An update on anticancer drug development and delivery targeting carbonic anhydrase IX

Submitted 2 August 2017 Accepted 30 October 2017 Published 23 November 2017 Corresponding author Daumantas Matulis, [email protected], [email protected] Academic editor Camillo Rosano Additional Information and Declarations can be found on page 15 DOI 10.7717/peerj.4068 Copyright 2017 Kazokait˙ e et al. Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS An update on anticancer drug development and delivery targeting carbonic anhydrase IX Justina Kazokait˙ e1,*, Ashok Aspatwar2,3,*, Seppo Parkkila2,3and Daumantas Matulis1 1Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania 2Faculty of Medicine and Life sciences, University of Tampere, Tampere, Finland 3Fimlab Ltd, Tampere, Finland *These authors contributed equally to this work. ABSTRACT The expression of carbonic anhydrase (CA) IX is up-regulated in many types of solid tumors in humans under hypoxic and acidic microenvironment. Inhibition of CA IX enzymatic activity with selective inhibitors, antibodies or labeled probes has been shown to reverse the acidic environment of solid tumors and reduce the tumor growth establishing the significant role of CA IX in tumorigenesis. Thus, the development of potent antitumor drugs targeting CA IX with minimal toxic effects is important for the target-specific tumor therapy. Recently, several promising antitumor agents against CA IX have been developed to treat certain types of cancers in combination with radiation and chemotherapy. Here we review the inhibition of CA IX by small molecule compounds and monoclonal antibodies. The methods of enzymatic assays, biophysical methods, animal models including zebrafish and Xenopus oocytes, and techniques of diagnostic imaging to detect hypoxic tumors using CA IX-targeted conjugates are discussed with the aim to overview the recent progress related to novel therapeutic agents that target CA IX in hypoxic tumors. Subjects Biochemistry, Biophysics, Drugs and Devices Keywords CA IX monoclonal antibodies, Hypoxic tumors, CA IX antitumor agents, Carbonic anhydrase IX, Drug development, Conjugated probes Introduction Recent advances in cancer therapy show that hypoxia is the major contributor to tumor development (Semenza, 2014;Hanahan & Weinberg, 2011). The poor and chaotic tumor angiogenesis leads to the insufficient oxygen and nutrient supply which drastically affects the cellular metabolism (Welti et al., 2013). Due to the up-regulated glycolysis, tumor cells produce increased amounts of lactate and protons. As a consequence of mTORC1&2 mediated functional and transcriptional activation of c-Myc, tumor cells tend to metabolize glucose preferably via glycolysis rather than oxidative phosphorylation despite sufficient levels of oxygen. This phenomenon is known as Warburg effect (Warburg, 1956;Vander Heiden, Cantley & Thompson, 2009). The resultant hypoxic and acidic extracellular milieu How to cite this article Kazokait˙ e et al. (2017), An update on anticancer drug development and delivery targeting carbonic anhydrase IX. PeerJ 5:e4068; DOI 10.7717/peerj.4068 significantly increases the resistance of cancer cells to chemotherapy and radiotherapy as well as promotes invasiveness and metastasis (Wojtkowiak et al., 2011;Good & Harrington, 2013). Hypoxia stimulates crucial pathways, one of which is implemented by the activation of the heterodimeric hypoxia-inducible factor (HIF) (Denko, 2008). This hypoxia-induced transcriptional program is important for tumor cells to survive harsh conditions. There are many downstream-target genes of HIF, which encode proteins, such as adhesion molecules (Ryu et al., 2010), matrix metalloproteinases (O’Toole et al., 2008), chemokine receptors (Li et al., 2009a), growth factors (Kotch et al., 1999), differentiation proteins (Takubo et al., 2010), glycolytic enzymes (Obach et al., 2004), lactate transporters (Ullah, Davies & Halestrap, 2006), and ion transporters (Parks, Chiche & Pouysségur, 2013). Some HIF-regulated proteins have been shown to be hypoxia-related anticancer targets and possess therapeutic applications (Wilson & Hay, 2011). Thus, HIF is critically essential for cancer cells to survive and metastasize in the hostile tumor environment