isoform Selectivity, and Potency of Sulfonamides and Coumarins Incorporating Arylsulfonylureido Groups

Sulfonamides and coumarins incorporating arylsulfonylureido tails were prepared and assayed as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Some derivatives incorporating 3-pyridinesulfonamide and arylsulfonylureoido fragments were low nanomolar inhibitors of isoforms CA II and XII (upregulated or overexpressed in glaucoma) and showed effective in vivo intraocular pressure lowering effects in an animal model of the disease, which were several times better compared to those of the antiglaucoma drug dorzolamide. By means of X-ray crystallography of adducts of several sulfonamides with CA II, the effective inhibitory properties were rationalized at the molecular level. The coumarins were ineffective as hCA I and II inhibitors but showed low nanomolar activity for the inhibition of the tumor-associated isoforms hCA IX and XII. The presence of arylsulfonylureido tails in these CA inhibitors possessing quite different mechanisms of action led to highly effective and isoform-selective compounds targeting enzymes involved in severe pathologies such as glaucoma or cancer.

Structural insights into carbonic anhydrase IX isoform specificity of carbohydrate-based sulfamates.

Carbonic anhydrase IX (CA IX) is an extracellular transmembrane homodimeric zinc metalloenzyme that has been validated as a prognostic marker and therapeutic target for several types of aggressive cancers. CA IX shares a close homology with other CA isoforms, making the design of CA IX isoform selective inhibitors challenging. In this paper, we describe the development of a new class of CA IX inhibitors that comprise a sulfamate as the zinc binding group, a variable linker, and a carbohydrate “tail” moiety. Seven compounds inhibited CA IX with low nM Ki values of 1–2 nM and also exhibited permeability profiles to preferentially target the binding of extracellular CA IX over cytosolic CAs. The crystal structures of two of these compounds in complex with a CA IX-mimic (a variant of CA II, with active site residues that mimic CA IX) and one compound in complex with CA II have been determined to 1.7 Å resolution or better and demonstrate a selective mechanism of binding between the hydrophilic and hydrophobic pockets of CA IX versus CA II. These compounds present promising candidates for anti-CA IX drugs and the treatment for several aggressive cancer types.

Ferrier sulfamidoglycosylation of glycals catalyzed by nitrosonium tetrafluoroborate: Towards new carbonic anhydrase glycoinhibitors

Ferrier sulfamidoglycosylation of glycals catalyzed by nitrosonium tetrafluoroborate allowed the preparation of hydroxysulfamide glycosides in good yields with a good α stereoselectivity. A variety of mono-saccharide derivatives was synthesized using this new methodology leading to selective and powerful glycoinhibitors of the tumor associated carbonic anhydrases (CA, EC 4.2.1.1) isoforms CA IX and CA XII.

Quinazoline–sulfonamides with potent inhibitory activity against the α-carbonic anhydrase from Vibrio cholerae

Thirteen novel sulfonamide derivatives incorporating the quinazoline scaffold were synthesized by simple, eco-friendly procedures. These compounds were tested for their ability to inhibit the α-carbonic anhydrases (CA, EC 4.2.1.1) from Vibrio cholerae (VchCA) as well as the human α-CA isoforms, hCA I and hCA II. Nine compounds were highly effective, nanomolar inhibitors of the pathogenic enzyme VchCA. Three of them were also highly effective sub-nanomolar inhibitors of the cytosolic isoform II. The best VchCA inhibitor had a KI of 2.7nM. Many of these developed compounds showed high selectivity for inhibition of the bacterial over the mammalian CA isoforms, with one compound possessing selectivity ratios as high as 97.9 against hCA I and 9.7 against hCA II. Compound 9d was another highly effective VchCA inhibitor presenting a selectivity ratio of 99.1 and 8.1 against hCA I and hCA II, respectively. These results suggest that sulfonamides with quinazoline backbone could be considered suitable tools to better understand the role of bacterial CAs in pathogenesis.

