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.     

Assessment of the antiproliferative and apoptotic roles of sulfonamide carbonic anhydrase IX inhibitors in HeLa cancer cell line

Carbonic anhydrase IX (CA IX) has recently been validated as an antitumor/antimetastatic drug target. In this study, we examined the underlying molecular mechanisms and the anticancer activity of sulfonamide CA IX inhibitors against cervical cancer cell lines. The effects of several sulfonamides on HeLa, MDA-MB-231, HT-29 cancer cell lines, and normal cell lines (HEK-293, PNT-1A) viability were determined. The compounds showed high cytotoxic and apoptotic activities, mainly against HeLa cells overexpressing CA IX. We were also examined for intracellular reactive oxygen species (ROS) production; intra-/extracellular pH changes, for inhibition of cell proliferation, cellular mitochondrial membrane potential change and for the detection of caspase 3, 8, 9, and CA IX protein levels. Of the investigated sulfonamides, one compound was found to possess high cytotoxic and anti-proliferative effects in HeLa cells. The cytotoxic effect occurred via apoptosis, being accompanied by a return of pHe/pHi towards normal values as for other CA IX inhibitors investigated earlier.     

Hypoxia activates the capacity of tumor-associated carbonic anhydrase IX to acidify extracellular pH

Acidic extracellular pH (pHe) is a typical attribute of a tumor microenvironment, which has an impact on cancer development and treatment outcome. It was believed to result from an accumulation of lactic acid excessively produced by glycolysis. However, metabolic profiles of glycolysis-impaired tumors have revealed that CO2 is a significant source of acidity, thereby indicating a contribution of carbonic anhydrase (CA). The tumor-associated CA IX isoform is the best candidate, because its extracellular enzyme domain is highly active, expression is induced by hypoxia and correlates with poor prognosis. This study provides the first evidence for the role of CA IX in the control of pHe. We show that CA IX can acidify the pH of the culture medium in hypoxia but not in normoxia. This acidification can be perturbed by deletion of the enzyme active site and inhibited by CA IX-selective sulfonamides, which bind only to hypoxic cells containing CA IX. Our findings suggest that hypoxia regulates both expression and activity of CA IX in order to enhance the extracellular acidification, which may have important implications for tumor progression.     

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: 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-tetrafluorophenylsulfonyl- 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.     

Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes

Carbonic anhydrase IX (CA IX) is an exceptional member of the CA protein family; in addition to its classical role in pH regulation, it has also been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes. To characterize the biochemical properties of this membrane protein, two soluble recombinant forms were produced using the baculovirus-insect cell expression system. The recombinant proteins consisted of either the CA IX catalytic domain only (CA form) or the extracellular domain, which included both the proteoglycan and catalytic domains (PG + CA form). The produced proteins lacked the small transmembrane and intracytoplasmic regions of CA IX. Stopped-flow spectrophotometry experiments on both proteins demonstrated that in the excess of certain metal ions the PG + CA form exhibited the highest catalytic activity ever measured for any CA isozyme. Investigations on the oligomerization and stability of the enzymes revealed that both recombinant proteins form dimers that are stabilized by intermolecular disulfide bond(s). Mass spectrometry experiments showed that CA IX contains an intramolecular disulfide bridge (Cys(119)-Cys(299)) and a unique N-linked glycosylation site (Asn(309)) that bears high mannose-type glycan structures. Parallel experiments on a recombinant protein obtained by a mammalian cell expression system demonstrated the occurrence of an additional O-linked glycosylation site (Thr(78)) and characterized the nature of the oligosaccharide structures. This study provides novel information on the biochemical properties of CA IX and may help characterize the various cellular and pathophysiological processes in which this unique enzyme is involved.     

