InhibitorâPolymer Conjugates as a Versatile Tool for Detection and

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Article Cite This: ACS Omega 2019, 4, 6746−6756

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Inhibitor−Polymer Conjugates as a Versatile Tool for Detection and Visualization of Cancer-Associated Carbonic Anhydrase Isoforms Klaŕ a Pospíšilova,́ †,⊥ Tomaś ̌ Knedlík,†,⊥ Pavel Š ać ha,† Libor Kostka,‡ Jirí̌ Schimer,† Jirí̌ Brynda,†,§ Vlastimil Kraĺ ,§ Petr Cígler,† Vać lav Navrat́ il,† Tomaś ̌ Etrych,‡ Vladimír Š ubr,‡ Michael Kugler,†,§ Milan Fab́ ry,§ Pavlína Ř ezać ǒ va,́ *,†,§ and Jan Konvalinka*,†,∥ †

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo n. 2, 16610 Prague 6, Czechia Institute of Macromolecular Chemistry of the Czech Academy of Sciences, Heyrovského n. 2, 16206 Prague 6, Czechia § Institute of Molecular Genetics of the Czech Academy of Sciences, v.v.i., Vídeňská 1083, 14220 Prague 4, Czechia ∥ Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, 12843 Prague, Czechia ACS Omega 2019.4:6746-6756. Downloaded from pubs.acs.org by 212.115.51.38 on 04/12/19. For personal use only.

‡

ABSTRACT: Carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze hydration of carbon dioxide to bicarbonate. They play a crucial role in a number of important physiological processes. CAIX is a tumor-associated transmembrane isoenzyme that is overexpressed on the cell surface in hypoxic solid tumors and represents a validated target for cancer therapy and diagnosis. Here, we describe development of an N-(2-hydroxypropyl)methacrylamide-based copolymer conjugate (iBody) containing a CAIX inhibitor for selective targeting of this enzyme on tumor cells. The iBody binds CAIX with sufficient affinity for use in biochemical applications. We show that the iBody can be used to isolate CAIX from cell lysates and to visualize it by flow cytometry and confocal microscopy. Our results demonstrate that the iBody is a valuable tool for targeting CAIX in vitro and with further development may serve as a platform for CAIX targeting in vivo.

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the epithelium of the major duodenal papilla,10 in the epithelial cells lining the body cavities, and in the male genital ducts, where CAIX participates in the formation of testicular fluid.11 This tissue expression pattern suggests that CAIX might serve as a good target for diagnosis, imaging, and specific drug delivery to CAIX-expressing tumors.5,12−15 CAIX positivity means a poor prognosis in lung, breast, cervical, brain and renal cancer, and neck cancer.16−21 CAIX positive tumors are poorly responsive to conventional anticancer therapy, the disease relapses repeatedly, and tumors also usually metastasize.22−24 Monoclonal antibodies have been used to achieve a selective recognition of CAIX. Two antibodies, M7525 and G250,26 are widely used as CAIX-specific immunological tools for clinical detection. Antibody M75 recognizes a linear epitope on the N-

INTRODUCTION

Carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze reversible hydration of carbon dioxide to bicarbonate and protons.1 The 16 different CA isoforms identified in humans are involved in various physiological processes, including gluconeogenesis, lipogenesis, and ureagenesis.2,3 Some isoforms also play important roles in the pathogenesis of diseases such as glaucoma, epilepsy, obesity, and cancer.4,5 The extracellular, membrane-bound CA IX (CAIX) isoform is overexpressed in cancer cells.6 This isoenzyme is involved in regulating pH in hypoxic tumor cells and contributes to intercellular adhesion through its proteoglycan (PG) domain.7 The physiological expression of CAIX is limited to the gastrointestinal tract, with the strongest expression on the stomach mucous membrane, where CAIX participates in the formation of gastric acid.8 To a lesser extent, CAIX is present on the bile duct mucous membrane, and a small amount of CAIX is expressed on the intestinal mucous membrane in rapidly proliferating cells.9 Minimal levels of CAIX are found in © 2019 American Chemical Society

Received: March 3, 2019 Accepted: March 26, 2019 Published: April 12, 2019 6746

DOI: 10.1021/acsomega.9b00596 ACS Omega 2019, 4, 6746−6756

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Figure 1. Inhibitor structures and binding to the CAIX active site. (a) Structural formulas of compounds 1, 2 and HPMA conjugate iBody 1 decorated with biotin, ATTO488 and CAIX inhibitor. (b) Crystal structure of compound 1 bound to the CAIX active site. Residues interacting with compound 1 are colored pink in stick (top) and surface (bottom) representations. Polar interactions between compound 1 and protein are indicated with black dashed lines, the zinc ion is depicted as a turquoise sphere. (c) Top view into the solvent accessible surface of the active site with overlay of compound 1 (green carbons) and 2 (yellow carbons). Compound 2 binds in two alternative positions (labeled by white letters A and B) and could not be fully modeled. Electron density maps were missing for due to dynamic disorder. Surface interacting with 1 and alternative position B of 2 is highlighted in pink, surface interacting with position A of 2 is colored cyan and residue numbers are indicated.

show that this iBody can be used for specific targeting of CAIX in a number of in vitro biochemical methods.

terminal PG-like domain of CAIX, whereas G250 recognizes a conformational epitope on the catalytic CA domain. Both antibodies are used widely for immunohistochemical detection of CAIX in human tumor tissue as well as in imaging CAIX in hypoxic tumors.27 Moreover, chimeric version of G250 (cG250) has been developed and extensively characterized as an anticancer immunotherapy.28−30 However, monoclonal antibodies have some inherent limitations: they are typically very costly, their modification is not quite straightforward, and their size and pharmacokinetic properties might limit their use in certain applications.31 Therefore, a number of different platforms have been investigated for synthetic or recombinant antibody replacement. Recently, we developed polymer-based antibody mimetics called iBodies that target proteins using selective ligands.32,33 The iBodies are conjugates of hydrophilic N-(2hydroxypropyl)methacrylamide (HPMA) copolymers decorated with protein-specific ligands, as well as fluorescent molecules and biotin for visualization and isolation of the protein of interest. Our previous works demonstrated that iBodies can be used to inhibit, visualize, and isolate glutamate carboxypeptidase II (GCPII), aspartic proteases including HIV-1 protease and pepsin, and fibroblast activated protein (FAP) and might exhibit several advantages in comparison to monoclonal antibodies: they are easily synthetically available and versatile and could be simply chemically modified for added specificity, stability, or pharmacokinetics.32,33 Besides the HPMA conjugates, also small molecule−drug conjugates, were recently developed; these were successfully used for targeting CAIX-positive tumor cells in vivo.34−36 In this report, we describe development of an anti-CAIX iBody using a CA selective inhibitor as the targeting ligand. We