due to the HIF-dependent activation of oncogenes and inactivation of tumor suppressor genes. As a consequence of HIF-mediated transcriptional response to tumor hypoxia, the intracellular and extracellular pH is unbalanced. Normal cells differ from cancer cells by the mechanisms of pH regulation, which create the reversed pH gradient in tumors. Physiologically the intracellular pH (pHi) is lower than the extracellular pH (pHe), which is ∼7.4. Pathologically pHiis higher than pHe, which is 6.7–7.1 (Hashim et al., 2011;Mazzio, Smith & Soliman, 2010). This phenomenon of extracellular acidification under hypoxic conditions is created by HIF-dependent induction of proteins, such as transmembrane enzymes, ion pumps, and transporters. They export lactate and protons and import bicarbonate ions to optimize the tumor progression. Key pH-regulators are V-ATPase, Na+/H+exchanger (NHE), monocarboxylate transporters (MCTs) and carbonic anhydrase (CA) IX. There are seven evolutionarily distinct CA gene families: α-, β-, γ-, δ-, ζ-η-, and θ-CAs (Prete et al., 2014;Supuran & Capasso, 2015;Krishnamurthy et al., 2008;Kikutani et al., 2016;Aggarwal et al., 2013;Capasso & Supuran, 2015). In humans, there are 15 α-CA isoforms, of which 12 are catalytically active and exhibit diverse enzymatic activity, various cellular distribution and physiological functions (Frost, 2014). Being a member of α-CA isoforms in human body, CA IX is a transmembrane homodimer, which catalyzes the reversible hydration of carbon dioxide to bicarbonate and proton outside the cell. The intracellular pH of cancer cells is regulated by the export of lactate and protons and on the import of bicarbonate ions generated by the hydration of CO2. The acidic metabolites accumulate pericellularly because of the ineffective tumor vasculature and extracellular acidosis. To reduce changes of intracellular pH, the bicarbonate is transported into the cell through the bicarbonate transport metabolon composed of CA IX and bicarbonate transporters. Thereby CA IX is important for cancer cell proliferation because of the participation in both processes: the extracellular acidification and the intracellular alkalinization (Aggarwal et al., 2013;Alterio et al., 2009). CA IX is relevant not only for the cancer cell survival, but also to several other biological processes, such as the maintenance of cancer stem cell (CSC) function, migration, and Kazokait˙ e et al. (2017), PeerJ, DOI 10.7717/peerj.4068 2/31 invasion. Cell migration depends on the formation of lamellipodia, which have been shown to be partially produced by activation of CA IX and its interaction with bicarbonate transporters (Svastova et al., 2012). In addition, acidosis under hypoxic conditions activates proteolytic enzymes, which degrade the extracellular matrix and promote metastasis formation. Thus, CA IX targeting compounds have shown to significantly diminish the cancer stem cell population, inhibit the growth of primary tumors, and reduce metastatic burden (Swietach et al., 2010;Pastorek & Pastorekova, 2015;Sedlakova et al., 2014;Lock et al., 2013;McDonald et al., 2010). In normal tissues, the expression of CA IX is negligible with the exception of the stomach and gallbladder epithelia (Pastorekova et al., 1997). There is a broad spectrum of aggressive malignancies, where CA IX is predominantly overexpressed, namely, neuroblastoma (Ameis et al., 2016), breast tumor (Betof et al., 2012), head and neck tumors (Yang et al., 2014), ovarian tumor (Choschzick et al., 2011), pancreatic tumor (Couvelard et al., 2005), hepatocellular carcinoma (Huang et al., 2015), etc. In addition, there are several reviews, which summarize the significance of CA IX as a promising biomarker for the tumor development (Van Kuijk et al., 2016). Thus, CA IX has emerged as the clinically relevant biomarker and a potential anticancer-drug target. At the core of α-CA active site, the metal ion, Zn (II), is tetrahedrally coordinated to three imidazole rings from His94, 96, and 119 (numbering according to CA II) and a water/hydroxide anion (Fisher et al., 2007). The catalytic site is located at approximately 15 Å depth conical cavity which consists of hydrophobic (Val121, Val143, Leu198, Val207, Trp209) as well as hydrophilic (Tyr7, Asn62, His64, Asn67, Thr199, Thr200) regions and provides the accessibility to the solvent (Krishnamurthy et al., 2008;Eriksson, Jones & Liljas, 1988;Pocker & Sarkanen, 1978). A high conservation of amino acids in the active site and surrounding faces has been found among the 12 catalytically active human CA isoforms (Aggarwal et al., 2013;Pinard et al., 2015). Thus, the design of CA isoform-selective inhibitors has been the challenging goal for many researchers. In 1954, acetazolamide was approved in clinic as the first CA-targeting antiglaucoma drug (Supuran, 2012). In the next decades, a vast collection of CA inhibitors with various affinities and selectivities has been designed and has been extensively reviewed (Lomelino & McKenna, 2016;Supuran, 2016;Supuran, 2017;Alterio et al., 2012;Monti, Supuran & De Simone, 2013). It is a challenging task to design inhibitors that would be not only highly selective to CA IX, but also safe for use in humans for the treatment and diagnosis of hypoxic tumors. Many aspects need to be considered to achieve the final goal of developing the promising drugs, that could selectively inhibit CA IX in hypoxic tumors. The knowledge about the active site structure of the protein and permeability of the inhibitor across the cell membrane is essential for designing the CA IX specific inhibitors. An inhibitor may be selective for CA IX, but it may need to be attached to a conjugate to make it impermeable through the membrane. Similarly, the potential inhibitors need to go through the physical and biochemical screening and various modifications to develop as CA IX isoform specific compounds. The most promising CA IX inhibitors have to be screened for safety and toxicity in vivo using Kazokait˙ e et al. (2017), PeerJ, DOI 10.7717/peerj.4068 3/31 animal models, such as zebrafish, before subjecting them to preclinical characterization. In addition to chemical compounds, CA IX-selective biological molecules, such as monoclonal antibodies (mAbs), are at various stages of preclinical and clinical trials as potential anticancer agents targeting CA IX in hypoxic tumors. In addition, the anticancer agents based on CA IX selective inhibitors can be conjugated with various probes for the diagnosis of hypoxic tumors. SURVEY METHODOLOGY A wide variety of chemical compounds have been described in the literature that target tumor-associated CA IX. In this review, we selectively describe only aromatic sulfonamides that have been demonstrated to bind and inhibit the catalytic domain of recombinant human CA IX by at least two experimental approaches, such as inhibition of enzymatic activity and biophysical assays including the fluorescent thermal shift assay (FTSA), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). We emphasize the use of non-mammalian animal models, such as zebrafish and Xenopus oocytes for the toxicity, affinity, and selectivity studies of CA IX targeting sulfonamides. Published in 2016–2017, these studies suggest possibilities that could help in the development of antitumor agents prior to preclinical characterization in mice models. For reviewing the information, we identified the articles containing information about different biological and chemical antitumor agents that target CA IX in hypoxic tumors. The literature search was performed using the relevant keywords in PubMed. For example, the antibody section was compiled with all available articles published since 1986 up to 2017, in which the use of antibodies for the detection of CA IX in patients was described. Publications were retained if they contained relevant information about the promising agents that target CA IX in humans and also during the development of these agents in human cell lines and mice models. Priority was given to the antitumor agents that have been developed either for the treatment or imaging of the tumors using novel strategies. The focus of this review is also to present recent developments in the treatment and diagnosis of solid tumors under hypoxic conditions that express CA IX. We present the recent achievements on the 8 diagnostic tools including chemical and biological antitumor agents targeting CA IX that are at various stages of preclinical and clinical trials for