Carbonic Anhydrase IX Inhibitors Research Articles

Synthesis and biological investigation of new carbonic anhydrase IX (CAIX) inhibitors

In this report, we describe the synthesis, characterization, in vitro anticancer activity and Carbonic anhydrase IX (CAIX) inhibition of new sulfamate conjugates of Betulin and Betulinic acid (BA). The betulinyl sulfamates were subjected to inhibit carbonic anhydrases (CA), e.g. CAIX, an attractive target for tumor-selective therapy strategies in cancer cells. Data on combined in vitro antitumor activity with CAIX inhibition are very rare. The betulinyl sulfamates were tested against five tumor cell lines and normal human skin fibroblasts. The mode of cell death on MCF7 breast cancer cells induced by the most active compounds CAI1, CAI3 and CAI6 was investigated by Fluorescence Activated Cell Sorting (FACS) experiments. The compounds showed inhibitory activity towards CAIX, which was determined via in vitro enz-yme assay. Our preliminary investigations revealed that all compounds showed potent anticancer properties with IC50 values below 20 μM against all tumor cells. Interestingly, among the panel of sulfamate conjugates, CAI3 found to be highly cytotoxic (average IC50 = 5–10 μM) and possess high inhibitory activity (Ki = 1.25 nM) towards CAIX. Our results suggest that betulinyl sulfamates seem to be attractive substances, due to their possibility of targeted drug delivery they deserve to be proceeded for further pre-clinical (kinetic studies) and in vivo investigations.

Carbonic Anhydrase IX (CAIX), Cancer, and Radiation Responsiveness.

Carbonic anhydrase IX has been under intensive investigation as a therapeutic target in cancer. Studies demonstrate that this enzyme has a key role in pH regulation in cancer cells, allowing these cells to adapt to the adverse conditions of the tumour microenviroment. Novel CAIX inhibitors have shown efficacy in both in vitro and in vivo pre-clinical cancer models, adversely affecting cell viability, tumour formation, migration, invasion, and metastatic growth when used alone. In co-treatments, CAIX inhibitors may enhance the effects of anti-angiogenic drugs or chemotherapy agents. Research suggests that these inhibitors may also increase the response of tumours to radiotherapy. Although many of the anti-tumour effects of CAIX inhibition may be dependent on its role in pH regulation, recent work has shown that CAIX interacts with several of the signalling pathways involved in the cellular response to radiation, suggesting that pH-independent mechanisms may also be an important basis of its role in tumour progression. Here, we discuss these pH-independent interactions in the context of the ability of CAIX to modulate the responsiveness of cancer to radiation.

CAIX furthers tumour progression in the hypoxic tumour microenvironment of esophageal carcinoma and is a possible therapeutic target

The hypoxic tumour microenvironment of solid tumours represents an important starting point for modulating progression and metastatic spread. Carbonic anhydrase IX (CAIX) is a known HIF-1α-dependent key player in maintaining cell pH conditions under hypoxia. We show that CAIX is strongly expressed in esophageal carcinoma tissues. We hypothesize that a moderate CAIX expression facilitates metastases and thereby worsens prognosis. Selective inhibition of CAIX by specific CAIX inhibitors and a CAIX knockdown effectively inhibit proliferation and migration in vitro. In the orthotopic esophageal carcinoma model, the humanized HER2 antibody trastuzumab down-regulates CAIX, possibly through CAIX’s linkage with HER2 in the hypoxic microenvironment. Our results show CAIX to be an essential part of the tumour microenvironment and a possible master regulator of tumour progression. This makes CAIX a highly effective and feasible therapeutic target for selective cancer treatment.