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RESULTS AND DISCUSSION

Recently, we developed antibody mimetics, iBodies, based on HPMA copolymer conjugates decorated with three different low-molecular weight ligands.32,33 Water-soluble and biocompatible HPMA copolymers were chosen due to previously reported beneficial physicochemical and biological properties in the fields of drug delivery vectors and polymer-based imaging agents.37 The HPMA copolymers are multivalent synthetic biocompatible macromolecules that carry a number of reactive groups enabling covalent attachment of various ligands such as fluorescent probes, therapeutics, peptides, oligonucleotides, and other low molecular weight compounds. This approach was previously utilized for design and synthesis of first iBodies selective toward FAP32 and GCPII.33,38 In the present work, we designed and synthetized new iBody toward CAIX, another valuable cancer target. Effectivity of CAIX selective iBody in various biochemical applications was evaluated and it proved the potential of these antibody mimetics. Compound Design and Inhibition of CAIX. The first step in the design of a macromolecular conjugate capable of recognizing CAIX was development of a low-molecular-weight inhibitor with a suitable linker for attachment to the polymer backbone. Compound 1 (Figure 1a) was designed based on the structure of known CA inhibitors with specificity toward CAIX (ureido-substituted benzenesulfonamides).39 We attached a short substituent to the inhibitor hydroxyl group, which did not impair compound binding to the CA active site. We explored the binding mode of compound 1 to the CAIX 6747


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effect was observed for inhibition of CAII and CAXII. Interestingly, modification of compound 1 with a PEG linker increased the selectivity for CAIX, which becomes similar to that observed for CAII. The crystal structure of CAIX mimic in complex with 2 did not reveal any specific binding of the PEG linker. On the contrary, presence of PEG linker introduced a dynamic disorder and CH2CH2NH2 group the 2 could not be modeled into electron density maps (Figure 1c). The sulfonamide proximal part of the 2 (phenylsufonamide anchor and bridging urea moiety) binds the enzyme active site similarly to 1. Phenyl moiety of 2 acquires two alternative conformations A and B with equal half occupancy. Conformation B binds similarly to that of compound 1, while conformation A engages opposite site of the active site entrance, specifically hydrophobic pocket formed by V134 and P201 (Figure 1c). This alternative binding mode together with favorable entropy contribution of a flexible part of the inhibitor might contribute to the increased affinity of 2 toward CAIX. Conjugation of compound 2 to an HPMA copolymer, yielding iBody 1, resulted in an approximately 10-fold decrease in the inhibitory potency (Ki = 100 nM). As the selectivity of the iBody is determined by the selectivity of the targeting ligand, we can summarize that the iBody 1 selectivity for CAIX over CAII is better (1:2) than for the original small molecule inhibitor, compound 1 (1:6). Furthermore, the selectivity of iBody toward CAIX is ensured by the presence of CAIX on the cell surface, while CAII and other isoforms are located within the cells. We can hypothesize that the decrease of the inhibitory potency of compound 2 after the attachment to the polymer should be ascribed to the steric hindrance of the polymer, nevertheless the iBody synthesis enabled to connect three functional groupstargeting ligand, fluorescent dye, and biotin anchorin one molecule suitable for the biochemical analysis. CAIX Can Be Isolated from Cell Lysates with iBody 1. We used iBody 1 to isolate His-tagged CAIX spiked into an lymph node carcinoma of the prostate (LNCaP) cell lysate (Figure 2a) in a manner analogous to immunoprecipitation with antibodies. Using the same technique, we also isolated CAIX endogenously produced by HT29 cells (Figure 2b). We

active site by X-ray crystallography. Compound 1 was cocrystallized with CAII containing the following seven amino acid substitutions: A65S, N67Q, E69T, I91L, F130V, K169E, and L203A. This CAIX mimic variant is often used in structural studies, as it retains the good crystallization properties of CAII, while the active site resembles that of CAIX.40,41 The crystal structure of the CAIX mimic-compound 1 complex was determined at 1.35 Å resolution (Figure 1b) and revealed that compound 1 interacts with the enzyme active site through its sulfonamide group, which coordinates the active site Zn2+, forming the canonical active site interactions reported for other sulfonamide-containing inhibitors.42 The distal part of the inhibitor interacts with residues from the central β-sheet: L57, Q67, T69, F70, D72, Q92, and V121 (residues forming polar interactions are in bold). The inhibitor acetyl group points toward the opening of the active site and is solvent exposed, suggesting that modification at this position will not significantly affect compound binding into the CAIX active site. Compound 1 was modified with a “PEG5” linker to yield compound 2, which was then conjugated to an HPMA copolymer decorated with biotin and the fluorophore ATTO488 to serve as an affinity anchor and a fluorescent label, respectively, forming copolymer conjugate (iBody) 1 (Mn = 111 000 g/mol, Mw = 116 000 g/mol, Đ = 1.2; the content of 2 was 10.5 wt %, content of biotin was 3.3 wt % and content of ATTO488 was 3.7 wt %) (Figure 1a). Corresponding conjugate lacking CA inhibitor (iBody 2)32 was used as a negative control. To synthesize well-defined polymers with dispersity close to one, we used controlled radical polymerization. The obtained HPMA polymeric precursor with molecular mass 64 000 g/mol contained 45 reactive thialozidine-2-thione groups along the polymer chain. This allowed us to attach in average 13 molecules of the CAIX targeting ligand (compound 2), 5 molecules of a fluorescent dye, and 12 molecules of biotin to one polymer chain. The versatility and avidity of the conjugates represent the main advantages of the iBody concept, since it enables to combine several various molecules into one polymer and thus increases its functionality and the binding potency. The CA family encompasses a large number of isoenzymes that differ by tissue specificity and function. For potential use in cancer imaging and treatment, high specificity of CAtargeted molecules is required. To assess the selectivity of compounds 1, 2 and iBody 1, we determined inhibitory constants toward CAIX, CAII, and CAXII using a stopped-flow carbon dioxide hydration assay.43 CAII represents a cytosolic isoform ubiquitously expressed in all tissues, while CAXII is membrane-bound isoform overexpressed on tumor cells.1 Compound 1 potently inhibited CAIX and CAII with Ki values in the nanomolar range, while inhibition of CAXII was significantly lower (Table 1). Modification with a poly(ethylene glycol) (PEG) linker, yielding compound 2, resulted in a significant decrease in Ki toward CAIX, while no

Figure 2. Affinity isolation of CAIX from cell lysates. (a) Recombinant His-tagged CAIX was isolated from an LNCaP cell lysate spiked with the enzyme using iBody 1 (silver-stained gel). iBody 2, which lacks the CAIX inhibitor, and blank streptavidin agarose (NC(SA)) were used as negative controls. (b) Western blot of CAIX affinity isolation from a HT29 cell lysate (a cell line endogenously expressing CAIX). CAIX was visualized with M75 antibody followed with IRDye 800CW-conjugated goat anti-mouse immunoglobulin. Multiple bands in line 3 (specific elution of CAIX) correspond to different glycosylation forms of the enzyme.

