Transmembrane Carbonic Anhydrase Research Articles

Interactions of transmembrane carbonic anhydrase, CAIX, with bicarbonate transporters

Association of some plasma membrane bicarbonate transporters with carbonic anhydrase enzymes forms a bicarbonate transport metabolon to facilitate metabolic CO(2)-HCO(3)(-) conversions and coupled HCO(3)(-) transport. The transmembrane carbonic anhydrase, CAIX, with its extracellular catalytic site, is highly expressed in parietal and other cells of gastric mucosa, suggesting a role in acid secretion. We examined in transfected HEK293 cells the functional and physical interactions between CAIX and the parietal cell Cl(-)/HCO(3)(-) exchanger AE2 or the putative Cl(-)/HCO(3)(-) exchanger SLC26A7. Coexpression of CAIX increased AE2 transport activity by 28 +/- 7% and also activated transport mediated by AE1 and AE3 (32 +/- 10 and 37 +/- 9%, respectively). In contrast, despite a transport rate comparable to that of AE3, coexpressed CAIX did not alter transport associated with SLC26A7. The CAIX-associated increase of AE2 activity did not result from altered AE2 expression or cell surface processing. CAIX was coimmunoprecipitated with the coexpressed SLC4 polypeptides AE1, AE2, and AE3, but not with SLC26A7. GST pull-down assays with a series of domain-deleted forms of CAIX revealed that the catalytic domain of CAIX mediated interaction with AE2. AE2 and CAIX colocalized in human gastric mucosa, as indicated by coimmunofluorescence. This is the first example of a functional and physical interaction between a bicarbonate transporter and a transmembrane carbonic anhydrase. We conclude that CAIX can bind to some Cl(-)/HCO(3)(-) exchangers to form a bicarbonate transport metabolon.

Expression of transmembrane carbonic anhydrases IX and XII in ovarian tumours

Carbonic anhydrase (CA) isozymes IX and XII have been suggested to play a role in oncogenic processes. The aim of the present study was to investigate CA IX and XII expression in patients with ovarian tumours. A series of ovarian tumours was immunostained for CA IX and XII and the results were correlated with histopathological and clinical parameters. Most cases of borderline mucinous cystadenomas, mucinous cystadenocarcinomas and serous cystadenocarcinomas were moderately or strongly positive for CA IX. In malignant tumours, the staining was most prominent in hypoxic regions. Expression of CA XII was detected in all tumour categories, although the mean staining intensity was weaker than for CA IX in all groups except for clear cell carcinomas. The wide expression of CA IX and XII in ovarian tumours suggests that these isozymes could represent potential targets in ovarian cancer therapy. The expression pattern of CA IX suggests that it could also serve as a useful histopathological marker protein for hypoxia in malignant ovarian tumours.

Inhibition of membrane-associated carbonic anhydrase isozymes IX, XII and XIV with a library of glycoconjugate benzenesulfonamides

A library of glycoconjugate benzenesulfonamides that contain diverse carbohydrate-triazole tails were investigated for their ability to inhibit the enzymatic activity of the three human transmembrane carbonic anhydrase (CA) isozymes hCA IX, hCA XII and hCA XIV. These isozymes have their CA domains located extracellularly, unlike the physiologically dominant hCA II, and are of immense current interest as druggable targets. Elevated expression of isozymes IX and XII is a marker for a broad spectrum of hypoxic tumors—this physiology may facilitate a novel approach to discriminate between healthy cells and cancerous cells. Many of these glycoconjugates were potent inhibitors (low nM), but importantly exhibited different isozyme selectivity profiles. The most potent hCA IX inhibitor was the glucuronic acid derivative 20 ( K i = 23 nM). This compound was uniquely hCA IX selective cf. all other isozymes (16.4-, 16.8- and 4.6-fold selective against hCA II, XII, and XIV, respectively). At hCA XII there were many inhibitors with K is < 10 nM that also demonstrated excellent selectivity (up to 344-fold) against other isozymes. Potent hCA XIV inhibitors were also identified, several with K is ∼ 10 nM, however no hCA XIV-selective derivatives were evidenced from this library. The sugar tails of this study have shown promise as a valuable approach to both solubilize the aromatic sulfonamide CA recognition pharmacophore and to deliver potent inhibition and isozyme differentiation of the transmembrane CAs.