Carbonic anhydrase inhibitors: transepithelial transport of thioureido sulfonamide inhibitors of the cancer-associated isozyme IX is dependent on efflux transporters

Sulfonamides and their derivatives inhibit the catalytic activity of carbonic anhydrases (CA, EC 4.2.1.1). Isozyme IX (CA IX) is a transmembrane isoform with the active site oriented toward the extracellular space. CA IX was recently shown to be a drug target, and it is highly overexpressed in hypoxic tumors with limited distribution in normal tissues. The present report deals with the drug design, synthesis, and biological investigation of a group of thioureido sulfonamides, which have been obtained by reaction of isothiocyanate-substituted aromatic sulfonamides with amines. These compounds have potent inhibitory properties against CA IX with K(I) values in the range of 10-37 nM and P(app)values > 0.34 x 10(-6) cm/s for the absorptive transepithelial transport in Caco-2 cells. In Caco-2 cells, one of these compounds (A6) was shown to be a substrate for efflux transporters such as P-glycoprotein (P-gp). P-gp activity is not likely to be rate-limiting for intestinal absorption, but might be useful when targeting hypoxic tumors expressing both P-gp and CA IX.     

Synthesis of an acridine orange sulfonamide derivative with potent carbonic anhydrase IX inhibitory action

Acridine orange (AO) a fluorescent cationic dye used for the management of human musculoskeletal sarcomas, due to its strong tumoricidal action and accumulation in the acidic environment typical of hypoxic tumors, was used for the preparation of a primary sulfonamide derivative. The rationale behind the drug design is the fact that hypoxic, acidic tumors overexpress carbonic anhydrase (CA, EC 4.2.1.1) isoforms, such as CA IX, which is involved in pH regulation, proliferation, cell migration and invasion, and this enzyme is strongly inhibited by primary sulfonamides. The AO-sulfonamide derivative was indeed a potent, low nanomolar CA IX inhibitor whereas its inhibition of the cytosolic isoforms CA I and II was in the micromolar range. A second transmembrane, tumor-associated isoform, CA XII, was also effectively inhibited by the AO-sulfonamide derivative, making this compound an interesting theranostic agent for the management of hypoxic tumors.     

The proteoglycan region of the tumor-associated carbonic anhydrase isoform IX acts as anintrinsic buffer optimizing CO2 hydration at acidic pH values characteristic of solid tumors

The enzymatic activities of carbonic anhydrase (CA, EC 4.2.1.1) isozymes CA I, II, IX (catalytic domain (cdCA IX) and catalytic domain plus proteoglycan, flCA IX), XII and XIV were investigated as a function of pH for the CO₂ hydration to bicarbonate and a proton. The cytosolic isoforms CA I and II as well as the catalytic domain of CA IX, together with the transmembrane isoforms CA XII and XIV showed sigmoid pH dependencies of k_cat/K_M, with a pK_a of 6.90–7.10, showing thus optimal catalytic efficiency around pH 7. The full length CA IX had a similar shape of the pH dependency curve but with a pK_a of 6.49, having thus maximal catalytic activity at pH values around 6.5, typical of hypoxic solid tumors in which CA IX is overexpressed. The proteoglycan domain of CA IX (present only in this transmembrane isoform) may thus act as an intrinsic buffer promoting efficient CO₂ hydration at acidic pH values found in hypoxic tumors.     

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.     

Carbonic anhydrase IX

Cell migration can be principally viewed as a chain of well-orchestrated morphological events that lead to dynamic reshaping of the cell body. However, behind the scene of such a “morphological theater” there are very complex, interrelated molecular and physiological processes that drive the cell movement. Among them, ion transport and pH regulation play a key role, with carbonic anhydrase IX (CA IX) emerging as one of the important “molecular actors.” CA IX is a highly active cell surface enzyme expressed in a broad range of solid tumors in response to hypoxia and explored as a clinically useful biomarker of hypoxia and as a therapeutic target. Its biological role is to protect tumor cells from hypoxia and acidosis in the tumor microenvironment. The study published recently by our group showed that CA IX actively contributes to cell migration and invasion. For the first time, we demonstrated CA IX accumulation in lamellipodia of migrating cells and its direct in situ interaction with bicarbonate transporters. Our findings indicate that tumor cells need CA IX not only as a pro-survival factor in hypoxia and acidosis, but also as a pro-migratory component of the cellular apparatus driving epithelial-mesenchymal transition.     

In Vivo Imaging and Quantification of Carbonic Anhydrase IX Expression as an Endogenous Biomarker of Tumor Hypoxia

Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%–70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6–24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.     