Table 1. In Vitro Inhibition of Human CAIX, CAII, and CAXII Isoforms compound

Ki (CAIX) [nM]

Ki (CAII) [nM]

Ki (CAXII) [nM]

1 2 iBody 1

35 ± 5 9±1 100 ± 10

6.3 ± 0.6 6.4 ± 0.8 49 ± 6

540 ± 80 600 ± 100 1000 ± 500 6748


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did not observe any nonspecific binding to the HPMA copolymer backbone decorated with biotin and ATTO488, as is evidenced by comparison with negative control iBody 2, which lacks the CAIX inhibitor (Figure 2). iBody 1 Facilitates CAIX Visualization by Flow Cytometry. To assess binding of iBody 1 to CAIX on the cell membrane, we evaluated CAIX expression on the human colorectal adenocarcinoma cell line HT29, which endogenously expresses CAIX, by flow cytometry. Anti-CAIX monoclonal antibody (mAb) M7525 was used for comparison. Nowadays, monoclonal antibodies are widely used for immunohistochemical detection of CAIX in human tumor tissue as well as in imaging of CAIX in hypoxic tumors. Specifically, antibody M75 is considered as a gold standard for immunohistochemical analysis because of its repetitive linear epitope whose affinity is not affected by protein denaturation. M75 binds the N-terminal PG domain of CAIX, specifically to GEEDLP motif,44 which occurs repeatedly within the PG domain.44 The affinity of M75 to its epitope is moderate with dissociation constant of 420 nM.45 In comparison, iBody 1 binds catalytic site of CAIX with affinity of 100 nM and inhibits the catalytic activity of the enzyme. Mouse fibroblasts (NIH3T3) served as CAIX-negative control cells. While we observed CAIX-specific fluorescent staining by both iBody 1 and M75, no staining was observed on the CAIX-negative cells (Figure 3a), demonstrating that iBody 1 binds selectively to CAIX expressed on the HT29 cell membrane. Interestingly, already low iBody 1 concentration (1 nM) enabled visualization of the CAIX on HT-29 cells. The difference between the fluorescence median of CAIX positive (HT-29) and negative (NIH3T3) cells was higher for M75 mAb in comparison to iBody 1. This difference is most probably caused by the different experimental setup used: while M75 mAb staining is further amplified by a fluorescently labeled secondary antibody, iBody 1 signal has not been amplified by any secondary agent. Together with a low CAIX expression on HT-29 cells, the signal of iBody 1 on HT-29 cells is quite low and does not increase significantly with an increasing iBody 1 concentration (Figure 3a). To confirm specific binding of iBody 1 to CAIX, we also conducted flow cytometry measurements with human cervix carcinoma C33 cells stably transfected with CAIX (C33CAIX).46 As a negative control, we used mock-transfected C33 cells (C33neo). Both C33CAIX and C33neo were incubated with iBody 1, iBody 2, or mAb M75. We observed strong fluorescent staining of C33CAIX cells incubated with as little as 10 nM iBody 1 compared to CAIX-negative C33neo cells (Figure 3b). No staining was observed with iBody 2. Similarly as in the previous experiment, the staining with M75 mAb provides higher fluorescence signal, probably due to amplification by a secondary antibody. However, since the CAIX expression on C33CAIX cells is higher compared to HT-29, iBody 1 provides much stronger staining of CAIX positive cells. Results of the flow cytometry analysis indicate that iBody 1 specifically recognizes CAIX expressing cells, enabling their potential separation from CAIX negative cells. iBody 1 Binding Enables Visualization of Endogenous CAIX by Confocal Microscopy. Binding of iBody 1 to CAIX on HT29 cell membranes was confirmed by confocal microscopy. The staining exhibited a clear membrane pattern, suggesting that CAIX did not undergo iBody 1-induced internalization (Figure 4). We observed the same localization of the fluorescence signal with CAIX-specific mAb M75

Figure 3. Detection of cell-surface CAIX with iBody 1 by flow cytometry. CAIX was incubated with various concentrations of iBody 1, iBody 2, or mAb M75 (90 nM) (followed by FITC-conjugated goat anti-mouse immunoglobulins, “GAM/FITC”). Median fluorescence intensity is shown; scales are logarithmic. (a) CAIX-positive HT29 cells (white) and CAIX-negative NIH3T3 cells (black). (b) CAIXpositive C33CAIX cell line (white) and CAIX-negative C33neo cell line (black).

followed with goat anti-mouse antibody conjugated with fluorescein isothiocyanate (FITC). Similarly to flow cytometry results, the staining of HT29 cells with mAb M75 was more intensive (Figure 4a). However, the direct comparison of the intensity of staining between iBody and M75 for the lowexpressing HT29 cells is misleading, since the M75 staining is amplified by the secondary antibody. We observed no binding of iBody 1 to CAIX-negative NIH3T3 cells. Additionally, iBody 2 did not bind to HT29 cells (Figure 4a). Finally, we also stained C33CAIX cells, expressing much higher levels of CAIX, which confirmed the membrane staining pattern of iBody 1. The C33_neo cells, not expressing CAIX, showed no staining, as well as iBody 2 applied to C33CAIX (Figure 4b). Together, these data support selective binding of iBody 1 to CAIX via the interaction of the inhibitor and the CAIX active site. To sum up, iBody 1 can serve as a single visualization system, which can ideally replace the two mAb-based visualization systems, thus reducing the number of the steps and cost of the staining experiment. If the stronger staining is required, the fluorescent signal of iBodies can be amplified by fluorescently labeled streptavidin, binding biotin conjugated to an HPMA scaffold. 6749


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We verified that anti-CAIX iBody 1 can be used effectively in a number of biochemical methods, showing it can substitute for the commonly used anti-CAIX antibodies. Especially in the confocal microscopy, iBody 1 can serve as a highly valuable visualization system, which can replace the mAb and simplify the method setup. A major advantage of polymer conjugates such as iBody 1 over monoclonal antibodies is their stability and their versatility in biochemical methods based on the unique composition and multivalency nature. iBodies can be easily modified with different affinity anchors and fluorescent labels. In summary, the iBody can be optimized for the given purpose not only in the composition, but also in the length and structure, bringing the versatility into the mentioned biochemical methods. Hypothetically, iBodies could be equipped with a cytotoxic cargo to serve as potential theranostic agents, capable of both visualizing and destroying cancer cells. High solubility, compatibility, non-immunogenicity, and modifiable size (i.e., modifiable pharmacokinetics) are additional advantages of the iBodies for the potential in vivo utilization. Our data indicate that the anti-CAIX iBody may serve as a system for the development of a theranostic nanomedicine for in vivo imaging and treatment of CAIXexpressing tumors.