Initiation of metamorphosis and control of ecdysteroid biosynthesis in insects: The interplay of absence of Juvenile hormone, PTTH, and Ca2+-homeostasis

The paradigm saying that release of the brain neuropeptide big prothoracicotropic hormone (PTTH) initiates metamorphosis by activating the Torso-receptor/ERK pathway in larval prothoracic glands (PGs) is widely accepted nowadays. Upon ligand–receptor interaction Ca2+ enters the PG cells and acts as a secondary messenger. Ecdysteroidogenesis results, later followed by apoptosis. Yet, some data do not fit in this model. In some species decapitated animals can still molt, even repeatedly, and metamorphose. PTTH does not universally occur in all insect species. PGs may also have other functions; PGs as counterpart of the vertebrate thymus? There are also small PTTHs. Finally, PTTH remains abundantly present in adults and plays a role in control of ecdysteroidogenesis (=sex steroid production) in gonads. This is currently documented only in males. This urges a rethinking of the PTTH–PG paradigm. The key question is: Why does PTTH-induced Ca2+ entry only result in ecdysteroidogenesis and apoptosis in specific cells/tissues, namely the PGs and gonads? Indeed, numerous other neuropeptides also use Ca2+ as secondary messenger. The recent rediscovery that in both invertebrates and vertebrates at least some isoforms of Ca2+-ATPase need the presence of an endogenous farnesol/juvenile hormone(JH)-like sesquiterpenoid for keeping cytosolic [Ca2+]i below the limit of apoptosis-induction, triggered the idea that it is not primarily PTTH, but rather the drop to zero of the JH titer that acts as the primordial initiator of metamorphosis by increasing [Ca2+]i. PTTH likely potentiates this effect but only in cells expressing Torso. PTTH: an evolutionarily ancient gonadotropin?

New series of sulfonamides containing amino acid moiety act as effective and selective inhibitors of tumor-associated carbonic anhydrase XII

New benzenesulfonamides incorporating water solubilizing moieties were synthesized using N-α-acetyl-l-lysine or γ-aminobutyric acid as scaffolds followed by the conversion of their terminal amino group to the guanidine one. Their inhibition activity was assessed by determining their KIs values against the human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, II, IX and XII. Some of these compounds were medium potency inhibitors of the cytosolic (CA I, II) and transmembrane (CA IX) isoforms and highly effective, nanomolar inhibitors of the second transmembrane isoform hCA XII. Some of these sulfonamides possessing good selectivity inhibition for the tumor-associated CA XII isoform over the cytosolic and physiologically dominant isoforms CA I and II may be used as tools to develop new anticancer agents.

Capsaicin: a potent inhibitor of carbonic anhydrase isoenzymes.

Carbonic anhydrase (CA, EC 4.2.1.1) is a zinc containing metalloenzyme that catalyzes the rapid and reversible conversion of carbon dioxide (CO2) and water (H2O) into a proton (H+) and bicarbonate (HCO3–) ion. On the other hand, capsaicin is the main component in hot chili peppers and is used extensively used in spices, food additives and drugs; it is responsible for their spicy flavor and pungent taste. There are sixteen known CA isoforms in humans. Human CA isoenzymes I, and II (hCA I and hCA II) are ubiquitous cytosolic isoforms. In this study, the inhibition properties of capsaicin against the slow cytosolic isoform hCA I, and the ubiquitous and dominant rapid cytosolic isozymes hCA II were studied. Both CA isozymes were inhibited by capsaicin in the micromolar range. This naturally bioactive compound has a Ki of 696.15 µM against hCA I, and of 208.37 µM against hCA II.

Dipotassium-trioxohydroxytetrafluorotriborate, K2[B3O3F4OH], is a potent inhibitor of human carbonic anhydrases

The boron heterocyclic compound dipotassium-trioxohydroxytetrafluorotriborate (K2[B3O3F4OH]) was investigated as inhibitor of the zinc enzyme, carbonic anhydrase (CA, EC 4.2.1.1). Eleven human (h) CA isoforms, hCA I–IV, VA, VI, VII, IX and XII–XIV, were included in the investigations. The anion, similar to tetraborate or phenylboronic acid, inhibited most of them. hCA III was not inhibited by K2[B3O3F4OH], whereas hCA VA, hCA VI, hCA IX and hCA XIII were inhibited in the submillimolar range, with KIs of 0.31–0.63 mM. hCA I and II (cytosolic, widespread isoforms), hCA IV (membrane-bound isoform), hCA XII (tumor-associated, transmembrane) and hCA XIV (transmembrane) were much more effectively inhibited by this anion, with inhibition constants ranging from 25 to 93 µM. hCA VII, a cytosolic enzyme present in the brain and associated to oxidative stress, was very effectively inhibited by K2[B3O3F4OH], with a KI of 8.0 µM. We propose that K2[B3O3F4OH] binds to the metal ion from the enzyme active site, coordinating to the Zn(II) ion monodentately through its B-OH functionality. We hypothesize that some of the beneficial antitumor effects reported for K2[B3O3F4OH] may be due to the inhibition of CAs present in skin tumors.

