Biochemical Characterization of CA IX, One of the Most Active Carbonic Anhydrase Isozymes

Hilvo, Mika; Baranauskienė, Lina; Salzano, Anna Maria; Scaloni, Andrea; Matulis, Daumantas; Innocenti, Alessio; Scozzafava, Andrea; Monti, Simona Maria; Fiore, Anna Di; Simone, Giuseppina De; Lindfors, M.; Jänis, Janne; Valjakka, Jarkko; Pastoreková, Silvia; Pastorek, Jaromı́r; Kulomaa, Markku S.; Nordlund, Henri R.; Supuran, Claudiu T.; Parkkila, Seppo

doi:10.1074/jbc.m800938200

Cited by 271 publications

(332 citation statements)

References 46 publications

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“…The potent therapeutic activity observed with compounds 4a and 5a suggests that these products may provide a benefit to patients with kidney cancer, as the majority of clear cell renal cell carcinomas are strongly positive for this target [32][33][34]. The biodistribution profiles of acetazolamide derivatives compares favourably with the ones previously reported for other ligands [23][24][25][26].…”

Section: Discussionsupporting

confidence: 69%

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Acetazolamide Serves as Selective Delivery Vehicle for Dipeptide-Linked Drugs to Renal Cell Carcinoma

Cazzamalli

Corso

Neri

2016

Molecular Cancer Therapeutics

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In most cases, cytotoxic drugs do not preferentially accumulate at the tumor site, causing unwanted toxicities and preventing dose escalation to therapeutically active regimens. Here, we show that acetazolamide derivatives, which bind to carbonic anhydrase IX (CAIX) on the surface of kidney cancer cells, selectively deliver payloads at the site of disease, sparing normal organs. Biodistribution studies, performed in tumor-bearing mice with acetazolamide derivatives bearing a technetium-99m chelator complex or a red fluorophore as payload, revealed a preferential tumor accumulation of the compound at doses up to 560 nmol/Kg. The percentage of injected dose per gram in the tumor was dose-dependent and revealed optimal tumor:organ ratios at 140 nmol/Kg, with a tumor:blood ratio of 80:1 at 6 h. Acetazolamide, coupled to potent cytotoxic drugs via a dipeptide linker, exhibited a potent antitumor activity in nude mice bearing SKRC-52 renal cell carcinomas, while drug derivatives devoid of the acetazolamide moiety did not exhibit any detectable anticancer activity at the same doses. The observation of tumor regression with a noninternalizing ligand and with different cytotoxic moieties (MMAE and PNU-159682) indicates a general mechanism of action, based on the selective accumulation of the product on tumor cells, followed by the extracellular proteolytic release of the cytotoxic payload at the neoplastic site and the subsequent drug internalization into tumor cells. Acetazolamide-based drug conjugates may represent a promising class of targeted agents for the treatment of metastatic kidney cancer, as the majority of human clear cell renal cell carcinomas are strongly positive for CAIX.

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Section: Discussionsupporting

confidence: 69%

“…CAIX is a membrane protein, expressed in the majority of clear cell renal cell carcinomas [32][33][34]. CAIX is virtually undetectable in most normal adult tissues, except for certain structures in the gastrointestinal tract (stomach, duodenum, gallbladder), which are strongly positive for the antigen.…”

Section: Introductionmentioning

confidence: 99%

Acetazolamide Serves as Selective Delivery Vehicle for Dipeptide-Linked Drugs to Renal Cell Carcinoma

Cazzamalli

Corso

Neri

2016

Molecular Cancer Therapeutics

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“…CA9 is expressed in the stomach and peritoneal lining, but it is ectopically induced and overexpressed in several types of solid tumours, predominantly owing to strong transcriptional activation of hypoxia-inducible transcription factor 1 (HIF1) 12,[15][16][17]28,29 . Interestingly, CA9 is the most strongly overexpressed gene in response to hypoxia in human cancer cells 28,30 , and it is also the most active isoform of carbonic anhydrase for the car bon dioxide hydration reaction 29,31 .…”