Figure 4. Visualization of CAIX on HT29 and C33CAIX cells with antiCAIX iBody 1 by confocal microscopy. CAIX was visualized on the membrane of (a) HT29 cells (endogenously expressing CAIX) and (b) C33CAIX (stable CAIX transfectants) using anti-CAIX iBody 1 (green). The anti-CAIX mAb M75 (followed by a FITC-conjugated goat anti-mouse secondary antibody; “GAM”) was used as a positive control and iBody 2 (lacking a CAIX inhibitor) was used as a negative control. C33_neo and NIH3T3 cells, which do not express CAIX, served as additional negative controls. The cell nuclei were stained with Hoechst H34580 dye (blue). For experimental details, see Materials and Methods.

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MATERIAL AND METHODS Chemical Syntheses. All chemicals were purchased from Sigma-Aldrich, unless stated otherwise. All inhibitors tested in the biological assays were purified using a preparative scale Waters Delta 600 HPLC (flow rate 7 mL/min, gradient shown for each compoundincluding RT) with a Waters SunFire C18 OBD Prep Column (5 μm, 19 × 150 mm). The purity of compounds was assessed on an analytical Jasco PU-1580 HPLC (flow rate 1 mL/min, invariable gradient 2−100% acetonitrile (ACN) in 30 min, RT shown for each compound) with a Watrex C18 analytical column (5 μm, 250 × 5 mm). The final inhibitors were all of at least 99% purity. Inhibitors were further characterized by high-resolution mass spectrometry (HRMS) on an LTQ Orbitrap XL (Thermo Fisher Scientific) and NMR (Bruker AVANCE I 500 MHz equipped with CryoProbe). All interaction constants are in hertz.

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CONCLUSIONS In this work, we designed and synthesized a novel CAIXtargeting molecule: an HPMA copolymer-based nanoprobe decorated with a selective sulfonamide-based CAIX inhibitor, fluorescence dye, and biotin anchor, designated as iBody 1. The unique composition of the iBody 1 gives the system variability for various applications in biochemistry and biology. Scheme 1. Synthesis of Compounds 1 and 2a

a

(a) tert-Butyl(4-hydroxybutyl)carbamate, (Ph)3P, diisopropyl azodicarboxylate (DIAD), THF; (b) 5 M NaOH, MeOH/H2O, reflux 6 h; (c) (1) diphenylphosphoryl azide (DPPA), N,N-diisopropylethylamine (DIEA), Tol, RT to 90 °C; (2) sulfanilamide, ACN, 60 °C; (d) TFA; (e) AcNhydroxysuccinimide (ONSU), DIEA, DMF; (f) (1) Boc-PEG5-COOH, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU), DIEA, DMF (2) TFA. 6750


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TFA was then removed by flow of nitrogen, and the product was used in subsequent steps without any characterization or purification. Compound 1, N-(4-(4-(3-(4-sulfamoylphenyl)ureido)phenoxy)butyl)acetamide: compound 104 (20 mg, 1 equiv, 41 μmol) was dissolved in 200 μL of dimethylformamide (DMF), and 8 mg (1.2 equiv, 49 μmol) of Ac-ONSU was added in one portion. The reaction was alkalized with 18 μL DIEA (2.5 equiv, 102 μmol). The reaction was left stirring for 3 h, after which all volatiles were evaporated. The reaction mixture was dissolved in 250 μL of AcOH and diluted to 25% with water, resulting in precipitation of the product. The product was then collected by filtration and washed with a small amount of cold 25% AcOH and then fully dried on vacuum overnight. Yield = 87% (18 mg). Analytical HPLC RT = 16.5 min. HRMS (ESI−): calcd for C19H24O5N4NaS [MNa]+, 443.13596. Found, 443.13588. 1H NMR (400 MHz, DMSO-d6): δ 8.98 (s, 1H), 8.59 (s, 1H), 7.84 (t, J = 5.8 Hz, 1H), 7.71 (d, J = 8.9 Hz, 2H), 7.59 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 9.0 Hz, 1H), 7.19 (s, 2H), 6.86 (d, J = 9.0 Hz, 1H), 3.92 (t, J = 6.4 Hz, 2H), 3.07 (td, J = 7.0, 5.6 Hz, 2H), 1.79 (s, 3H), 1.68 (dq, J = 8.5, 6.5 Hz, 2H), 1.58−1.47 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 168.95, 154.08, 152.39, 143.04, 136.59, 132.21, 126.77, 120.25, 117.28, 114.63, 67.30, 38.16, 26.26, 25.82, 22.61. Compound 2, 18-oxo-23-(4-(3-(4-sulfamoylphenyl)ureido)phenoxy)-3,6,9,12,15-pentaoxa-19-azatricosan-1-aminium 2,2,2-trifluoroacetate: Boc-PEG5-COOH (46 mg, 1 equiv, 112 μmol) was dissolved in 0.5 mL of DMF along with 36 mg (1 equiv, 112 μmol) of TBTU and 49 μL (2.5 equiv, 279 μmol) of DIEA. To this solution, 55 mg (1 equiv, 112 μmol) of compound 104 was added, and the mixture was stirred overnight. The solvent was then evaporated and the crude product dissolved in 10 mL of EtOAc. The organic phase was washed twice with saturated bicarbonate and twice with 10% KHSO4, then dried and evaporated, yielding 53 mg of solid. TFA (1 mL) was added, and the mixture was alternately sonicated and stirred for 15 min. TFA was then removed by flow of nitrogen, and the product was purified by preparative HPLC (gradient: 10−50% ACN in 40 min, RT 22 min). Yield = 31% (17 mg). Analytical HPLC RT = 16.5 min. HRMS (ESI−): calcd for C30H48O10N5S [MH]+, 670.31164. Found, 670.31164. Synthesis and Characterization of HPMA Copolymers and Conjugates. HPMA and 3-(3-methacrylamidopropanoyl)thiazolidine-2-thione (Ma-β-Ala-TT) monomers were synthesized as previously described.47 The chain transfer agent S-2-cyano-2-propyl S′-ethyl trithiocarbonate was synthesized as described.48 The copolymer precursor 1 (poly(HPMA-co-Ma-β-Ala-TT)) was prepared by reversible addition−fragmentation chain transfer copolymerization.49 Ma-βAla-TT (246 mg, 0.95 mmol) was dissolved in 1.7 mL of dimethylsulfoxide (DMSO), and the solution was diluted with tert-butanol (9.6 mL). HPMA (1.00 g, 6.98 mmol), S-2-cyano2-propyl S′-ethyl trithiocarbonate (2.33 mg, 1.13 × 10−2 mmol), and the initiator 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70; Wako Chemicals GmbH, Germany) (1.75 mg, 5.67 × 10−3 mmol) were added, and the solution was introduced into a polymerization ampule. The mixture was bubbled with argon for 10 min, and the ampule was sealed. Polymerization was carried out at 40 °C for 16 h. The polymer precursor was isolated by precipitation into a mixture of acetone/diethyl ether (3:1, 250 mL), filtered off, washed with