Selective inhibition of carbonic anhydrase-IX by sulphonamide derivatives induces pH and reactive oxygen species-mediated apoptosis in cervical cancer HeLa cells

Selective inhibition with sulphonamides of carbonic anhydrase (CA) IX reduces cell proliferation and induces apoptosis in human cancer cells. The effect on CA IX expression of seven previously synthesised sulphonamide inhibitors, with high affinity for CA IX, as well as their effect on the proliferation/apoptosis of cancer/normal cell lines was investigated. Two normal and three human cancer cell lines were used. Treatment resulted in dose- and time-dependent inhibition of the growth of various cancer cell lines. One compound showed remarkably high toxicity towards CA IX-positive HeLa cells. The mechanisms of apoptosis induction were determined with Annexin-V and AO/EB staining, cleaved caspases (caspase-3, caspase-8, caspase-9) and cleaved PARP activation, reactive oxygen species production (ROS), mitochondrial membrane potential (MMP), intracellular pH (pHi), extracellular pH (pHe), lactate level and cell cycle analysis. The autophagy induction mechanisms were also investigated. The modulation of apoptotic and autophagic genes (Bax, Bcl-2, caspase-3, caspase-8, caspase-9, caspase-12, Beclin and LC3) was measured using real time PCR. The positive staining using γ-H2AX and AO/EB dye, showed increased cleaved caspase-3, caspase-8, caspase-9, increased ROS production, MMP and enhanced mRNA expression of apoptotic genes, suggesting that anticancer effects are also exerted through its apoptosis-inducing properties. Our results show that such sulphonamides might have the potential as new leads for detailed investigations against CA IX-positive cervical cancers.

Biological evaluation of p-toluene sulphonylhydrazone as carbonic anhydrase IX inhibitors: An approach to fight hypoxia-induced tumors

To find potential inhibitors of human carbonic anhydrase IX (CAIX), we have successfully deigned, synthesized and characterized three p-toluene sulphonylhydrazone derivatives (1–3). Molecular docking studies provided the structural basis of CAIX inhibition and a deeper insight into the protein-ligand interactions. p-Toluene sulphonylhydrazone derivatives show a well organized conformational compatibility with the active site of CAIX. The protein-ligand complex was stabilized by several non-covalent interactions offered by residues present in the active site cavity. The actual binding affinity of synthesized compounds with CAIX was experimentally measured by fluorescence and isothermal titration calorimetry (ITC).Results of both fluorescence binding and ITC measurements show the binding affinity of p-Toluene sulphonylhydrazone derivatives to the CAIX in the μM range. CAIX enzyme inhibition assay showed the IC50 values in nM range. Though all the three compounds (1–3) showed a good binding with CAIX, compound 2 showed the best inhibition of CAIX activity. These compounds were non-toxic on normal cell lines (HEK-293) and significantly inhibit the proliferation of hypoxic cancer cells. All compounds induce apoptosis in the hypoxic cancer cells. These compounds may be further exploited as promising therapeutic agents to control the hypoxia-induced tumors.

Abstract 2931: Targeting membrane-bound carbonic anhydrases in breast cancer to intervene in the metastatic phenotype

Abstract Breast cancer is the second leading cause of cancer related deaths among women in the United States. Despite the tremendous progress that has been made towards treating localized tumors, nearly 40,000 women die each year, predominantly from metastatic drug resistance. The tumor microenvironment plays a pivotal role in determining tumor growth, invasion, metastasis, and therapeutic success or failure. Therefore, a therapy targeting the tumor microenvironment is needed to sufficiently preserve the quality of life of cancer patients, by inhibiting metastasis. Elevated levels of Carbonic anhydrase IX (CAIX) expression in primary breast cancers is a marker for highly aggressive and metastatic tumors, especially of the triple negative subtype (TNBC). It is also associated with hypoxia, extracellular acidification, and poor prognosis. Low pH (values of ~6.5-6.8) is toxic to normal cells in the tumor microenvironment while enhancing cancer cell proliferation and tumor growth. Our goal was to compare the structure of a CAIX-mimic bound to ureidosulfonamide inhibitors with the biological activity of these inhibitors in triple negative and estrogen receptor positive (ER+) breast cancer cell lines. CAIX is a reversible enzyme and at low pH (high proton concentration), the enzyme will consume protons, raising pH. Our hypothesis is that CAIX inhibition, in the context of an acidic microenvironment, will dysregulate its ability to maintain the acidic pH preferred by cancer cells which favors their growth and migration. In this study, we have shown the interaction of sulfonamide-based inhibitors with a CAIX-mimic using X-ray crystallography. These structures show that the inhibitors make multiple contacts within the active site cavity. This is consistent with the inhibitor-induced decrease in CAIX activity and to some extent expression. We have also investigated the effect of CA inhibition on breast cancer cell growth, proliferation, activation of cell death pathways and migration. This reveals that, although CA inhibition with the sulfonamide-based compounds inhibits cell growth and migration, it does not activate apoptotic pathways. In total, these observations indicate that CAIX is a viable small molecular drug target for the treatment of metastatic breast cancer. Citation Format: Mam Y. Mboge, Zhijuan Chen, Brian P. Mahon, Chingkkuang Tu, Alyssa S. Wolff, John V. Mathias, Fabrizio Carta, Claudiu T. Supuran, Rob McKenna, Susan C. Frost. Targeting membrane-bound carbonic anhydrases in breast cancer to intervene in the metastatic phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2931. doi:10.1158/1538-7445.AM2017-2931