Carbonic Anhydrase Inhibitors: Synthesis and Inhibition of Cytosolic/Tumor‐Associated Carbonic Anhydrase Isozymes I, II, and IX with Sulfonamides Incorporating Thioureido‐Sulfanilyl Scaffolds.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Carbonic anhydrase inhibitors: Inhibition of the human transmembrane isozyme XIV with a library of aromatic/heterocyclic sulfonamides

The first inhibition study of the transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozymes hCA XIV with a library of aromatic and heteroaromatic sulfonamides synthesized earlier is reported. Most of the inhibitors were sulfanilamide, homosulfanilamide and 4-aminoethyl-benzenesulfonamide derivatives, to which tails that would induce diverse physicochemical properties have been attached at the amino moiety. Several of these compounds were metanilamide, benzene-1,3-disulfonamide or the 1,3,4-thiadiazole/thiadiazoline-2-sulfonamide derivatives. The tails incorporated in these molecules were of the alkyl/aryl-carboxamido/ sulfonamido-, ureido- or thioureido-types. The sulfanilamides acylated at the 4-amino group with short aliphatic/aromatic moieties incorporating 2-6 carbon atoms showed modest hCA XIV inhibitory activity ( K I-s in the range of 1.25–4.2 μM) which were anyhow better than that of sulfanilamide ( K I of 5.4 μM). Better activity showed the homosulfanilamide and 4-aminoethyl-benzenesulfonamide derivatives bearing arylsulfonamido/ureido and thioureido moieties, with K I’s in the range of 203–935 nM. The best activity was observed for the heteroaromatic compounds incorporating 1,3,4-thiadiazole/thiadiazoline-2-sulfonamide and 5-arylcarboxamido/sulfonamido moieties, with K I’s in the range of 10–85 nM. All these compounds were generally also much better inhibitors of the other two transmembrane CA isozyme, hCA IX and XII. Thus, highly potent hCA XIV inhibitors were detected, but isozyme-specific inhibitors were not discovered for the moment.

Carbonic anhydrase inhibitors: Inhibition of the tumor-associated isozymes IX and XII with polyfluorinated aromatic/heterocyclic sulfonamides

The tumor-associated transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozymes IX (CA IX) and XII (CA XII) are involved in acidification of hypoxic tumors, a process correlated with poor prognosis and clinical outcome of patients harboring such tumors. This process may be reversed by inhibiting these enzymes with potent sulfonamide/sulfamate inhibitors. A series of such aromatic/heterocyclic sulfonamides incorporating 2,3,5,6-tetrafluorobenzoyl-, 2,3,5,6-tetrafluoro- phenylsulfonyl- and pentafluorophenylureido moieties has been investigated for its interaction with the catalytic domain of the human isozymes hCA IX and hCA XII. Some of these compounds showed excellent inhibitory properties against both isozymes IX and XII, with several subnanomolar inhibitors detected for the first time. These sulfonamides may constitute valuable candidates for the development of novel antitumor therapies based on the inhibition of such tumor-associated CA isozymes.

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides incorporating thioureido-sulfanilyl scaffolds

The tumor-associated transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isozyme IX (CA IX) is overexpressed in hypoxic tumors and appears to be involved in acidification of the tumor microenvironment, a process correlated with cancer progression and bad prognosis. The acidification may be reduced by inhibiting the enzyme with potent sulfonamide/sulfamate CA inhibitors. A series of such aromatic sulfonamides incorporating thioureido-sulfanilyl moieties has been prepared and investigated for its interaction with the catalytic domain of the human isozyme hCA IX. The key intermediates in the synthesis were obtained by reacting sulfanilamide, homosulfanilamide, or 4-aminoethylbenzenesulfonamide with 4-acetamido-benzenesulfonyl chloride followed by deacetylation and reaction with thiophosgene. The obtained isothiocyanato sulfonamides were reacted with aliphatic or aromatic primary amines or hydrazines, leading to the corresponding thioureas. Some of these compounds showed excellent inhibitory properties against isozymes I, II, and IX, with several inhibitors also presenting selectivity for the inhibition of CA IX over that of the ubiquitous isozyme CA II. Such sulfonamides may constitute interesting candidates for the development of novel antitumor therapies based on the inhibition of the CA isozymes overexpressed in hypoxic tumors. Due to the highest expression of CA IX in clear renal cell carcinoma and its chemo/radioresistance, our efforts are first of all directed to generate effective therapeutic strategies for the cure of this malignancy.