treating the hypoxic tumors. This review combines the information about animal models, enzymatic, biophysical methods used in CA field, as summarized in Fig. 1, with the latest references of novel anticancer agents that are currently applied to target CA IX for the diagnosis and treatment. CA INHIBITOR ASSAYS CA enzymatic activity inhibition assay To evaluate the potency of CA-targeting inhibitor, the stopped-flow CO2hydration assay (SFA) has been widely applied for more than five decades since the discovery of the method to measure CA catalyzed CO2hydration rate by Gibbsons and Edsall and by Khalifah (Gibbons & Edsall, 1963;Gibbons & Edsall, 1964;Khalifah, 1971). This approach Kazokait˙ e et al. (2017), PeerJ, DOI 10.7717/peerj.4068 4/31 Figure 1 Methods which might be applied for developing CA IX-targeting compounds before preclinical characterization in tumor cells and mice. Full-size DOI: 10.7717/peerj.4068/fig-1 is based on the monitoring of the changes in absorbance of pH sensitive indicator upon CA catalyzed CO2hydration reaction. The half-maximal inhibitory concentration, IC50, is determined by fitting the compound dose curve according to the Hill model or Morrison equation (Morrison, 1969). The inhibition constant, Ki, can be obtained from IC50 value by Cheng-Prusoff equation (Cheng & Prusoff, 1973). Supuran and co-authors have developed a large library of CA inhibitors by SFA and divided them into five groups according to CA inhibition mechanisms: (1) the zinc binders (sulfonamides and their isosteres, dithiocarbamates and their isosteres, hydroxamates, etc.) (Supuran, 2012;Alterio et al., 2012;Carta et al., 2013;Innocenti, Scozzafava & Supuran, 2010;Carta et al., 2012;Supuran, 2013);(2)compoundsthatanchortothezinc-coordinated water molecule/hydroxide ion (phenols, polyamines, sulfocoumarins, etc.) (Nocentini et al., 2016;Davis et al., 2014;Carta et al., 2010;Innocenti et al., 2008;Santos et al., 2007); (3) inhibitors which occlude the entrance to the CA active site (coumarins and their isosteres) (Nocentini et al., 2015;Bozdag et al., 2017;Tars et al., 2013); (4) compounds which bind out of the active site (carboxylic acid derivates) (D’Ambrosio et al., 2015); (5) inhibitors which bind in an unknown way (secondary/tertiary sulfonamides, imatinib, nilotinib, etc.) (Parkkila et al., 2009;Supuran, 2016;Métayer et al., 2013). Since these various compounds have been subject of numerous recent reviews, here we concentrate only on aromatic sulfonamides as CA inhibitors. Supuran’s group also measured the affinity of monoclonal antibodies to target CA isoforms using SFA (Dekaminaviči¯ ut˙ e et al., 2014). In addition to other previously synthesized compounds containing fluorine, our group has identified a series of fluorinated benzenesulfonamides as strong CA IX inhibitors by SFA and have shown a correlation between parameters obtained by enzymatic and biophysical assays (Dudutien˙ e et al., 2014). Importantly, CA isoforms share not only hydratase, but also esterase activity which was discovered in early 1960s (Tashian, Douglas & Yu, 1964). Both reactions occur in the same catalytic pocket suggesting similarities in their mechanisms. The method to determine Kazokait˙ e et al. (2017), PeerJ, DOI 10.7717/peerj.4068 5/31 esterase activity is a high-throughput colorimetric assay with various applications, such as screening chemical molecules or antibodies against CA isozymes (Akıncıoğlu et al., 2013; Uda et al., 2015). Biophysical assays of inhibitor binding to CAs Advantages and limitations of enzymatic inhibition versus biophysical assays of inhibitor binding have been assessed and are compared in our recent manuscript (Smirnovien˙ e, Smirnovas & Matulis, 2017). Biophysical methods not only determine the thermodynamic parameters of ligand binding to CAs, but also provide insight into numerous significant factors, which influence the binding: local water structure, hydrogen bonding, hydrophobic interactions, and desolvation. The thermodynamic profiles of drug candidate binding to CA have been widely used. Here we will focus on biophysical techniques, such as fluorescent thermal shift assay (FTSA), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR), which have been applied in the rational drug