Redox State and Carbonic Anhydrase Isozyme IX Expression in Human Renal Cell Carcinoma: Biochemical and Morphological Investigations

Clear renal cell carcinomas (RCC) frequently express carbonic anydrase IX (CA IX) because of non-functional mutation of von Hippel Lindau (VHL) tumor suppressor gene. CA IX is a tumor-associated transmembrane antigen, which catalyzes the extracellular, reversible hydration of carbon dioxide to bicarbonate and proton and thereby contributes to acidification of extracellular milieu. Extracellular acidic pH facilitates tumor growth and progression. CA IX expression is upregulated by Hypoxia Inducible Factor-1 (HIF-1), which is negatively controlled by oxygen via wild type VHL protein and is also regulated by the cell redox state. We investigated the immunohistochemical pattern of distribution of CA IX in a small series (14 cases) of RCCs. CA IX expression was matched with the redox state of RCC, stratifying our series in relation to clinical and histopathological parameters, such as Fuhrman grade, staging, proliferation markers expression, and particularly, the presence of necrosis. Our results show for the first time the existence of a perivascular pattern of CA IX distribution in RCC. We also found a significant relationship between CA IX expression and the presence of necrosis. Tumors with higher CA IX expression exhibited higher degree of necrosis [Formula: See Text] Notably, an almost significant relationship between the redox state and CA IX expression was detected in RCC patients with 5 years disease-free survival, most of them showing organ-confined disease. Tumors with lower redox state showed an algebraically higher degree of CA IX expression. On the contrary, tumors with higher redox state exhibited an algebraically lower CA IX expression [Formula: See Text] The observed relationship of CA IX expression and necrosis suggests a role for CA IX in RCC. Further investigations are necessary to further establish the role of the redox state in regulation of CA IX expression in RCC.     

Redox state and carbonic anhydrase isozyme IX expression in human renal cell carcinoma: biochemical and morphological investigations

Clear renal cell carcinomas (RCC) frequently express carbonic anydrase IX (CA IX) because of non-functional mutation of von Hippel Lindau (VHL) tumor suppressor gene. CA IX is a tumor-associated transmembrane antigen, which catalyzes the extracellular, reversible hydration of carbon dioxide to bicarbonate and proton and thereby contributes to acidification of extracellular milieu. Extracellular acidic pH facilitates tumor growth and progression. CA IX expression is upregulated by Hypoxia Inducible Factor-1 (HIF-1), which is negatively controlled by oxygen via wild type VHL protein and is also regulated by the cell redox state. We investigated the immunohistochemical pattern of distribution of CA IX in a small series (14 cases) of RCCs. CA IX expression was matched with the redox state of RCC, stratifying our series in relation to clinical and histopathological parameters, such as Fuhrman grade, staging, proliferation markers expression, and particularly, the presence of necrosis. Our results show for the first time the existence of a perivascular pattern of CA IX distribution in RCC. We also found a significant relationship between CA IX expression and the presence of necrosis. Tumors with higher CA IX expression exhibited higher degree of necrosis (p < 0.05). Notably, an almost significant relationship between the redox state and CA IX expression was detected in RCC patients with 5 years disease-free survival, most of them showing organ-confined disease. Tumors with lower redox state showed an algebraically higher degree of CA IX expression. On the contrary, tumors with higher redox state exhibited an algebraically lower CA IX expression (p = 0.057). The observed relationship of CA IX expression and necrosis suggests a role for CA IX in RCC. Further investigations are necessary to further establish the role of the redox state in regulation of CA IX expression in RCC.     