Carbonic Anhydrase Inhibitors: The First Selective, Membrane‐Impermeant Inhibitors Targeting the Tumor‐Associated Isozyme IX.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Carbonic anhydrase inhibitors: the first QSAR study on inhibition of tumor-associated isoenzyme IX with aromatic and heterocyclic sulfonamides

QSAR study on the tumor-associated transmembrane carbonic anhydrase IX (CA IX) isoenzyme has been made using a large pool of distance-based topological indices : W, Sz, PI, 0χ, 1χ, 2χ, 0χv, 1χv, and 2χv. A combined set of 32 aromatic and heterocyclic compounds, including the six clinically used derivatives: acetazolamide, methazolamide, ethoxyzolamide, dichlorophenamide, dorzolamide, and brinzolamide are used for this purpose. The results have shown that the inhibition of the tumor-associated isoenzyme IX with aromatic and heterocyclic sulfonamides can be modeled excellently in multiparametric regression after introduction of indicator parameters. The predictive power of the models is discussed using probable error of correlation (PE), variance-inflation factor (VIF), and cross-validation parameters: PRESS, SSY, r2cv, SPRESS, and PSE. This is the first report on QSAR study on inhibition of tumor-associated isoenzyme IX.

Carbonic anhydrase inhibitors: the first QSAR study on inhibition of tumor-associated isoenzyme IX with aromatic and heterocyclic sulfonamides

QSAR study on the tumor-associated transmembrane carbonic anhydrase IX (CA IX) isoenzyme has been made using a large pool of distance-based topological indices : W, Sz, PI, 0χ , 1χ , 2χ , 0χ v , 1χ v , and 2χ v . A combined set of 32 aromatic and heterocyclic compounds, including the six clinically used derivatives: acetazolamide, methazolamide, ethoxyzolamide, dichlorophenamide, dorzolamide, and brinzolamide are used for this purpose. The results have shown that the inhibition of the tumor-associated isoenzyme IX with aromatic and heterocyclic sulfonamides can be modeled excellently in multiparametric regression after introduction of indicator parameters. The predictive power of the models is discussed using probable error of correlation (PE), variance-inflation factor (VIF), and cross-validation parameters: PRESS, SSY, r 2 cv, S PRESS, and PSE. This is the first report on QSAR study on inhibition of tumor-associated isoenzyme IX.

Carbonic anhydrase inhibitors: The first selective, membrane-impermeant inhibitors targeting the tumor-associated isozyme IX

The inhibition of the tumor-associated transmembrane carbonic anhydrase IX (CA IX) isozyme possessing an extracellular active site has been investigated with a series of positively-charged, pyridinium derivatives of sulfanilamide, homosulfanilamide and 4-aminoethylbenzenesulfonamide. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and IV (membrane-bound) were also provided for comparison. A very interesting inhibition profile against CA IX with these sulfonamides has been observed. Several nanomolar (Ki's in the range of 6–54 nM) CA IX inhibitors have also been detected. Because CA IX is a highly active isozyme predominantly expressed in tumor tissues with bad prognosis of disease progression, this finding is very promising for the potential design of CA IX-specific inhibitors with applications as anti-tumor agents. This is the first report of inhibitors that may selectively target CA IX, due to their membrane-impermeability and high affinity for this clinically relevant isozyme.

Transmembrane carbonic anhydrase isozymes IX and XII in the female mouse reproductive organs

BackgroundCarbonic anhydrase (CA) classically catalyses the reversible hydration of dissolved CO2 to form bicarbonate ions and protons. The twelve active CA isozymes are thought to regulate a variety of cellular functions including several processes in the reproductive systems.MethodsThe present study was designed to investigate the expression of transmembrane CAs, CA IX and XII, in the mouse uterus, ovary and placenta. The expression of CA IX and XII was examined by immunoperoxidase staining method and western blotting. CA II and XIII served as positive controls since they are known to be present in the mouse reproductive tract.ResultsThe data of our study indicated that CA XII is expressed in the mouse endometrium. Only very faint signal was observed in the corpus luteum of the ovary and the placenta remained mainly negative. CA IX showed weak reaction in the endometrial epithelium, while it was completely absent in the ovary and placenta.ConclusionThe conservation of CA XII expression in both mouse and human endometrium suggests a role for this isozyme in reproductive physiology.