A new affinity gel for the purification of α-carbonic anhdrases

The new affinity gel reported in this study was prepared using EUPERGIT C250L as a chromatographic bed material, to which etylenediamine spacer arms were attached to prevent steric hindrance between the matrix and ligand, and to facilitate effective binding of the CA-specific ligand, of the aromatic sulfonamide type for the purification of α-carbonic anhydrases (Cas; EC 4.2.1.1). Indeed, the aminoethyl moieties of the affinity gel were derivatized by reaction with 4-isothiocyanatobenzenesulfonamide, with the formation of a thiourea-based gel, having inhibitory effects against CAs. Both bovine erythrocyte carbonic anhydrase BCA and human (h) erythrocyte CA isoforms I, II (hCA I and II) have been purified from hemolysates, by using this affinity gel. The greatest purification fold and column yields for BCA and for cytosolic (hCA I + II) enzymes were of 181-fold (21.07%) and 184-fold (9.49%), respectively. Maximum binding was achieved at 15 °C and I = 0.3 ionic strength for α-carbonic anhydrases.

Dual carbonic anhydrase/matrix metalloproteinase inhibitors incorporating bisphosphonic acid moieties targeting bone tumors

A set of bisphosphonate matrix metalloproteinase (MMP) inhibitors was investigated for inhibitory activity against several carbonic anhydrase (CA, EC 4.2.1.1) isozymes, some of which are overexpressed in hypoxic tumors. Some of the bisphosphonate revealed to be very potent inhibitors (in the low nanomolar range) of the cytosolic isoform CA II and the membrane-bound CA IX, XII and XIV isozymes, a feature useful for considering them as interesting compounds for bone resorption inhibition applications. We suggest here that it is possible to develop dual enzyme inhibitors bearing bisphosphonate moieties that may target both MMPs and CAs, two families of enzymes involved in tumor formation, growth, and metastasis.

Toward the Identification of Novel Carbonic Anhydrase XIV Inhibitors using 3D-QSAR Pharmacophore Model, Virtual Screening and Molecular Docking Study

Carbonic anhydrases XIV (CA XIV) is responsible for health issue and a therapeutic target of many disorders like epilepsy, and retinopathy. Here we described the first pharmacophore model for human CA XIV to identify the new scaffolds compounds. Hypotheses 1, with the highest cost difference, best correlation coefficient as well as the lowest RMSD, was validated by test set and Fischer method. Then, Hypotheses 1 was used for virtual screening in 718361 databases compounds. After Lipinski’s rule of five and ADMET properties, 148 compounds with high potential activity were filtered and estimated by docking. Finally, 6 new scaffold compounds turned out to be potential as new class CA XIV antagonists. In addition, we also analyzed the interaction between compound 7 and ten CA isoforms and explained various inhibitions caused by their distinct residues. Therefore, this paper could be helpful in novel CA XIV inhibitors discovery and its selective inhibitors design.

Pyrazolo[4,3-e][1,2,4]triazine sulfonamides as carbonic anhydrase inhibitors with antitumor activity

A series of sildenafil analogues and aniline substituted pyrazolo[4,3-e][1,2,4]triazine sulfonamides were prepared and evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors and for their anticancer activity against two human breast cancer cell lines (MCF-7, MDA-MB-231). The new compounds were ineffective as CA I inhibitors, poorly inhibited CA II, but were more effective against the tumor-associated isoforms CA IX and XII, with some compounds acting as low nanomolar inhibitors. Evaluation of the cytotoxicity by using an MTT assay, the inhibition of [3H]thymidine incorporation into DNA as well as collagen synthesis inhibition, demonstrated that these sulfonamides exhibit cytotoxic effects on breast cancer cell lines ex vivo.

Crystal structure and conformational flexibility of the unligated FK506-binding protein FKBP12.6.