Synthesis of Compounds 1 and 2. Compounds 1 and 2 were synthesized according to Scheme 1. Compound 101, methyl 4-(4-((tert-butoxycarbonyl)amino)butoxy)benzoate: DIAD (312 μL, 1.5 equiv, 1.59 mmol), was added in one portion to a solution of 161 mg (1 equiv, 1.06 mmol) of methyl 4-hydroxybenzoate, 300 mg (1.5 equiv, 1.59 mmol) of tert-butyl (4-hydroxybutyl)carbamate, and 400 mg (1.5 equiv, 1.59 mmol) of triphenylphosphine in 10 mL of tetrahydrofuran (THF). (Note: methyl 4-hydroxybenzoate has an identical RF with the product; therefore, 1.5 equiv of other reactants were used) The reaction was left stirring overnight. The reaction mixture was then evaporated to remove solvent, and the crude product was purified by column chromatography (He/EtOAc 4:1, RF = 0.25) to yield 260 mg of white powder. Yield = 75%. MS (ESI+): calcd for C17H25O5N [MNa]+, 346.17. Found, 346.2. 1H NMR (400 MHz, CDCl3): δ 7.95 (d, J = 8.9 Hz, 2H), 6.87 (d, J = 8.9 Hz, 2H), 4.71 (s, 1H), 3.99 (t, J = 6.2 Hz, 2H), 3.85 (s, 3H), 3.17 (dd, J = 12.8, 6.3 Hz, 2H), 1.86−1.75 (m, 2H), 1.69−1.61 (m, 2H), 1.42 (s, 9H). 13C NMR (101 MHz, CDCl3): δ 166.92, 162.78, 156.10, 131.64, 122.57, 114.12, 79.20, 67.73, 51.89, 40.29, 28.49, 26.86, 26.49. Compound 102, 4-(4-((tert-butoxycarbonyl)amino)butoxy)benzoic acid: compound 101 (270 mg) was dissolved in 5 mL of methanol, and 5 mL of 5 M NaOH was added. The mixture was refluxed until thin-layer chromatography analysis showed complete disappearance of compound 101 (6 h). The reaction mixture was diluted with EtOAc (20 mL), the aqueous phase was acidified with 10% KHSO4 and extracted twice with 20 mL of EtOAc. An oily product, which turned to crystalline white after removal of solvent traces, was obtained. Yield 95% (240 mg). MS (ESI−): calcd for C16H22O5N [M]−, 308.16. Found, 308.2. 1H NMR (400 MHz, CDCl3): δ 8.03 (d, J = 8.9 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 4.65 (s, 1H), 4.04 (t, J = 6.2 Hz, 2H), 3.27−3.20 (m, 2H), 1.91−1.78 (m, 2H), 1.69 (dd, J = 14.8, 7.2 Hz, 2H), 1.44 (s, 9H). 13C NMR (101 MHz, CDCl3): δ 171.51, 163.46, 156.20, 132.42, 121.92, 114.28, 79.42, 67.86, 40.36, 28.56, 26.89, 26.53. Compound 103, tert-butyl (4-(4-(3-(4-sulfamoylphenyl)ureido)phenoxy)butyl)carbamate: compound 102 (720 mg, 1 equiv, 2.33 mmol) was dissolved in 15 mL of dry toluene, and 810 μL (2 equiv, 4.65 mmol) of DIEA was added. DPPA (552 μL, 1.1 equiv, 2.56 mmol) was added to the reaction mixture in one portion, and the temperature was raised to 90 °C for 2 h. The reaction mixture was then evaporated and dissolved in dry ACN. Sulfanilamide (601 mg, 1.5 equiv, 3.49 mmol) was added in one portion, and the reaction was heated to 60 °C overnight while stirring. All volatiles were evaporated after 12 h, and the crude product was purified by column chromatography on silica (He/EtOAc, 2:5, RF = 0.25). Isolated yield 30% (340 mg). MS (ESI+): calcd for C22H30O6N4S [MNa]+, 501.17. Found, 501.2. 1H NMR (400 MHz, DMSO): δ 8.98 (s, 1H), 8.59 (s, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 8.9 Hz, 2H), 7.34 (d, J = 9.0 Hz, 2H), 7.20 (s, 2H), 6.91−6.81 (m, 3H), 3.91 (t, J = 6.4 Hz, 2H), 2.96 (dd, J = 12.9, 6.7 Hz, 2H), 1.71−1.61 (m, 2H), 1.51 (dt, J = 13.1, 6.5 Hz, 2H), 1.37 (s, 9H). 13C NMR (101 MHz, DMSO): δ 155.37, 154.02, 152.16, 142.99, 136.40, 132.04, 126.61, 120.14, 117.12, 114.50, 77.06, 67.05, 40.35, 27.77, 26.85, 25.73. Compound 104, 4-(4-(3-(4-sulfamoylphenyl)ureido)phenoxy)butan-1-aminium 2,2,2-trifluoroacetate: compound 103 (500 mg) was dissolved in 1 mL of trifluoroacetic acid (TFA) and was sonicated and stirred alternately for 15 min. 6751