design of isoform-selective CA inhibitors. Isothermal titration calorimetry Since the invention of first analog of an isothermal titration calorimeter in 1966 (Izatt et al., 1966;Christensen et al., 1966) and its modifications for biological applications in 1980s (Ramsay, Prabhu & Freire, 1986;Schön & Freire, 1989), ITC has become the method of choice to study protein target-ligand interactions. During the experiment, in the current commercial titration calorimeters, the inhibitor solution from the syringe is injected at constanttemperature into the proteinsolution preloaded to thecalorimeter cell until allbinding sites of the protein become occupied by the ligand. Importantly, ITC does not require the inhibitor or protein to be labeled or immobilized and allows the determination of the affinity, the binding enthalpy and the stoichiometry in a single titration experiment (Klebe, 2015;Krimmer & Klebe, 2015;Geschwindner, Ulander & Johansson, 2015;Falconer, 2016). Numerous studies of interactions between diverse ligands and target CA isoforms have been performed by ITC (Krishnamurthy et al., 2008;DiTusa et al., 2001;Khalifah et al., 1993). The binding of anions to CA II was evaluated using ITC, X-ray crystallography, and molecular dynamics simulations by Whitesides group (Fox et al., 2015). For the deeper understanding of structure–activity relationships, the analysis of buffer ionization effects was performed by ITC upon an inhibitor binding to recombinant human CA isoforms, including CA I (Mork¯ unait˙ e et al., 2015), CA II (Mork¯ unait˙ e et al., 2015), CA VB (Kasiliauskait˙ e et al., 2015), CA VI (Kazokait˙ e et al., 2015), CA VII (Pilipuityt˙ e & Matulis, 2015), CA IX (Linkuvien˙ e et al., 2016), CA XII (Jogait˙ e et al., 2013), and CA XIII (Baranauskien˙ e & Matulis, 2012). In addition, ITC standard and displacement titrations were combined with the X-ray crystallographic structures to determine the intrinsic, buffer-independent affinity of para substituted tetrafluorobenzenesulfonamides binding to several human CA isoforms (Zubrien˙ e et al., 2015). Fluorescent thermal shift assay FTSA, also called differential scanning fluorimetry and, in high-throughput format, ThermoFluor R , has been widely applied by numerous researchers and companies, such as Kazokait˙ e et al. (2017), PeerJ, DOI 10.7717/peerj.4068 6/31 Johnson & Johnson, New Brunswick, United States. It is a rapid screening method in the drug discovery to measure the binding affinities of chemical compounds to targets (Kranz & Schalk-Hihi, 2011;Lo et al., 2004;Pantoliano et al., 2001;Niesen, Berglund & Vedadi, 2007). FTSA monitors the equilibrium of a protein between its folded and unfolded states by detecting the fluorescence of solvatochromic probes, such as 1,8-anilinonaphthalene sulfonate or SYPRO R orange, while the temperature is steadily increased. This method determines the protein melting temperature which can be highly affected by the affinity of ligand and its concentration (Cimmperman & Matulis, 2011;Cimmperman et al., 2008). In addition, FTSA is a convenient technique to characterize protein thermal stabilities at various conditions including diverse buffers, excipients, etc (Mezzasalma et al., 2007; Cummings, Farnum & Nelen, 2006). FTSA has been widely applied in the search of CA inhibitors. The binding of sulfamate and sulfamide derivatives to human CA II was investigated using FTSA by Klinger et al. (2006). FTSA was also applied by our group to investigate the interactions between human CA isoforms and various series of inhibitors, including triand tetrafluorobenzenesulfonamides (Dudutien˙ e et al., 2013;Dudutien˙ e et al., 2015), benzenesulfonamide derivatives with pyrimidine moieties (Čapkauskait˙ e et al., 2013), saccharin sulfonamides (Mork¯ unait˙ e et al., 2014), benzenesulfonamides with benzimidazole moieties (Zubrien˙ e et al., 2014), 4-amino-substituted benzenesulfonamides (Rutkauskas et al., 2014). In addition, the profiles of thermal stabilities of recombinant human CA VB (Kasiliauskait˙ e et al., 2015), CA VI (Kazokait˙ e et al., 2015), CA IX (Linkuvien˙ e et al., 2016), and CA XII (Jogait˙ e et al., 2013) was described using FTSA. Surface plasmon resonance SPR was first demonstrated for the monitoring of