Ординатура / Офтальмология / Английские материалы / Carbonic Anhydrase Its Inhibitors and Activators_Supuran, Scozzafava, Conway_2004

CA IX

CA XII

FIGURE 9.3 Amino acid alignment and domain composition of CA IX and CA XII. Signal peptides (SP), proteoglycan-related region (PG), carbonic anhydrase domains (CA), transmembrane regions (TM) and intracellular tails (IC) are designated by different background colors. Acidic tripeptides present in the PG region of CA IX are enclosed in medium gray rectangles and cysteine residues are encircled. Exon borders are indicated by arrowheads. Schemes of the proteins below the alignment illustrate the domain composition of their monomeric forms.

active-site clefts facing the extracellular space (Whittington et al. 2001). CA domains associate to form an isologous dimer that is presumably further stabilized by two putative amino acid signature GXXXG and GXXXS motifs in the transmembrane portion of the protein. CA XII contains two extracellular glycosylation sites (N52 and N136), and a single disulfide linkage is present between C23 and C203 [numbered

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according to Whittington et al. (2001)]. An additional cysteine residue is localized close to the transmembrane region. The intracellular domain contains potential phosphorylation sites with a possible role in regulating structure, enzyme activity and signal transduction.

CA IX differs from CA XII in that it forms trimers linked by disulfidic bonds, as revealed by mobility in nonreducing PAGE (Pastoreková et al. 1992). Putative transmembrane dimerization motifs of CA XII are absent from the CA IX sequence. There is only one predicted extracellular glycosylation site (N346), but the CA domain contains four cysteine residues and at least some of them appear to be required for intermolecular oligomerization. The cytoplasmic tail of CA IX shows high homology to that of CA XII and also contains putative phosphorylation sites. Deletion of this region results in loss of proper transport and plasma membrane localization of CA IX despite the presence of the transmembrane anchor (Svastova et al. submitted), suggesting involvement of this region in protein–protein interactions at the cytoplasmic side of the plasma membrane.

However, the major difference between the two cancer-related CA isozymes is in the presence of a unique PG-like region located at the N-terminus of CA IX but not of CA XII (Figure 9.3). This domain contains an imperfect repetitive sequence of six amino acids and has been implicated in cell adhesion. Zavada et al. (2000) have shown that it is required for CA IX-mediated cell adhesion to a solid support, suggesting its possible role in cell–matrix interactions. This adhesion was abrogated by the monoclonal antibody M75, which binds to the epitope within the repetitive region and by the peptides derived from this region. The PG domain also appears to be important for the capacity of hCA IX to reduce cell–cell adhesion of MDCK cells. Svastova and coworkers (2003) have recently shown that in these cells, CA IX colocalizes with a key cell–cell adhesion molecule E-cadherin, responds by internalization to same external stimuli (i.e., calcium depletion and hypoxia) as E- cadherin and weakens intercellular adhesion by destabilization of E-cadherin links to the cytoskeleton via a mechanism that involves direct binding of CA IX to β-catenin. The effect of CA IX is pronounced in hypoxia, wherein it increases dissociation of MDCK cells. This observation conforms to a recent proposal that hypoxia can initiate tumor invasion via decreased E-cadherin-mediated cell–cell adhesion and offers a possibility that CA IX is functionally implicated in this process (Beavon 1999).

Involvement of CA IX in cell adhesion represents a novel aspect of CA function, the concept related RPTPβ, expressed in the form of a proteoglycan, regulates

cellular processes linked to cell adhesion and differentiation. However, this function of RPTPβ is executed by the inactive CA domain via binding of the neuronal cell

recognition molecule contactin. It cannot be excluded that the CA domain of CA IX is used in a manner analogous to that for RPTPβ and that an interplay between

PG and CA domains is needed for its full biological activity.

Cooperation between PG and CA domains might be relevant also for the enzyme activity of CA IX. PG domain contains many acidic amino acid residues arranged in tripeptide clusters (Figure 9.3). Similar peptides, externally added to CA II isozyme, were shown to substantially enhance CA II catalytic performance (Scozzafava and Supuran 2002). Thus, it is possible that the acidic PG domain might work as an intrinsic modulator of CA IX, cross-talking with the catalytic domain.