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obtained CAIX-mimic by introducing the following mutations into the CAII coding sequence: A65S, N67Q, E69T, I91L, F130V, K169E, and L203A. The CAXII construct encodes the extracellular catalytic domain of CAXII. Gene coding for CAXII amino acids 30−291 was inserted into a pET-based vector. CAII and CAIX-mimic and CAXII were expressed in Escherichia coli BL21(DE3) and purified on p-aminomethylbenzene sulfonamide-agarose affinity columns (Sigma Life Science Aldrich).51,52 The CAIX construct used encodes the extracellular part of CAIX comprising the PG and CA domains (residues 38−391) with a single amino acid replacement (C174S).53 The construct contains a C-terminal polyhistidine sequence (Histag) for easier purification and an N-terminal signal peptide for protein secretion into the medium. The construct was cloned into a pTT5 vector for expression in human embryonal kidney cells. CAIX was purified by the HisTrap HP column (GE Healthcare). The column was equilibrated with buffer A (50 mM NaH2PO4, pH 8.3; 300 mM NaCl). The sample was loaded at a flow rate 0.5 mL/min. A two-step wash was performed at a flow rate of 2 mL/min. In the first wash step, the column was washed with 50 mL of buffer A, and in the second with 50 mL of 98% buffer A and 2% of buffer B (50 mM NaH2PO4, pH 8.3; 300 mM NaCl; 500 mM imidazole). CAIX was eluted with buffer B at a flow rate of 1 mL/min. We collected fractions of 5 mL; the whole purification was carried out at 4 °C. Purified fractions were collected and dialyzed overnight at 4 °C against 20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) pH 7.5, 100 mM Na2SO4·10H2O. CA Inhibition Assay. A stopped-flow instrument (Applied Photophysics) was used to measure CA-catalyzed CO2 hydration activity in the presence of inhibitors.43 The assay buffer consisted of 0.2 mM phenol red (pH indicator with maximum absorbance change at 557 nm), 20 mM HEPES, pH 7.5, and 20 mM Na2SO4. The CA concentrations in the reaction mixture ranged from 2 to 38 nM. To stabilize CAIX during measurements, 0.0025% dodecyl-β-D-maltopyranosid (Anatrace) was included into the reaction mixture. The substrate (CO2) concentration in the reactions was 8.5 mM. Rates of the CA-catalyzed CO2 hydration reaction were followed for a period of 30 s at 25 °C. Four traces of the initial 5−10% of the reaction were used to determine the initial reaction velocity for each inhibitor. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitors (100 mM) were prepared in DMSO, and up to 100 nM dilutions were made with DMSO. To allow the formation of the E−I complex, inhibitor and enzyme solutions were pre-incubated for 5 min at room temperature prior to measurement. The inhibition constants were obtained by nonlinear least-squares methods using EXCEL spreadsheets.54 Protein X-ray Crystallography. For X-ray studies, CAII with amino acid substitutions A65S, N67Q, E69T, I91L, F130V, K169E, and L203A was used as a CAIX mimic.40 Cocrystals of the CAIX mimic with 1 and 2, respectively, were obtained by the vapor-diffusion hanging drop method at 18 °C. The complex was prepared by mixing equimolar amounts of inhibitor (dissolved in pure DMSO) and protein (25 mg/mL in 50 mM Tris-HCl, pH 7.8). Drops containing 2 μL of protein−inhibitor complex were mixed with 1 μL of a precipitant solution containing 1.6 M sodium citrate, 50 mM Tris H2SO4, pH 7.8, and equilibrated over a 1 mL reservoir of

acetone (20 mL) and diethyl ether (20 mL), and dried in vacuo. The terminating trithiocarbonate group was removed as described by Perrier.50 The copolymer precursor 1 with molecular weight Mn = 58 500 g/mol, Mw = 64 400 g/mol, dispersity Đ = 1.1 and content of reactive thiazolidine-2-thione groups of 10.3 mol % was obtained. The HPMA copolymer conjugates (iBodies 1 and 2) were prepared by a conjugation of the copolymer precursor 1 containing thiazolidine-2-thione reactive groups (TT) with a combination of a targeting ligand (absent in negative control iBody 2 lacking the targeting ligand), fluorophore (ATTO488NH2), and affinity anchor N-(2-aminoethyl)biotinamide hydrobromide (biotin-NH2), according to the procedure described previously.33 Synthesis of iBody 1. The copolymer precursor 1 (25 mg), compound 2 (3.6 mg), biotin-NH2 (2.8 mg), and ATTO488-NH2 (2.0 mg) were dissolved in 0.5 mL DMSO. Then, 13.0 μL of N,N-diisopropylethylamine was added. The reaction was carried out for 4.5 h at room temperature, then 5 μL of 1-aminopropan-2-ol was added, and the reaction was stirred for 10 min. The resulting solution of the copolymer conjugate poly(HPMA-co-Ma-β-Ala-2-co-Ma-β-Ala-ATTO488co-Ma-β-Ala-NH-biotin) was diluted with 1.5 mL of methanol and purified on a Sephadex LH-20 chromatography column in methanol. Methanol was evaporated, and the iBody 1 was dissolved in 1.5 mL of distilled water, purified on a Sephadex G-25 chromatography column and lyophilized. The yield of iBody 1 (Mn = 111 000 g/mol, Mw = 116 000 g/mol, Đ = 1.2) was 17.5 mg; the content of 2 was 10.5 wt %, content of biotin was 3.3 wt %, and content of ATTO488 was 3.7 wt %. The content of the targeting ligand (compound 2) was determined from NH2-PEG5-OH degradation product with pre-column o-phtaldialdehyde (OPA)/3-mercaptopropionic acid derivatization on an HPLC system (Shimadzu) with an RF-20A fluorescence detector (Ex = 229 nm/Em = 450 nm) using a reverse-phase Chromolith RP18e column (100 × 4.6 mm). The flow rate was 1 mL/min in a gradient of sodium acetate buffer and methanol. Prior to analysis, samples were hydrolyzed with 6 M HCl at 115 °C for 16 h. The hydrolysate was dried and dissolved in water. We assumed that hydrolysis of free compound 2 (used as a standard) and compound 2 bound to the conjugate (iBody 1) occurred to the same extent. Synthesis of iBody 2. The synthesis of iBody 2, a negative control conjugate lacking the CAIX inhibitor, was described previously by Dvořaḱ ová et al.32 Briefly, iBody 2 (Mn = 80 900 g/mol, Mw = 131 000 g/mol, Đ = 1.6) contains 5.4 wt % of biotin and 4.1 wt % of ATTO488. The weight-average molecular weights (Mw), number average molecular weights (Mn), and dispersity (Đ) of the polymer precursor and conjugates were determined using an HPLC Shimadzu system equipped with a UV detector, an OptilabrEX differential refractometer, a multi-angle light scattering DAWN 8 (Wyatt Technology, USA) detector, and a size-exclusion chromatography TSKgel G4000SW column. The Mw, Mn, and D̵ were calculated using the Astra V software. The refractive index increment dn/dc = 0.167 mL/g was used for calculation. For these experiments, a 20% 300 mM sodium acetate, pH 6.5: 80% methanol (v/v) buffer was used. The flow rate was 0.5 mL/min. The TT content in polymer precursor, ATTO488, and biotin content were determined spectrophotometrically as described recently by Dvořaḱ ová et al.32 CA Expression and Purification. The CAII expression plasmid (pET-based) was kindly provided by McKenna.40 We 6752