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confidence: 99%

“…Interestingly, CA9 is the most strongly overexpressed gene in response to hypoxia in human cancer cells 28,30 , and it is also the most active isoform of carbonic anhydrase for the car bon dioxide hydration reaction 29,31 . The X-ray crystal structure of CA9 reveals that it is a dimeric enzyme 32 , unlike all other carbonic anhydrase isoforms known so far, which are monomeric.…”

mentioning

confidence: 99%

Interfering with pH regulation in tumours as a therapeutic strategy

Neri¹,

Supuran

2011

Nat Rev Drug Discov

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1,375

1,269

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The growth of solid tumours is characterized not only by the uncontrolled proliferation of cancer cells but also by changes in the tumour environment that support the growth of the neoplastic mass and the metastatic spread of cancer cells to distant sites 1 . The formation of new blood vessels from pre-existing ones (angiogenesis) provides oxygen and nutrients to the tumour, which are essential for tumour growth 2 . A complex dynamic interplay exists between the expanding neoplastic mass and the tumour environment, which is affected by the metabolic requirements of cancer cells and by their products, such as secreted proteins (for example, extracellular matrix components and proteases) and metabolites.Upregulated glucose metabolism is a hallmark of invasive cancers 3 . In normal cells glucose is converted to glucose-6-phosphate and subsequently to pyruvate, which is then oxidized in the mitochondria to carbon dioxide and water; this releases 38 moles of ATP per mole of glucose 3 . However, inadequate oxygen delivery to some regions of tumours leads to hypoxia, which restricts oxidative phosphorylation. As a consequence, hypoxic tumour cells shift their metabolism towards glycolysis so that the pyruvate generated in the first step of the process is reduced to lactate, generating only 2 moles of ATP per mole of glucose. This is a less energy-efficient process but it does not depend on oxygen. Furthermore, glycolysis often persists even after reoxygenation because the obtained metabolic intermediates (that is, lactate and pyruvate) can be used for the biosynthesis of amino acids, nucleotides and lipids, thus providing a selective advantage to proliferating tumour cells. This explains Warburg's observation of high glucose consumption and high lactate production in tumour tissues 3-5 (known as the Warburg effect).Oncogenic metabolism also generates an excess of protons and carbon dioxide, which are kept in equilibrium with carbonic acid by the enzyme carbonic anhydrase 3-9 . Thus, increased glucose metabolism in tumour cells leads to enhanced acidification of the extracellular milieu, which is frequently accompanied by various levels of hypoxia. This phenotype confers a substantial Darwinian growth advantage to tumour cells over normal cells, which undergo apoptosis in response to such an acidic extracellular environment 3 .Tumour cells have evolved various mechanisms to cope with the acidic and hypoxic stress mentioned above. They eliminate acidic catabolites by ion transporters and pumps to preserve a slightly alkaline intracellular pH (pH i ), which is optimal for cell proliferation and tumour survival 3-10 . Acid export leads to a reduction in the extracellular pH (pH e ) to values as low as 6.0 (the usual pH e in tumours is in the range of 6.5-7.0) 3 , which is a salient feature of the tumour microenvironment 3-9 . As well as triggering the overexpression of many proteins involved in glucose metabolism -such as the glucose transporter GLUT1 (also known as SLC2A1) and pH-regulating proteins such as carbonic anhyd...

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“…The peak of the d 111 reflection has been deconvoluted to the Lorentzian curve for determining the full width at half-maximum (FMWH) value. The crystalline domain has been calculated from the Debye-Scherrer formula using the FWMH value of the corresponding index peaks giving an average † Ecole Nationale Supérieure 12 Inhibition data of 3a,b, GNP-1/2, the standard, clinically used CAI acetazolamide AZA (5-acetamido-1,3,4-thiadiazole-2-sulfonamide), and Au NPs (Au@) as control, against isoforms hCA I and II (cytosolic isozymes) and the transmembrane, tumor-associated isozyme hCA IX (a construct incorporating the catalytic domain and proteoglycan regions of the enzyme), 13 are shown in Table 1. 14 A stopped-flow method has been used for assaying the CA-catalyzed CO 2 hydration activity with Phenol red as indicator, working at the absorbance maximum 557 nm, following the initial rates of the CA-catalyzed CO 2 hydration reaction for 10-100 s. For each inhibitor at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity.…”