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account, despite not being directly obtained with CA IX and CA XII. During the last few years, there has been remarkable progress in understanding how CAs function to ensure efficient ion flux and pH regulation. Sterling and coworkers (2001, 2002a, 2002b) have shown that CAs are directly involved in the transport metabolon, an operative protein complex consisting of a membrane-bound CA IV that interacts with an extracellular part of transmembrane anion exchangers (AE1, AE2, AE3 and DRA), which in turn bind to cytosolic CA II at the intracellular side. The interaction between CA II and AE1 requires the acidic binding motif LDADD in the C-terminal region of AE1, which also seems to be responsible for the stimulation of CA II catalytic activity (Vince and Reithmeier 2000). The external peptides enhancing CA II activity were derived from this motif (Scozzafava and Supuran 2002). These data show that CAs are closely associated functionally with bicarbonate transport proteins, including anion exchangers and natrium/bicarbonate cotransporters (Sterling et al. 2002b; Li et al. 2002). They potentiate the bicarbonate transport activity by a push–pull mechanism, in which the CA-mediated production of bicarbonate on one side of the membrane provides push for its movement across the membrane and the CA-mediated conversion to CO2 on the other side provides pull by bicarbonate minimization (Sterling et al. 2002a).

There is no reason to deny that CA IX and CA XII might participate in the analogous metabolon in tumor cells. Actually, many tumor cells maintain the expression of CA II, which might serve as an intracellular pull component of the metabolon (Parkkila et al. 1995a, 1995b; Bekku et al. 2000). The performance of such a tumor metabolon might then be regulated by availability of either ion transporter or CA IX/CA XII. In support of the possible functional cooperation between CA IX/CA XII and ion transporters in tumor cells, Karumanchi and coworkers (2001) identified AE2 as a candidate pVHL target whose mRNA and protein levels were upregulated by wild-type pVHL, but its transport activity was reduced to 50%. They proposed that downregulation of the AE2 exchange activity might represent secondary adaptation to pVHL-mediated downregulation of CA IX/CA XII, leading then to compensatory upregulation of the AE2 level. This proposal is in accordance with the observation of Sterling and coworkers (2002a) that inhibition of CA IV abrogated activity of AEs in the transport metabolon.

Translation of all this data into a simplified model relevant for the tumor phenotype suggests that loss of pVHL negative regulatory function either due to inactivating mutations or due to hypoxia might lead to upregulation of CA IX/CA XII expression or activity, or both; assembly of the putative transport metabolon via incorporation of transmembrane ion transporters and CA II; and finally to activation of ion transport. In this putative metabolon, extracellular CA IX/CA XII might preferentially catalyze hydration of CO2, producing bicarbonate, which is efficiently transported to the intracellular space and dehydrated back to CO2, which then leaves the cells by diffusion. The dehydration reaction relies on consumption of intracellular protons and might help neutralize the cell interior, whose pH is regulated by a very complex interplay of proton extrusion paths (Figure 9.4). On the other hand, outside CO2 hydration generates not only bicarbonate but also protons that remain in extracellular milieu and can contribute to its acidification.

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FIGURE 9.4 Mechanisms of pH regulation and ion transport in tumors. Lactate ion and H+ produced by glycolysis are extruded to the extracellular space by the monocarboxylate carrier

(a) and acidify extracellular pH (pHe). The Na+/H+ antiport (b) activated in transformed cells exports H+ and imports Na+. Vacuolar H+ pump (c) and ATP-dependent Na+/K+ antiport (d) further contribute to maintenance of neutral intracellular pH (pHi). According to a recent view, HCO3– generated from interstitial CO2 via catalysis by extracellular CAs enters the cell through the HCO3–/Cl– anion exchanger (e). In the cytoplasm, CA II catalyzes conversion of HCO3– to CO2, consuming intracellular H+. CO2 leaves the cell by diffusion and can be used for further hydration reactions catalyzed by extracellular CAs. H+ produced from this reaction appears to contribute to acidification of pHe. (Adapted from Stubbs, M. et al. (2000) Molecular Medicine Today 6, 15–19 and modified to include CA activity.)

In accord with this model, human CA IX protein ectopically expressed in MDCK cells elicited acidification of the culture medium. This acidification occurred only under hypoxia and was not observed in mock-transfected control cells. Its inhibition by the membrane impermeant sulfonamide supported involvement of CA IX enzyme activity (Svastova et al. submitted). Because CA IX was expressed from the constitutive promoter, this finding indicates that hypoxia can regulate also the catalytic performance of CA IX.