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precipitant solution. Crystals with dimensions of 0.3 × 0.1 × 0.2 mm grew within one week. Before data collection, the crystal was soaked for 5−10 s in a reservoir solution supplemented with 25% (v/v) glycerol and stored in liquid nitrogen. Diffraction data were collected at 100 K on our home diffractometer (MicroMax-007 HF microfocus equipped with PILATUS 300K detector, Rigaku) and integrated and reduced using XDS program55 and its graphical interface XDSAPP.56 The crystal structures were solved by molecular replacement with the MolRep program57 using the CAII structure (PDB entry 3PO658) as a model. Atomic coordinates of the inhibitor molecule were generated by PRODRG subroutine in COOT.59 The geometric library for the inhibitors was generated using the Libcheck program. Compound 1 was modeled into a welldefined electron density map with full occupancy. COOT was used for inhibitor fitting, model rebuilding, and addition of water molecules. Refinement was carried out with Refmac5,60 with more than 2000 of the reflections reserved for crossvalidation. The structures were first refined with isotropic atomic displacement parameters (ADPs). After adding solvent atoms and zinc ions, building inhibitor molecules in the active site, and exploring several alternate conformations for a number of residues, anisotropic ADPs were refined for nearly all atoms (with the exception of spatially overlapping atoms in segments with alternate conformations; also, oxygen atoms of water molecules with an unrealistic ratio of ellipsoid axes were refined with isotropic ADPs), including atoms in the inhibitor molecules. The quality of the crystallographic model was assessed with MolProbity.61 All the figures representing structures were created using PyMOL [Schrodinger, LLC (2010) The PyMOL Molecular Graphics System, Version 1.3r1]. Atomic coordinates and structure factors were deposited in the PDB with accession code 6GXB and 6GXE. Crystal parameters and data collection and refinement statistics are summarized in Table 2. Cell Cultures. HT29, NIH3T3, C33, and transfected derivatives were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS) at 37 °C under a 5% carbon dioxide atmosphere (Sanyo CO 2 incubator, MCO-ATAI) and passaged in a standard way. The C33 cell line constitutively expressing CAIX was a generous gift from Dr. Silvia Pastoreková from the Institute of Virology of the Slovak Academy of Sciences, Bratislava, Slovakia. Flow Cytometry. To analyze the utility of iBody 1 for visualizing CAIX-expressing cells, we used HT29 and C33CAIX cell lines with NIH3T3 and C33 neo as negative controls. We incubated the cells with iBody 1 and iBody 2, at concentrations ranging from 0.1 to 1000 nM. Furthermore, we used the anti-CAIX antibody M7525 (14 μg/mL) as a positive control of CAIX expression. As secondary antibodies, we used FITC-conjugated goat anti-mouse immunoglobulin (Jackson Laboratories, #115-095-164) for cells labeled with M75 antibody and the anti-c-myc/FITC (Exbio Praha, Czechia) for cells labeled with the single-chain fragment variable fragments, both at 0.02 mg/mL concentration. We used propidium iodide (0.5−1 μg/mL) to distinguish live and dead cells. A standard protocol for flow cytometry staining was used for all samples. Data were collected on BD LSR Fortessa (laser 488 nm detector 530/30); FlowJo software 7.6.5 was used for data evaluation.

Table 2. Crystal Data and Diffraction Data Collection and Refinement Statistics complex of CAIX mimic with wavelength (Å) space group cell parameters (Å, deg) number of molecules in AU resolution range (Å) number of unique reflection multiplicity completeness Rmerge (%) Rmeas (%) CC(1/2) (%) average I/σ(I) Wilson B (Å2) resolution range (Å) no. of reflection in working set no. of reflection in test set Rwork value (%) Rfree value (%) RMSD bond length (Å) RMSD angle (deg) number of atoms in AU number of water mol. in AU mean ADP value (Å2) residues in favored regions (%) residues in allowed regions (%) PDB code

compound 1

compound 2

Data Collection Statistics 1.54187 P21 41.8, 41.2, 72.0; 90.0, 103.8, 90.0 1

1.54187 P21 41.9, 41.2, 71.1; 90.0, 103.9, 90.0 1

50−1.35 (1.39−1.35) 41 518 (2062)

50−1.3 (1.33−1.3) 51 503 (2250)

3.7 (2.86) 0.791 (0.538) 3 (29.9) 3.5 (36.5) 100 (87.2) 28.17 (3.36) 11.8 Refinement Statistics 40.6−1.35 (1.385−1.350) 40 480 (2009)

5.06 (2.53) 0.872 (0.517) 3 (20.9) 3.2 (26.8) 100 (88.8) 27.83 (3.85) 17.5

1038 (51)

1031 (45)

11.3 (16.5) 15.4 (22.9) 0.011

12.5 (15.5) 15.4 (19.2) 0.010

1.55 2433

1.62 2366

319

261

39.6−1.3 (1.33−1.30) 50 472 (2199)

13.2 15.3 Ramachandran Plot Statistics 97.5 98.2 2.5

1.8

6GXB

6GXE

Visualization of CAIX on Cells Using Confocal Microscopy. Confocal microscopy was performed using the cell lines HT29 (endogenously expressing CAIX) and NIH3T3 (not expressing CAIX). Cells were grown in 4chamber 35 mm glass bottom dishes (In Vitro Scientific) in DMEM supplemented with 10% FBS and 4 mM L-glutamine to approximately 60% confluence. The next day, the anti-CAIX iBody 1 was added to a final concentration of 100−1000 nM, and cells were incubated for 1 h at 37 °C. The anti-CAIX antibody M75 (14 μg/mL) was used as a positive control, and iBody 2 (lacking the CAIX inhibitor) was used as a negative control. The medium was removed, and after washing (2 × 1 mL tris-buffered saline, TBS), polyclonal FITC-labeled goat anti-mouse antibody (20 μg/mL) was used to visualize cells treated with M75. Cell nuclei were stained using Hoechst stain solution H34580 (Sigma; 0.5 μg/mL; 10 min). Confocal images were obtained with a Zeiss LSM 780 confocal microscope using an inverse oil-immersion objective (Plan-Apochromat 63×/1.40 Oil DIC M27). Images were taken using a 405 nm laser diode (4.5%; max. output 25 mW) 6753


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ACKNOWLEDGMENTS We would like to thank Jana Starková and Karolı ́na Š rámková for their excellent technical support, Jana Dvořanová Š těpánková for helpful training, Petro Khoroshyy for help with confocal microscopy, and Marek Roesel and Hillary Hoffman for language editing. This work was supported by grant no. GA16-02938S from the Grant Agency of the Czech Republic, by the Ministry of Education of the Czech Republic, projects LO1304 and LO1302, and in part by RVO 68378050 and 61388963 awarded by the Czech Academy of Sciences. The work of P.C. was supported by the European Regional Development Fund; OP RDE; Projects: “Chemical biology for drugging undruggable targets (Chem-BioDrug)” (No. CZ.02.1.01/0.0/0.0/16_019/0000729). The work of P.C. and J. Sch. was supported by the European Regional Development Fund; OP RDE; Project: “Carbon allotropes with rationalized nanointerfaces and nanolinks for environmental and biomedical applications” (No. CZ.02.1.01/0.0/ 0.0/16_026/0008382).