biomolecular interactions by Lundstrom et al. in 1983 (Liedberg, Nylander & Lunström, 1983) and the first commercial SPR instrument was launched by Pharmacia Biosensors AB in 1991 (Jönsson et al., 1991). During the last decades, SPR biosensors have become the state-of-the-art technology in diagnostics and biomedical research to determine a real-time kinetics and binding affinities of ligand-protein interactions. To screen lead compounds, one of the binding partners, usually the target protein, is immobilized on a metal surface and the ligand flows over that surface by microfluidic system. SPR is a label-free optical method, which measures the changes in refractive index at the metal surface upon the binding reaction. Studies of SPR application in CA research used recombinant human CA I (Jecklin et al., 2009) or mostly CA II (Myszka, 2004;Navratilova & Hopkins, 2010;Papalia et al., 2006) isoform as a model for the screening of numerous inhibitors. In contrast, Talibov et al. immobilized six human recombinant CA isoforms (full-length CA I, CA II, CA VII, CA XIII, catalytic domains of CA IX and CA XII) and analyzed their interactions with 17 benzenesulfonamide ligands by SPR. Interestingly, results revealed one compound from investigated series to be as a tight binder to recombinant CA IX with the dissociation rates too slow to be determined by SPR (Talibov et al., 2016). Kazokait˙ e et al. (2017), PeerJ, DOI 10.7717/peerj.4068 7/31 Zebrafish model for compound toxicity Phenotype-based screening using zebrafish has become a promising high-throughput assay for the drug discovery. This approach revealed that 62%, of drugs approved from 1999 till 2008, were discovered by phenotype-based screens despite that they represented only a small fraction of all screens (MacRae & Peterson, 2015). Phenotypic screens possess many significant advantages over target-based screens including the identification of drugs withoutavalidatedtargetorthecharacterizationofthetherapeuticprofileofthecompound, which affects several targets simultaneously. Zebrafish has emerged as a powerful model system for phenotypic screens of drug-candidates in vivo because of many advantages that include high homology between zebrafish and mammalian CAs, low cost, and avoidance of most ethical issues associated with the use of other animals. However, zebrafish lack lung, prostate, and mammary glands, heart septation, limbs, and it is necessary to grow zebrafish at 30 ◦C, while compounds against mammalian targets are usually optimized for 37 ◦C (Lin, Chiang & Tsai, 2016;Rennekamp & Peterson, 2015). Zebrafish can be particularly useful to carry out toxicological studies of CA inhibitors. Toxic effects of two fluorinated benzenesulfonamides as CA IX inhibitors were investigated on zebrafish development (Kazokait˙ e et al., 2016b). LC50 values showed that one compound exhibited 10-fold lower toxicity than ethoxzolamide (EZA), a compound used as a drug in humans. In addition, light-field microscopy and histological analysis revealed that EZA induced side effects such as pericardial edema, unutilized yolk sac and abnormal body shape of zebrafish. In contrast, developmental abnormalities were not detected in embryos treated with the fluorinated benzenesulfonamides (Table 1). Thus, this study showed that CA IX inhibitors did not have adverse effects on phenotype and morphology of zebrafish larvae. Such toxicological screenings of the compounds using zebrafish could provide information on the safety of lead molecule that could be useful for further development into a drug. Oocyte system for heterologous expression of CAs to determine compound affinity and selectivity Since 1960s, the Xenopus laevis has been widely used as a convenient animal model in various biomedical fields including molecular and physiological research. The Xenopus oocytes have many advantages including a large number of offspring, easy manipulations because of their big size (1.1–1.3 mm) and easy maintenance. Furthermore, oocytes feature highly efficient translation of heterologous RNA into protein. Native Xenopus oocytes do not possess any CA activity and thus have become a convenient in vivo model system to investigate CA inhibitors. The enzymatic activity of CA can be evaluated