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9.7INFLUENCE OF EXTRACELLULAR PH ON TUMOR PROGRESSION AND POSSIBLE IMPLICATION OF CA ACTIVITY

It had been believed for long that acidification of the tumor microenvironment is caused by accumulation of lactic acid produced during aerobic and anaerobic glycolysis, coupled with the poor removal of this catabolite by the inadequate tumor vasculature (Stubbs et al. 2000). However, experiments with glycolysis-deficient cells indicated that the production of lactic acid via glycolysis is not the only mechanism leading to tumor acidity. The deficient cells did not produce lactic acid and did not acidify culture medium when exposed to either aerobic or hypoxic conditions, but formed acidic tumors in vivo (Newell et al. 1993). Moreover, a recently published comparison of the metabolic profiles of glycolysis-impaired and parental cells in both in vitro and in vivo models revealed that CO2, in addition to lactic acid, was a significant source of acidity in tumors (Helmlinger et al. 2002). These results strongly imply the contribution of CAs that is now emerging as a new paradigm.

Low extracellular pH has been associated with tumor progression via multiple effects, including upregulation of growth factors (e.g., IL-8, VEGF, bFGF) and proteases (e.g., MMPs, cathepsin B), degradation of the interstitial matrix, loss of intercellular adhesion, increased migration and metastasis, increased rate of mutations and reduced nucleotide excision repair (Xu and Fidler 2000; Fukumura et al. 2001; D’Arcangelo et al. 1999; Kato et al. 1992; Martinez-Zaguilan et al. 1996; Reynolds et al. 1996). It is a prominent feature of the reaction-diffusion model of cancer invasion that conveys a selective advantage to tumor cells invading normal tissues (Gatenby and Gawlinski 1996). Acidic pH can also influence the uptake of anticancer drugs exhibiting weakly electrolytic properties and impose varying effects on the response of tumor cells to conventional anticancer therapy. Manipulation of the extracellular pH was found to improve the cytotoxic efficacy of the weakly basic drugs mitoxantrone, paclitaxel and topotecan (Vukovic and Tannock 1997). Raghunand and coworkers. (1999) have shown that the tumor growth delay induced by the weak base doxorubicin was enhanced by increasing the extracellular pH of tumors following chronic ingestion of a sodium bicarbonate solution. This short summary suggests that modulation of interstitial tumor pH might have therapeutic value.

9.8CA INHIBITION AS AN APPROACH TO ANTICANCER THERAPY

Although there are no complete data that would link the phenotypic or therapeutic effects of CA inhibition as a means of tumor pH manipulation to perturbed activity of particular CA isozymes, the literature available so far clearly indicates that this is an exploitable avenue toward treating cancer. Teicher and co-workers (1993) showed that acetalozamide, a prototype carbonic anhydrase inhibitor (CAI) of several CA isozymes, reduced in vivo growth of tumors when given alone and produced additive tumor growth delays when administered in combination with various chemotherapeutic agents. Parkkila and co-workers (2000b) investigated the effect of

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acetazolamide on the invasive capacity of renal carcinoma cell lines and found that a 10-μM concentration in the culture medium inhibited relative cell invasion rate through the matrigel membrane by 18 to 74%. Based on the levels of CA isoenzymes, this effect was attributed to the inhibition of CA II or CA XII, or both. The other two examined isoforms were either absent (CA IV) or present in only in one cell line (CA IX). However, the possible role of CA IX in this process warrants further investigation, especially in light of the current knowledge that its level and activity are regulated by hypoxia, whereas the previous experiments were performed under normoxic conditions.

There is also extensive literature showing in vitro antiproliferative activities of CAIs in a broad range of human tumor cell lines. Inhibition of human cancer cell proliferation by classical sulfonamide CAIs was reported by Chegwidden and Spencer (1995), who demonstrated that methazolamide (0.4 mM) and ethoxzolamide (10 μM) inhibited growth of a human lymphoma cell line U937. Interestingly, no or only weak inhibition was observed in cells cultured in a medium containing the nucleotide precursors hypoxanthine and thymidine, assuming that sulfonamides inhibited synthesis of nucleotides. This explanation was deduced from involvement of CA activity in production of bicarbonate that is required by carbamoyl phosphate synthetase I for synthesis of pyrimidines, but other mechanisms have not been excluded (Chegwidden et al. 2000).