Monoclonal antibodies generated in carbonic anhydrase IX-deficient mice recognize different domains of tumour-associated hypoxia-induced carbonic anhydrase IX

Transmembrane carbonic anhydrase IX (CA IX) is frequently expressed in human tumours in response to hypoxia and may serve as a tumour marker and therapeutic target. So far, only a single monoclonal antibody (MAb) M75 with an epitope in the N-terminal proteoglycan (PG)-like region has been available for detection purposes. Attempts to produce MAbs against other parts of CA IX were unsuccessful due to the immunodominance of the PG region that significantly differs between human and mouse homologues. To overcome this problem, we used various forms of human CA IX antigen to immunize CA IX-deficient mice recently produced by targeted disruption of Car9 gene. Here, we describe new MAbs that react with human, but not mouse CA IX in different immunodetection settings, and show no cross-reactivity with CA I, II and XII. MAb IV/18 is directed to the PG region, while the other six antibodies bind to the CA domain, as determined by CA IX deletion variants. IV/18 recognizes a linear epitope, while anti-CA MAbs V/10, V/12, VII/20, VII/28, VII/32 and VII/38 react with conformational epitopes clustered into three antigenic sites. The new antibodies represent important tools for improving our knowledge of structure–function relationships in the CA IX molecule and a better understanding of the role of CA IX in cancer development. Moreover, the availability of the MAbs specific for distinct antigenic regions on two separate extracellular domains offers an opportunity to elaborate a sensitive assay that could be particularly important for CA IX detection in body fluids of cancer patients.

Expression of cell surface transmembrane carbonic anhydrase genes CA9 and CA12 in the human eye: overexpression of CA12 (CAXII) in glaucoma

Purpose: Carbonic anhydrase enzymes (CAs) are universally involved in many fundamental physiological processes, including acid base regulation and fluid formation and movement. In glaucoma patients, CA inhibitors are very effective...

Carbonic anhydrase inhibitors: inhibition of the tumor-associated isozyme IX with aromatic and heterocyclic sulfonamides

The inhibition of the tumor-associated transmembrane carbonic anhydrase IX (CA IX) isozyme has been investigated with a series of aromatic and heterocyclic sulfonamides, including the six clinically used derivatives acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide and brinzolamide. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and IV (membrane-bound) were also provided for comparison. A very interesting and unusual inhibition profile against CA IX with these sulfonamides has been observed. Several nanomolar ( K I-s in the range of 14–50 nM) CA IX inhibitors have been detected, both among the aromatic (such as orthanilamide, homosulfonilamide, 4-carboxy-benzenesulfonamide, 1-naphthalenesulfonamide and 1,3-benzenedisulfonamide derivatives) as well as the heterocylic (such as 1,3,4-thiadizole-2-sulfonamide, etc.) sulfonamides examined. Because CA IX is a highly active isozyme predominantly expressed in tumor tissues with poor prognosis of disease progression, this finding is very promising for the potential design of CA IX-specific inhibitors with applications as anti-tumor agents.

Effects of von Hippel-Lindau gene mutation and methylation status on expression of transmembrane carbonic anhydrases in renal cell carcinoma.

Our primary goal was to determine whether increased expression of the transmembrane carbonic anhydrase (CA) isozymes in renal cell carcinoma (RCC) is regulated by the von Hippel-Lindau (VHL) gene. We studied mRNA expression of all three transmembrane CAs - CA IX, XII, and XIV - in 17 RCC cell lines, transformants of the wild-type VHL gene, and normal kidney tissue cultures, and then compared them with the mutation status in the VHL gene. Northern blot analysis showed no detectable signal for CA XIV mRNA expression in normal and cancer cells. CA XII mRNA was ubiquitously expressed except in two cell lines. Although CA XII expression levels tended to be lower in RCC cell lines without the VHL mutation and in transformants of the wild-type VHL gene, the results were not conclusive. Significant expression of CA IX mRNA was seen in eight of 17 RCC cell lines. Among five cell lines which had no VHL mutation, four lines showed no detectable signal and one cell line showed a low amount of CA IX mRNA expression. In patients with RCC, VHL mutations and significant CA IX expression were seen in established tumor cell lines but not in primary tissue cultures from normal counterparts. Further study of methylation status showed that the 5' region in the CA9 gene was hypomethylated in all CA IX-positive cell lines and hypermethylated in all CA IX-negative cell lines. Especially, methylation status at -74 and -6 CpG sites perfectly correlated with CA IX expression. These findings indicate that VHL suppresses CA IX expression but has no conclusive effect on CA XII and XIV expressions in RCC. CA IX expression is also driven by the methylation status of the CA9 gene.