for excitation with emission collected from 410 to 585 nm (detector voltage 570 V) for Hoechst 34580 and a 488 nm argon laser (4.0%; max. output 25 mW) for excitation with emission collected from 517 to 534 nm (detector voltage 870 V) for ATTO488. All images were taken using the same settings. The obtained images were processed with the ZEN 2011 software package (Carl Zeiss Microscopy). Affinity Isolation of CAIX from Cell Lysates. Lysates of HT29 cells and LNCaP cells were prepared by 5 min sonication in 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 (TBS), with 1% Tween 20. The solutions were then centrifuged at 16 100g for 10 min, and the supernatants were transferred into new tubes. First, 1 μM solutions of iBody 1 or iBody 2 in 20 mM TrisHCl, 150 mM NaCl, pH 7.4, 0.1% Tween 20 (TBST) were incubated with 30 μL of Streptavidin Agarose Ultra Performance (Solulink) for 1 h at 4 °C. As a negative control, blank streptavidin agarose (i.e., not containing any iBody) was used. The resins were then washed twice with 1 mL of TBST. The resins were then incubated with either His-tagged CAIX-spiked LNCaP cell lysate in TBST (CAIX: 3 μg/mL, total protein: 400 μg/mL) or HT29 cell lysate (total protein: 500 μg/mL) in TSBT and incubated for 1 h 30 μL of reducing sodium dodecyl sulfate (SDS) sample buffer was added to each sample, and the mixtures were heated to 98 °C for 10 min. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and visualized by either silver staining or western blot. Western Blot. Proteins were resolved by SDS-PAGE and blotted on a nitrocellulose membrane (1 h, 100 V, cooled). The membrane was then blocked with 0.55% (w/v) casein in TBS (Casein Buffer 20×−4× concentrate, Stereospecific detection technologies (SDT); 1 h at RT). CAIX was visualized using CAIX-specific M75 monoclonal antibody (200 ng/mL, diluted in casein solution; 1 h at RT), followed by goat anti-mouse secondary antibody conjugated with IRDye 800CW [LI-COR Biosciences; 1:15 000 in phosphate-buffered saline with 0.1% (w/v) Tween 20; 1 h at RT]. CAIX was visualized using an Odyssey CLx Infrared Imaging System (LICOR).

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REFERENCES

(1) Supuran, C. T. Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat. Rev. Drug Discovery 2008, 7, 168−181. (2) Sly, W. S.; Hu, P. Y. Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu. Rev. Biochem. 1995, 64, 375−401. (3) Chegwidden, W. R.; Dodgson, S. J.; Spencer, I. M. The roles of carbonic anhydrase in metabolism, cell growth and cancer in animals. Carbonic Anhydrases 2000, 90, 343−363. (4) Supuran, C. T.; Scozzafava, A. Carbonic anhydrases as targets for medicinal chemistry. Bioorg. Med. Chem. 2007, 15, 4336−4350. (5) Cianchi, F.; Vinci, M. C.; Supuran, C. T.; Peruzzi, B.; De Giuli, P.; Fasolis, G.; Perigli, G.; Pastorekova, S.; Papucci, L.; Pini, A.; Masini, E.; Puccetti, L. Selective Inhibition of Carbonic Anhydrase IX Decreases Cell Proliferation and Induces Ceramide-Mediated Apoptosis in Human Cancer Cells. J. Pharmacol. Exp. Ther. 2010, 334, 710−719. (6) Pastorekova, S.; Parkkila, S.; Zavada, J. Tumor-associated Carbonic Anhydrases and Their Clinical Significance. Adv. Clin. Chem. 2006, 42, 167−216. (7) Benej, M.; Pastorekova, S.; Pastorek, J. Carbonic anhydrase IX: regulation and role in cancer. Subcell. Biochem. 2014, 75, 199−219. (8) Pastorekova, S.; Parkkila, S.; Parkkila, A.; Opavsky, R.; Zelnik, V.; Saarnio, J.; Pastorek, J. Carbonic anhydrase IX, MN/CA IX: analysis of stomach complementary DNA sequence and expression in human and rat alimentary tracts. Gastroenterology 1997, 112, 398−408. (9) Saarnio, J.; Parkkila, S.; Parkkila, A.-K.; Waheed, A.; Casey, M. C.; Zhou, X. Y.; Pastoreková, S.; Pastorek, J.; Karttunen, T.; Haukipuro, K.; Kairaluoma, M. I.; Sly, W. S. Immunohistochemistry of carbonic anhydrase isozyme IX (MN/CA IX) in human gut reveals polarized expression in the epithelial cells with the highest proliferative capacity. J. Histochem. Cytochem. 1998, 46, 497−504. (10) Kivelä, A. J.; Parkkila, S.; Saarnio, J.; Karttunen, T. J.; Kivelä, J.; Parkkila, A. K.; Pastoreková, S.; Pastorek, J.; Waheed, A.; Sly, W. S.; Rajaniemi, H. Expression of transmembrane carbonic anhydrase isoenzymes IX and XII in normal human pancreas and pancreatic tumours. Histochem Cell Biol. 2000, 114, 197−204. (11) Ivanov, S.; Liao, S.-Y.; Ivanova, A.; Danilkovitch-Miagkova, A.; Tarasova, N.; Weirich, G.; Merrill, M. J.; Proescholdt, M. A.; Oldfield, E. H.; Lee, J.; Zavada, J.; Waheed, A.; Sly, W.; Lerman, M. I.; Stanbridge, E. J. Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrases in human cancer. Am. J. Pathol. 2001, 158, 905−919. (12) Swietach, P.; Wigfield, S.; Cobden, P.; Supuran, C. T.; Harris, A. L.; Vaughan-Jones, R. D. Tumor-associated carbonic anhydrase 9

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +420 220 183 144 (P.Ř .). *E-mail: [email protected]. Phone: +420 220 183 218 (J.K.). ORCID

Tomás ̌ Knedlík: 0000-0001-7162-6631 Pavel Š ácha: 0000-0001-6198-9826 Libor Kostka: 0000-0002-7770-1855 Petr Cígler: 0000-0003-0283-647X Tomás ̌ Etrych: 0000-0001-5908-5182 Jan Konvalinka: 0000-0003-0695-9266 Author Contributions ⊥

K.P. and T.K. contributed equally.

Notes

The authors declare the following competing financial interest(s): A patent application (PCT/CZ2016/05003) has been filed related to this work. 6754


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