The structure of the regulatory domain of the adenylyl cyclase Rv1264 from Mycobacterium tuberculosis with bound oleic acid

The universal secondary messenger cAMP is produced by adenylyl cyclases (ACs). Most bacterial and all eukaryotic ACs belong to class III of six divergent classes. A class III characteristic is formation of the catalytic pocket at a dimer interface and the presence of additional regulatory domains. Mycobacterium tuberculosis possesses 15 class III ACs, including Rv1264, which is activated at acidic pH due to pH-dependent structural transitions of the Rv1264 dimer. It has been shown by X-ray crystallography that the N-terminal regulatory and C-terminal catalytic domains of Rv1264 interact in completely different ways in the active and inhibited states. Here, we report an in-depth structural and functional analysis of the regulatory domain of Rv1264. The 1.6 A resolution crystal structure shows the protein in a tight, disk-shaped dimer, formed around a helical bundle, and involving a protein chain crossover. To understand pH regulation, we determined structures at acidic and basic pH values and employed structure-based mutagenesis in the holoenzyme to elucidate regulation using an AC activity assay. It has been shown that regulatory and catalytic domains must be linked in a single protein chain. The new studies demonstrate that the length of the linker segment is decisive for regulation. Several amino acids on the surface of the regulatory domain, when exchanged, altered the pH-dependence of AC activity. However, these residues are not conserved amongst a number of related ACs. The closely related mycobacterial Rv2212, but not Rv1264, is strongly activated by the addition of fatty acids. The structure resolved the presence of a deeply embedded fatty acid, characterised as oleic acid by mass spectrometry, which may serve as a hinge. From these data, we conclude that the regulatory domain is a structural scaffold used for distinct regulatory purposes.     

Glycosidic carbonic anhydrase IX inhibitors: A sweet approach against cancer

Targeting tumour associated carbonic anhydrase (CA, EC 4.2.1.1) isoforms IX and XII is now considered as a pertinent approach for the development of new cancer therapeutics against hypoxic tumours. In the last period, with the help of X-ray crystallography, much progress has been achieved for the drug-design of selective CA IX inhibitors, by considering the three main structural elements that govern both potency and selectivity, that is, a zinc binding group (ZBG), an organic scaffold, and one or more side chains substituting the scaffold. The use of sugar moiety in the structure of sulfonamide-based CA inhibitors (CAIs), has allowed the discovery of very potent CA IX inhibitors able to impair the growth of both primary tumors and metastases. The search for specific CA IX inhibitors by using the sugar approach has become an important research field, leading to sulfonamides, sulfamates, sulfamides and coumarins with excellent in vitro activity and relevant potency in vivo, in animal models of cancer. This paper will review the latest development in this hot topic.     

The effects of CA IX catalysis products within tumor microenvironment

Solid tumors are composed of both cancer cells and various types of accessory cells, mainly fibroblasts, that collectively compose the so called tumor-microenvironment. Cancer-associated fibroblasts have been described to actively participate in cancer progression by establishing a cytokine-mediated as well as metabolic crosstalk with cancer cells. In the present paper we show that activated human fibroblasts are able to boost tumor cells proliferation and that this effect is greatly dependent on stromal carbonic anhydrase IX (CA IX) activity. In fact fibroblasts show a strong upregulation of CA IX expression upon activation by cancer cells, while CA IX products, protons and bicarbonate, exert differential effects on cancer cells proliferation. While acidification of extracellular pH, a typical condition of rapidly growing solid tumors, is detrimental for tumor cells proliferation, bicarbonate, through its organication, supplies cancer cells with intermediates useful to sustain their high proliferation rate. Here we propose a new kind of fibroblasts/tumor cells crosstalk within tumor microenvironment, mediated by stromal CA IX products, aimed to favor cancer cells growth, opening new perspectives on CA IX role in tumor microenvironment.     

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 anhydrase inhibitors: copper(II) complexes of polyamino-polycarboxylamido aromatic/heterocyclic sulfonamides are very potent inhibitors of the tumor-associated isoforms IX and XII

Reaction of EDTA/DTPA dianhydride with aromatic/heterocyclic sulfonamides afforded a series of derivatives incorporating polyaminopolycarboxylate tails and benzenesulfonamide or 1,3,4-thiadiazole-2-sulfonamide heads. These compounds have been used as ligands to prepare Cu(II) complexes. Both parent sulfonamides as well as their copper complexes behaved as potent inhibitors of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, and transmembrane CA IX and XII. Some Cu(II) complexes showed subnanomolar affinities and some selectivity for the inhibition of the tumor-associated isoforms IX and XII and might be used as PET hypoxia markers of tumors.