Activation of carbonic anhydrase IX by alternatively spliced tissue factor under late-stage tumor conditions

Molecules of the coagulation pathway predispose patients to cancer-associated thrombosis and also trigger intracellular signaling pathways that promote cancer progression. The primary transcript of tissue factor, the main physiologic trigger of blood clotting, can undergo alternative splicing yielding a secreted variant, termed asTF (alternatively spliced tissue factor). asTF is not required for normal hemostasis, but its expression levels positively correlate with advanced tumor stages in several cancers, including pancreatic adenocarcinoma. The asTF-overexpressing pancreatic ductal adenocarcinoma cell line Pt45.P1/asTF+ and its parent cell line Pt45.P1 were tested for growth and mobility under normoxic conditions that model early-stage tumors, and in the hypoxic environment of late-stage cancers. asTF overexpression in Pt45.P1 cells conveys increased proliferative ability. According to cell cycle analysis, the major fraction of Pt45.P1/asTF+ cells reside in the dividing G2/M phase of the cell cycle, whereas the parental Pt45.P1 cells are mostly confined to the quiescent G0/G1 phase. asTF overexpression is also associated with significantly higher mobility in cells plated under either normoxia or hypoxia. A hypoxic environment leads to upregulation of carbonic anhydrase IX (CAIX), which is more pronounced in Pt45.P1/asTF+ cells. Inhibition of CAIX by the compound U-104 significantly decreases cell growth and mobility of Pt45.P1/asTF+ cells in hypoxia, but not in normoxia. U-104 also reduces the growth of Pt45.P1/asTF+ orthotopic tumors in nude mice. CAIX is a novel downstream mediator of asTF in pancreatic cancer, particularly under hypoxic conditions that model late-stage tumor microenvironment.

Inhibition of carbonic anhydrase IX in glioblastoma multiforme

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme upregulated in several types of tumors including glioblastoma multiforme (GBM). GBM is among the most aggressive tumors among gliomas. Temozolomide (TMZ) therapy combined with surgical or radiation approaches is the standard treatment but not effective in long term. In this study we tested the treatment with acetazolamide (ATZ), an inhibitor of CAIX, alone or combined with TMZ. The experiments were performed in 2D and 3D cultures (spheroids) using glioblastoma U251N and human brain tumor stem cells (BTSCs). Several proteins implicated in tumor cell death were also investigated. The key results from these studies suggest the following: (1) Cell death of human glioblastoma spheroids and BTSC is significantly increased with combined treatment after 7 days, and (2) the effectiveness of ATZ is significantly enhanced against BTSC and U251N when incorporated into nano-carriers. Collectively, these results point toward the usefulness of nano-delivery of CAIX inhibitors and their combination with chemotherapeutics for glioblastoma treatment.

The Sulfamate Small Molecule CAIX Inhibitor S4 Modulates Doxorubicin Efficacy.