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confidence: 99%

Carbonic Anhydrase Inhibitor Coated Gold Nanoparticles Selectively Inhibit the Tumor-Associated Isoform IX over the Cytosolic Isozymes I and II

Stiti

Cecchi²,

Rami³

et al. 2008

J. Am. Chem. Soc.

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102

106

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The carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes with five independently evolved (R, ,γ,δ,ς) classes reported up to date. [1][2][3] These enzymes catalyze the reversible hydration of carbon dioxide to bicarbonate and protons by means of a metalhydroxide (Lig 3 M 2+ (OH) -) mechanism. 2 In addition to the established role of the carbonic anhydrase inhibitors (CAIs) as diuretics and antiglaucoma drugs, it has recently emerged that CAIs could have potential as novel antiobesity, anticancer, and anti-infective drugs. 1,2 A critical problem in the design of CAIs is related to the high number of isoforms in mammals (15), their diffuse localization in many tissues/organs, and the lack of isozyme selectivity of the presently available inhibitors. [1][2][3][4] Recently, nanoparticles received great attention for their biomedical applications both for the site-specific delivery of drugs 7 or for imaging purposes. 7,8 Several recent such examples include HIV inhibition with a CCR5 antagonist attached to multivalent gold nanoparticles (Au NPs), tumor targeting with NPs loaded with hydroxycamptothecin, paclitaxel, or fumagillin, and magnetic resonance imaging (MRI) techniques of tumors based on integrins targeting with Fe 3 O 4 NPs, computer tomography (CT) or MRI imaging with gadolinium chelate Au NPs, etc. 7,8 Human CA IX (hCA IX) is an extracellular, transmembrane isoform which was recently shown to constitute a novel and interesting target for the anticancer therapy due to its overexpression in many cancer tissues and not in their normal counterparts. 1b,9 Its expression is strongly induced by hypoxia present in many tumor tissues and correlated with a bad response to classical chemo-and radiotherapies. 1b,9 CA IX was shown to acidify the extratumoral medium leading both to the acquisition of metastasic phenotypes and to chemoresistance with many anticancer drugs, these processes being reverted by inhibition of the enzyme catalytic activity with sulfonamide inhibitors. 1b,9 The development of selective CA IX inhibitors might provide useful tools for highlighting the exact role of CA IX in hypoxic cancers, to control the pH imbalance of tumor cells and lead to novel diagnostic or therapeutic applications for the management of such tumors. 1b,9 Although many sulfonamide/sulfamate/sulfamide potent CA IX inhibitors were reported, few of them show an acceptable level of selectivity for inhibiting the transmembrane-tumor associated target isoform IX over the cytosolic, ubiquitous isozymes hCA I and II. 1b,9 Considering the extracellular localization of the target CA isoform and the fact that nanomaterials generally show membrane impermeability, 10 we report here the synthesis of CAI coated Au NPs which show excellent CA IX inhibitory properties and selectivity for the inhibition of the tumor-associated isoform over hCA I and II.The key intermediate 3a was synthesized by coupling lipoic acid 1 with 4-aminoethylbenzene sulfonamide 2a in the presence of EDCI/ DMAP, as outlined in Scheme 1. The CAI coated...

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Biochemical Characterization of CA IX, One of the Most Active Carbonic Anhydrase Isozymes

Cited by 271 publications

References 46 publications

Acetazolamide Serves as Selective Delivery Vehicle for Dipeptide-Linked Drugs to Renal Cell Carcinoma

Acetazolamide Serves as Selective Delivery Vehicle for Dipeptide-Linked Drugs to Renal Cell Carcinoma

Interfering with pH regulation in tumours as a therapeutic strategy

Carbonic Anhydrase Inhibitor Coated Gold Nanoparticles Selectively Inhibit the Tumor-Associated Isoform IX over the Cytosolic Isozymes I and II

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