Supuran and collaborators, motivated by the emerging role of CAs in cancer and by the possibility of using them as therapeutic targets, synthesized and tested several hundreds of potent sulfonamide CAIs containing the aromatic or heterocyclic moiety, or both (Supuran and Scozzafava 2000a, 2000b, 2000c; Scozzafava and Supuran 2000; Supuran et al. 2001). These compounds were subjected to screening for their ability to inhibit the growth of tumor cells in vitro by using a panel of 60 cancer cells lines. The screening performed at the National Cancer Institute in the U.S. led to the identification of lead compounds that exhibited considerably higher inhibitory properties (in the low micromolar range) than did classical sulfonamides (Supuran and Scozzafava 2000a, 2000b, 2000c). These leads were used to design novel classes of derivatives with enhanced antitumor activities by using the tail approach, in which new tails were attached to precursor sulfonamides (Supuran and Scozzafava 2000c, 2001; Casini et al. 2002). The active compounds showed GI50 values (i.e., 50% inhibition of tumor cell growth after 48 h of exposure) in micromolar to nanomolar concentrations. Tumor cell lines used in the study were derived from different types of cancers, including leukemia, nonsmall cell lung carcinoma; melanoma; and carcinoma of the colon, ovary, kidney, prostate and breast (Supuran et al. 2001; Casini et al. 2002). Many, but not all, of these cell lines are known to express either CA IX or CA XII, or both; therefore, it is not impossible that at least in some cases, the observed growth-restricting effect of sulfonamides was mediated by inhibition of these cancer-related isozymes. However, CA IX and CA XII appear to be functionally linked with the tumor metabolism and microenvironment in vivo rather than with growth dynamics in a two-dimensional culture in vitro; future experiments designed to address these aspects of tumor phenotype might reveal whether they are targets of these anticancer sulfonamides. Tumor cell growthinhibiting effects of the examined drugs might be related to other isozymes, such

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as cytosolic CA II and mitochondrial CA V, which donate bicarbonate to carboxylation reactions in biosynthetic processes and might be required to uphold increased tumor cell growth.

In addition to drugs developed for CA inhibition and then tested for antitumor effects, a new and very potent anticancer sulfonamide E-7070 (indisulam) has been discovered by elaborate preclinical screening (Owa et al. 1999, Owa and Nagasu 2000). Although it was selected regardless of CA-inhibitory capacity, it has been recently shown to act as a nanomolar CA inhibitor (Abbate et al. 2004). Its anticancer effects were shown to involve decrease in the S-phase fraction along with cell cycle perturbations in G1 or G2, or both; downregulation of the cyclins E, A, B1, H, CDK2 and CDC2; reduction of CDK2 activity; inhibition of pRb phosphorylation; and differential expression of many additional molecules known to participate in metabolism, the immune response, signaling and cell adhesion (Fukuoka et al. 2001; Yokoi et al. 2002). In animal models in vivo, intraperitoneally or intravenously administered E7070 exhibited significant antitumor effect against human tumor xenografts generated from various colon and lung carcinoma cell lines (Ozawa et al. 2001). Based on the high antitumor potency in preclinical studies, E7070 has progressed to Phase I and Phase II clinical trials for treating solid tumors and holds promise as a novel therapeutic strategy. For the CA research field, it will be of great interest to examine whether cancer-related or other CA isozymes belong to molecular targets of E7070 in tumor cells.

9.9 INHIBITION PROFILE OF CA IX

To learn more about the role of cancer-related CAs and consequences of their inhibition, it is also important to obtain information on their inhibition profile with different sulfonamides, particularly with those exerting an anticancer effect. For CA XII, such comprehensive information is missing. However, recent analyses of CA IX inhibition profiles by using several dozens of aromatic and heterocyclic sulfonamides reveal very interesting data (Vullo et al. 2003; Ilies et al. 2003). These studies report inhibition of recombinant CA IX catalytic domain by six classical sulfonamides that have been used as either systemic or topical antiglaucoma drugs (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide and brinzolamide) in parallel with the newly synthesized compounds. In addition, inhibition of CA I, CA II and CA IV by the same collection of sulfonamides was determined for comparing isozyme-related inhibition patterns. CA I, CAII and CA IV isozymes showed a general preference for the heterocyclic compounds. The overall range of CA I and CA IV inhibition constants differed by three to four orders of magnitude. In contrast, CA IX behaved as a sulfonamide-avid enzyme, with a relatively narrow range of inhibition constants (14 to 305 nM). In that respect it was similar to CA II, considered up to now to be responsible for the majority of the pharmacological effects of sulfonamides. In addition, CA IX was effectively inhibited by all the examined sulfonamides, including the aromatic compounds. Most importantly, these studies identified several strong inhibitors of CA IX that are weak or medium inhibitors of CA I, CA II and CA IV, including orthanilamide derivatives, 1,3-benzene-disulfonamide derivatives and dihalogenated sulfanilamide derivatives

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FIGURE 9.5 CA IX inhibition constant (Ki) values obtained with the prototype CA inhibitor acetazolamide (upper formula) and four newly synthesized aromatic sulfonamides selected from the collections tested by Vullo, D. et al. (2003) Bioorganic and Medicinal Chemistry Letters, and Ilies et al. (2003) Journal of Medicinal Chemistry. The new compounds efficiently inhibit CA IX while acting as weak or medium inhibitors of CA I, CA II and CA IV isozymes. a Human cloned isozyme, analyzed by the esterase method. b Bovine isozyme isolated from lung microsomes, esterase method. c Catalytic domain of human cloned isozyme analyzed by the CO2 hydration method.