Transmembrane carbonic anhydrase, MN/CA IX, is a potential biomarker for biliary tumours

Background/Aims : Carbonic anhydrase isoenzyme IX (MN/CA IX) is a transmembrane protein with a suggested function in maintaining the acid–base balance and intercellular communication. Previous studies have demonstrated that MN/CA IX is expressed in the basolateral plasma membrane of normal biliary epithelial cells, but not in hepatocytes. This study was designed to examine the expression of MN/CA IX in hepatobiliary neoplasms and to elucidate its value as a marker for biliary differentiation. Methods : Fifty-seven hepatobiliary lesions were immunostained for MN/CA IX using biotin–streptavidin complex method. Twenty samples containing normal biliary epithelium and five containing normal liver tissue were used as controls. Results : In the biliary epithelial tumours, immunostaining for MN/CA IX was mainly localized at the basolateral surface of the epithelial cells, like in normal mucosa. All non-invasive dysplastic lesions and 57% of invasive lesions of gall-bladder expressed MN/CA IX. In liver, 78% of cholangiocellular malignant lesions showed a positive reaction for MN/CA IX, whereas only 33% of hepatocellular carcinomas showed a weak immunoreaction. Conclusions : Our results suggest that abnormal expression of MN/CA IX may be linked to malignant transformation of hepatobiliary cells. In addition, it seems to be a promising marker for biliary differentiation in hepatobiliary neoplasms.

Expression of the transmembrane carbonic anhydrases, CA IX and CA XII, in the human male excurrent ducts.

Testicular fluid is concentrated and acidified during its passage through the excurrent ducts. These processes involve bicarbonate absorption, in which carbonic anhydrases are implicated. In this study, the distribution of two transmembrane carbonic anhydrase isozymes (CA IX and CA XII) in the human excurrent ducts was investigated using isozyme-specific antibodies in conjunction with immunohistochemical and immunoblotting techniques. Specific staining for CA XII was present in the basolateral plasma membrane of the epithelial cells in the efferent ducts, predominantly in the non-ciliated cells. In the epididymal duct, CA XII was detected only in sporadic cells, which also contained CA II, thus suggesting that they are apical mitochondria-rich cells. CA IX was also localized to the basolateral plasma membrane of the epithelium in the efferent ducts, but its staining was weaker and less uniform compared to CA XII. No signal for CA IX was detected in the epididymal duct. Western blot analysis from efferent duct samples revealed specific bands for CA IX and CA XII, confirming that the immunohistochemical stainings represent these isozymes. The expression of CA XII and CA IX in the excurrent duct system and co-expression of CA XII with Aquaporin-1 in the same efferent duct epithelial cells suggest their functional involvement in ion transport and concentration processes of testicular fluid.

Expression of Hypoxia-Inducible Cell-Surface Transmembrane Carbonic Anhydrases in Human Cancer

An acidic extracellular pH is a fundamental property of the malignant phenotype. In von Hippel-Lindau (VHL)-defective tumors the cell surface transmembrane carbonic anhydrase (CA) CA9 and CA12 genes are overexpressed because of the absence of pVHL. We hypothesized that these enzymes might be involved in maintaining the extracellular acidic pH in tumors, thereby providing a conducive environment for tumor growth and spread. Using Northern blot analysis and immunostaining with specific antibodies we analyzed the expression of CA9 and CA12 genes and their products in a large sample of cancer cell lines, fresh and archival tumor specimens, and normal human tissues. Expression was also analyzed in cultured cells under hypoxic conditions. Expression of CA IX and CA XII in normal adult tissues was detected only in highly specialized cells and for most tissues their expression did not overlap. Analysis of RNA samples isolated from 87 cancer cell lines and 18 tumors revealed high-to-moderate levels of expression of CA9 and CA12 in multiple cancers. Immunohistochemistry revealed high-to-moderate expression of these enzymes in various normal tissues and multiple common epithelial tumor types. The immunostaining was seen predominantly on the cell surface membrane. The expression of both genes was markedly induced under hypoxic conditions in tumors and cultured tumor cells. We conclude that the cell surface trans-membrane carbonic anhydrases CA IX and CA XII are overexpressed in many tumors suggesting that this is a common feature of cancer cells that may be required for tumor progression. These enzymes may contribute to the tumor microenvironment by maintaining extracellular acidic pH and helping cancer cells grow and metastasize. Our studies show an important causal link between hypoxia, extracellular acidification, and induction or enhanced expression of these enzymes in human tumors.

Carbonic anhydrase isozymes in the human pancreas

Recently, an increasing number of carbonic anhydrase (CA) isozymes have been discovered in the human pancreas. These isozymes are classified as the CA family with various molecular structures and different subcellular localizations: cytoplasmic CA II, mitochondrial CA VB, secretory CA VI, membrane-bound CA IV, and transmembrane CA IX and XII. However, there is little evidence concerning their pathophysiological roles. Here, we reviewed the expression of CA isozymes in the human pancreas and proposed hypotheses related to their physiological and pathological roles.