with microelectrodes while monitoring the intracellular and extracellular acidification. Results can be confirmed by mass spectrometric gas analysis of lysed or intact oocytes (Becker, 2014). The transfection of Xenopus oocytes with cRNA of CA isozymes has been published by Deitmer’s group (Klier et al., 2016;Schneider et al., 2013). They showed the complete inhibition of CA IX enzymatic activity with 30 µM EZA according to the rates of cytosolic pH changes and amplitudes of pH changes at the outer membrane side (Klier et al., 2016). The same effect was found in CA IX expressing Kazokait˙ e et al. (2017), PeerJ, DOI 10.7717/peerj.4068 8/31 Table 1 Biological model systems for the investigation of CA IX inhibitors. The compounds did not show any significant toxicity on zebrafish and possessed nanomolar IC50 for heterologous CA IX expressed in Xenopus oocytes. In addition, the selectivity of compounds toward CA isoforms was evaluated according to the effect of compounds on the reduction of extracellular (CA IX, CA IV, and CA XII) and intracellular (CA II) CA-induced acidification in oocytes (Kazokait˙ e et al., 2016a;Kazokait˙ e et al., 2016b). Table 1. Biological model systems for the investigation of CA IX inhibitors. The compounds did not show any significant toxicity on zebrafish and possessed nanomolar IC50 for heterologous CA IX expressed in Xenopus oocytes. In addition, the selectivity of compounds toward CA isoforms was evaluated according to the effect of compounds on the reduction of extracellular (CA IX, CA IV, and CA XII) and intracellular (CA II) CA-induced acidification in oocytes [119, 123]. Inhibitor Type of study VD11-4-2 VD12-09 Toxicolo gy LC 50 = 120 µM LC 50 = 13 µM Methods: 1. light-field microscopy 2. histological analysis Affinit y and selectivit y CA IX: IC 50 = 25 nM CA II: <5.0% effect on pH at 10 µM CA IV: 57.8% effect on pH at 10 µM CA XII: 28.0% effect on pH at 50 nM CA IX: 25.5% effect on pH at 10 µM CA II: <5.0% effect on pH at 10 µM Methods: 1. pH monitoring with microelectrodes 2. mass spectrometric gas analysis IC50 - the concentration causing 50% inhibition of target activity, LC50 - 50% lethal concentration. SO O NH2 F F SO2 OH N H F SO O NH2 F FF S N H OH oocytes treated with 1 µM fluorinated benzenesulfonamide targeting CA IX (Kazokait˙ e et al., 2016a). The IC50 was found to be in the range of 15–25 nM for both intracellularly and extracellularly expressed CA IX. Moreover, the compound exhibited strong selectivity over CA II, CA IV or CA XII in oocytes expressing a particular CA isoform (Table 1). This novel in vivo approach allows the identification of the affinity and selectivity of CA IX inhibitors in the living eukaryotic cell with fully matured target CA isozyme. CA IX-TARGETED STRATEGIES Targeting CA IX enzyme is a promising approach for the development of new therapeutics against hypoxic tumors. There are several agents that can selectively target CA IX by using different strategies. Here, we present therapeutic agents that have been used against CA IX for diagnosis and treatment of hypoxic tumors in humans (Table 2). Monoclonal antibodies for CA IX-targeted therapy M75 and chimeric G250 (cG250) are two widely-applied monoclonal antibodies (mAbs) recognizing human CA IX. These mAbs have been used for clinical detection or therapy (Oosterwijk et al., 1986;Závada et al., 1993). The M75 targets the PG-domain of CA IX and is used for the detection of CA IX in human tissues (Chrastina, Pastoreková & Pastorek, 2003;Chrastina et al., 2003;Zatovicova et al., 2010). cG250 has been successfully developed Kazokait˙ e et al. (2017), PeerJ, DOI 10.7717/peerj.4068 9/31 Author Contributions •Justina Kazokait˙ e and Ashok Aspatwar conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. •Seppo Parkkila and Daumantas Matulis conceived and designed the experiments, analyzed the data, wrote the paper, reviewed drafts of the paper. Data Availability The following information was supplied regarding data availability: This is a review article that did not generate any additional previously unpublished raw data. REFERENCES Aggarwal M, Boone CD, Kondeti B, McKenna R. 2013. Structural annotation of human carbonic anhydrases. 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