The primary known physiological function of FKBP12.6 involves its role in regulating the RyR2 isoform of ryanodine receptor Ca(2+) channels in cardiac muscle, pancreatic β islets and the central nervous system. With only a single previously reported X-ray structure of FKBP12.6, bound to the immunosuppressant rapamycin, structural inferences for this protein have been drawn from the more extensive studies of the homologous FKBP12. X-ray structures at 1.70 and 1.90 Å resolution from P2₁ and P3₁21 crystal forms are reported for an unligated cysteine-free variant of FKBP12.6 which exhibit a notable diversity of conformations. In one monomer from the P3₁21 crystal form, the aromatic ring of Phe59 at the base of the active site is rotated perpendicular to its typical orientation, generating a steric conflict for the immunosuppressant-binding mode. The peptide unit linking Gly89 and Val90 at the tip of the protein-recognition `80s loop' is flipped in the P2₁ crystal form. Unlike the >30 reported FKBP12 structures, the backbone conformation of this loop closely follows that of the first FKBP domain of FKBP51. The NMR resonances for 21 backbone amides of FKBP12.6 are doubled, corresponding to a slow conformational transition centered near the tip of the 80s loop, as recently reported for 31 amides of FKBP12. The comparative absence of doubling for residues along the opposite face of the active-site pocket in FKBP12.6 may in part reflect attenuated structural coupling owing to increased conformational plasticity around the Phe59 ring.

Combining the tail and the ring approaches for obtaining potent and isoform-selective carbonic anhydrase inhibitors: Solution and X-ray crystallographic studies

5-(3-Tosylureido)pyridine-2-sulfonamide and 4-tosylureido-benzenesulfonamide (ts-SA) only differ by the substitution of a CH by a nitrogen atom, but they have very different inhibitory properties against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). By means of X-ray crystallography on the human CA II adducts of the two compounds these differences have been rationalized. As all sulfonamides, the two compounds bind in deprotonated form to the Zn(II) ion from the enzyme active site and their organic scaffolds extend throughout the cavity, participating in many interactions with amino acid residues and water molecules. However the pyridine derivative undergoes a tilt of the heterocyclic ring compared to the benzene analog, which leads to a very different orientation of the two scaffolds when bound to the enzyme. This tilt also leads to a clash between a carbon atom from the pyridine ring of the first inhibitor and the OH moiety of Thr200, leading to less effective inhibitory properties of the pyridine versus the benzene sulfonamide derivative. Indeed, ts-SA is a promiscuous, low nanomolar inhibitor of 7 out of 10 human (h) CA isoforms, whereas the pyridine sulfonamide is a low nanomolar inhibitor only of the tumor-associated hCA IX and XII, being less effective against other 9 isoforms. Thus, a difference of one atom (N vs CH) in two isostructural sulfonamides leads to drastic differences of activity, phenomenon understood at the atomic level through the high resolution crystallographic structure and kinetic measurements reported in the paper. Combining the tail and the ring approaches in the same chemotype leads to isoform-selective, highly effective sulfonamide CA inhibitors.

Mitochondrial Ca2+-Handling in Fast Skeletal Muscle Fibers from Wild Type and Calsequestrin-Null Mice

Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum (SR) in muscle tissue are subject of lively debate. In this study we aimed to clarify how the SR determines mitochondrial calcium handling using dCASQ-null mice which lack both isoforms of the major Ca2+-binding protein inside SR, calsequestrin. Mitochondrial free Ca2+-concentration ([Ca2+]mito) was determined by means of a genetically targeted ratiometric FRET-based probe. Electron microscopy revealed a highly significant increase in intermyofibrillar mitochondria (+55%) and augmented coupling (+12%) between Ca2+ release units of the SR and mitochondria in dCASQ-null vs. WT fibers. Significant differences in the baseline [Ca2+]mito were observed between quiescent WT and dCASQ-null fibers, but not in the resting cytosolic Ca2+ concentration. The rise in [Ca2+]mito during electrical stimulation occurred in 20−30 ms, while the decline during and after stimulation was governed by 4 rate constants of approximately 40, 1.6, 0.2 and 0.03 s−1. Accordingly, frequency-dependent increase in [Ca2+]mito occurred during sustained contractions. In dCASQ-null fibers the increases in [Ca2+]mito were less pronounced than in WT fibers and even lower when extracellular calcium was removed. The amplitude and duration of [Ca2+]mito transients were increased by inhibition of mitochondrial Na+/Ca2+ exchanger (mNCX). These results provide direct evidence for fast Ca2+ accumulation inside the mitochondria, involvement of the mNCX in mitochondrial Ca2+-handling and a dependence of mitochondrial Ca2+-handling on intracellular (SR) and external Ca2+ stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia. The differences in structure and in mitochondrial function observed relative to WT may represent compensatory mechanisms for the disease-related reduction of calcium storage capacity of the SR and/or SR Ca2+-leakage.