(Figure 9.5, Vullo et al. (2003); Ilies et al. 2003). The best CA IX inhibitor was the very simple dihalogenated compound 3-fluoro-5-chloro-4-aminobenzenesulfona- mide (KI = 12 nM), which was twice as active as acetazolamide (KI = 25 nM; Ilies et al. 2003). These findings indicate the feasibility of using such differentially acting potent CA IX inhibitors as lead compounds to design CA IX-specific sulfonamides that can be potentially used as novel therapeutic tools against cancer. This strategy can be further refined by another approach that has led to the generation of mem- brane-impermeable sulfonamides (Supuran et al. 2000; Scozzafava et al. 2000), thus combining enzyme specificity with spatial selectivity.

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9.10 CONCLUSIONS

Contributions from several streams of investigation are required to achieve the major goal of exploiting therapeutically CA activity, which is presumably involved in tumor development and progression. The principal and interdependent tasks include a better understanding of the functional relevance of CAs for malignant phenotype and metabolism by characterizing inhibition profiles of these CAs, developing isozymespecific inhibitors and determining in vitro and in vivo consequences of the selective isozyme inhibition. This chapter allows us to foresee that the research rapidly progressing in all these directions will soon approach the point of convergence and answer the question of whether inhibitory targeting of CA isozymes can benefit cancer patients.

ACKNOWLEDGMENTS

We thank Dr. Juraj Kopacek (Institute of Virology, Bratislava) for help with graphical illustrations. Our research is supported by the Bayer Corporation and by APVT (51005802) and VEGA (2/2025,2/3055) Slovak Grant Agencies.

REFERENCES

Abbate, F., Casini, A., Owa, T., Scozzafava, A., Supuran, C.T. (2004) Carbonic anhydrase inhibitors: E7070, a sulfonamide anticancer agent, potently inhibits cytosolic isoenzymes I and II, and transmembrane, tumor-associated isozyme IX. Bioorganic and Medicinal Chemistry Letters 14, 217–223.

Ashida, S., Nishimori, I., Tanimura, M., Onishi, S., and Shuin, T. (2002) Effects of von HippelLindau gene mutation and methylation status on expression of transmembrane carbonic anhydrases in renal cell carcinoma. Journal of Cancer Research and Clinical Oncology 128, 561–568.

Bartosova, M., Parkkila, S., Pohlodek, K., Karttunen, T.J., Galbavy, S., Mucha, V., Harris, A.L., Pastorek, J., and Pastorekova, S. (2002) Expression of carbonic anhydrase IX in breast is associated with malignant tissues and related to overexpression of c-erbB2.

Journal of Pathology 197, 1–8.

Beasley, N.J.P., Wykoff, C.C., Watson, P.H., Leek, R., Turley, H., Gatter, K., Pastorek, J., Cox, G.J., Ratcliffe, P., and Harris, A.L. (2001) Carbonic anhydrase IX, an endogenous hypoxia marker, expression in head and neck squamous cell carcinoma and its relationship to hypoxia, necrosis and microvessel density. Cancer Research 61, 5262–5267.

Beavon, I.R.G. (1999) Regulation of E-cadherin: does hypoxia initiate the metastatic cascade?

Journal of Clinical and Molecular Pathology 52, 179–188.

Bekku, S., Mochizuki, H., Yamamoto, T., Ueno, H., Takayama, E., and Tadakuma, T. (2000) Expression of carbonic anhydrase I or II and correlation to clinical aspects of colorectal cancer. Hepatogastroenterology 47, 998–1001.

Casini, A., Scozzafava, A., Mastrolorenzo, A., and Supuran, C.T. (2002) Sulfonamides and sulfonylated derivatives as anticancer agents. Current Cancer Drug Targets 2, 55–75.

Chegwidden, W.R., Carter, N., and Edwards, Y. (Eds.) (2000) The Carbonic Anhydrases: New Horizons. Birkhäuser Verlag, Basel.

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