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

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Abstract

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 (KI-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.

Introduction

Among the zinc enzymes extensively studied in the last period, the carbonic anhydrases (CAs, EC 4.2.1.1) occupy a special place for several reasons: (i) these enzymes are ubiquitous in all kingdoms, starting with Archaea, Bacteria, algae and green plants, and ending with superior animals, including vertebrates;1, 2, 3, 4 (ii) their physiological function is essential for these organisms, as CAs catalyze a fundamental physiological reaction, the interconversion between carbon dioxide and bicarbonate.1, 2, 3, 4 This reaction is critical for respiration and transport of CO2 between metabolizing tissues and excretion sites, secretion of electrolytes in a variety of tissues and organs, pH regulation and homeostasis, CO2 fixation (for algae and green plants), several metabolic biosynthetic pathways (in vertebrates), and so on;1, 2, 3, 4 (iii) inhibition (but also activation) of these enzymes may be exploited clinically in the treatment or prevention of a variety of disorders.1, 2, 3 In consequence, CA inhibitors (CAIs) and to a less extent up to now, CA activators possess a variety of applications in therapy.1, 2 Four such pharmacological agents, acetazolamide AAZ, methazolamide MZA, ethoxzolamide EZA, and dichlorophenamide DCP, have been used for more than 40 years as systemic CAIs, whereas two additional drugs dorzolamide DZA (clinically launched in 1995) and the structurally-related brinzolamide BRZ (used since 1999) are topically acting antiglaucoma CAIs.1, 2

CAs are encoded by three distinct, evolutionarily unrelated gene families: the α-CAs (present in vertebrates, Bacteria, algae and cytoplasm of green plants), the β-CAs (predominantly in Bacteria, algae and chloroplasts of both mono- as well as dicotyledons) and the γ-CAs (mainly in Archaea and some Bacteria), respectively.1, 2, 5 In higher vertebrates, including humans, 14 different CA isozymes or CA-related proteins (CARP) were described, with very different subcellular localization and tissue distribution.1, 2, 5 Basically, there are several cytosolic forms (CA I–III, CA VII), four membrane-bound isozymes (CA IV, CA IX, CA XII and CA XIV), one mitochondrial form (CA V) as well as a secreted CA isozyme, CA VI.1, 2, 5 Not much is known about the cellular localization of the other isozymes.

Some of the isozymes mentioned above, such as CA IX and CA XII, are overexpressed in cancer cells.6 The first tumor-associated CA isozyme discovered was CA IX, a transmembrane protein with a suggested function both in maintaining acid-base balance and in intercellular communication. It consists of an N-terminal proteoglycan-like domain that is unique among the CAs, a highly active CA catalytic domain, a single transmembrane region and a short intracytoplasmic tail.7 CA IX is particularly interesting for its ectopic expression in a multitude of carcinomas derived from cervix uteri, kidney, lung, esophagus, breast, colon and so on, contrasting with its restricted expression in normal tissues, namely in the epithelia of the gastrointestinal tract.7, 8, 9, 10, 11, 12, 13, 14

It has recently been demonstrated that such tumor-associated CAs (mainly CA IX) may be of considerable value as markers of tumor progression. This is mostly due to their induction by hypoxia, a clinically important factor of tumor biology that significantly affects treatment outcome and disease progression.9 Strong association between CA IX expression and intratumoral hypoxia (either measured by microelectrodes, or detected by incorporation of a hypoxic marker pimonidazole, or by evaluation of extent of necrosis) has been demonstrated in the cervical, breast, head and neck, bladder and non-small cell lung carcinomas (NSCLC).10, 11, 12, 13 Moreover, in NSCLC and breast carcinomas, correlation between CA IX and a constellation of proteins involved in angiogenesis, apoptosis inhibition and cell–cell adhesion disruption has been observed, possibly contributing to strong relationship of this enzyme to a poor clinical outcome.13 Hypoxia is linked with acidification of extracellular milieu that facilitates tumor invasion and CA IX is believed to play a role in this process via its catalytic activity.14 Thus, inhibition of this enzyme may constitute a novel approach to the treatment of cancers in which CA IX is expressed.

In fact, acetazolamide, one of the best-studied, classical CAI used clinically, was shown to function as a modulator in anticancer therapies, in combination with different cytotoxic agents (such as alkylating agents; nucleoside analogues; platinum derivatives, etc.), to suppress tumor metastasis and to reduce the invasive capacity of several renal carcinoma cell lines (Caki-1, Caki-2, ACHN, and A-498).15, 16 Such valuable studies constituted a proof-of-concept demonstration that CAIs may be used in the management of tumors that overexpress one or more CA isozymes. It should also be mentioned that our group reported the design and in vitro antitumor activity of several classes of sulfonamide CAIs, shown to act as nanomolar inhibitors against the classical isozymes known to possess critical physiological roles, such as CA I, CA II and CA IV. These compounds were also shown to exert potent inhibition of cell growth in several leukemia, non-small cell lung, ovarian, melanoma, colon, CNS, renal, prostate and breast cancer cell lines, with GI50 values of 10–75 nM in some cases.17, 18, 19, 20 On the other hand, no data regarding inhibition of tumor-associated CA IX with different types of sulfonamides are available up to now, although inhibition of this particular isozyme might be clinically exploited for designing novel anticancer therapies. In this paper we report the first CA IX inhibition study with a series of aromatic and heterocyclic sulfonamides, as well as with the six clinically used CAIs mentioned above.

Section snippets

Chemistry

Sulfonamides investigated for the inhibition of the tumor-associated isozyme CA IX, of types 1, fx2

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are shown below. Compounds 16, 1112, 20 and 26 are commercially available, whereas 710,21 131922 and 212523 were prepared as reported earlier. The six clinically used compounds were also assayed, since no such data are available in the literature.

CA Inhibition Data

Inhibition data against four CA isozymes, CA I, II, IV and IX,24, 25, 26 with the above mentioned compounds fx2, fx2 and the six clinically used inhibitors, are shown in Table 1.

These data are rather surprizing, since the inhibition profile of isozyme CA IX is very different from that of the classical isozymes CA I and II (cytosolic) as well as CA IV (membrane-bound). The following particular features may be noted: (i) all the 32 sulfonamides investigated here act as CA IX inhibitors, with

Conclusion

We report here the first inhibition study of the tumor-associated, transmembrane isozyme CA IX with a series of aromatic and heterocyclic sulfonamides, including also 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) are also provided for comparison. Very interesting inhibition profile against CA IX with

Acknowledgements

This research was financed by a grant from the Italian CNR—target project Biotechnology. JP and SP are recipients of the grants from Bayer Corporation and from the Slovak Grant Agency.

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