Carbonic anhydrase IX (CAIX) is a tumor-specific protein that is upregulated during hypoxic conditions where it is involved in maintaining the pH balance. CAIX causes extracellular acidification, thereby limiting the uptake of weak basic chemotherapeutic agents, such as doxorubicin, and decreasing its efficacy. The aim of this study was to determine if doxorubicin efficacy can be increased when combined with the selective sulfamate CAIX inhibitor S4. The effect of S4 on doxorubicin efficacy was tested in vitro using cell viability assays with MDA-MB-231, FaDu, HT29 –CAIX high and HT29 –CAIX low cell lines. In addition, the efficacy of this combination therapy was investigated in tumor xenografts of the same cell lines. The addition of S4 in vitro increased the efficacy of doxorubicin in the MDA-MB-231 during hypoxic exposure (IC50 is 0.25 versus 0.14 µM, p = 0.0003). Similar results were observed for HT29—CAIX high with S4 during normoxia (IC50 is 0.20 versus 0.08 µM, p<0.0001) and in the HT29 –CAIX low cells (IC50 is 0.09 µM, p<0.0001). In vivo doxorubicin treatment was only effective in the MDA-MB-231 xenografts, but the efficacy of doxorubicin was decreased when combined with S4. In conclusion, the efficacy of doxorubicin treatment can be increased when combined with the selective sulfamate CAIX inhibitor S4 in vitro in certain cell lines. Nevertheless, in xenografts S4 did not enhance doxorubicin efficacy in the FaDu and HT29 tumor models and decreased doxorubicin efficacy in the MDA-MB-231 tumor model. These results stress the importance of better understanding the role of CAIX inhibitors in intratumoral pH regulation before combining them with standard treatment modalities, such as doxorubicin.

Abstract B25: Characterization, targeting, and modulation of carbonic anhydrase IX activity for the development of small-molecule inhibitors to treat triple-negative breast cancer

Abstract The microenvironment within a solid tumor is usually heterogeneous with certain regions being acidic and hypoxic. These acidic and hypoxic regions arise from rapidly proliferating cells combined with poor tumor perfusion. Cancer cells cope with these hostile changes in their microenvironment by expressing genes that are essential for survival. One of the coping mechanisms is an upregulation of pH regulatory factors, including carbonic anhydrase IX (CAIX). The action of CAIX helps to maintain physiological pH inside the cell (pHi) while regulating extracellular acidification (pHe). Extracellular acidification of the tumor microenvironment promotes local invasion, metastasis and decreases the effectiveness of adjuvant therapies, thus contributing to poor clinical outcome. Our goal was to compare the structure of a CAIX-mimic bound to ureidosulfonamide inhibitors with the biological activity of these inhibitors in a triple negative breast cancer cell line. CAIX is a reversible enzyme and at low pH (high proton concentration), the enzyme will consume protons, raising pH. Our hypothesis is that CAIX inhibition, in the context of an acidic microenvironment, will dysregulate its ability to maintain the acidic pH preferred by cancer cells which favors their growth and migration. In this study, we have shown the interaction of sulfonamide-based inhibitors using X-ray crystallography methods. These structures show the inhibitors make multiple contacts within the active site cavity. This is consistent with the inhibitor-induced decrease in CAIX activity measured as 18O exchange between H2O16 and H13C18O3 . We have also investigated the effect CAIX inhibition on cancer cell metabolism and extracellular acidification using Seahorse technology. This reveals that CAIX may contribute to the “non-glycolytic” acidification process. In total, these observations indicate that CAIX is a viable small molecular drug target and contribute to our understanding of the function of CAIX in modulating pH in cancer cells. Citation Format: Mam Y. Mboge, Zhijuan Chen, Brian P. Mahon, Nicole Lamas, Shingkuang Tu, Fabrizio Carta, Claudiu T. Superan, Robert McKenna, Susan C. Frost. Characterization, targeting, and modulation of carbonic anhydrase IX activity for the development of small-molecule inhibitors to treat triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B25.

Abstract 754: Targeting carbonic anhydrase IX in multiple pancreatic ductal adenocarcinoma models results in tumor growth inhibition and increased survival

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by aggressive growth, metastatic spread and poor 5 year survival rates of less than 5%. PDAC tumors develop extensive regions of hypoxia that contribute to its aggressive behavior and pose a significant hurdle to therapeutic response. Hypoxia-mediated adaptive responses for tumor cell survival and metastasis include metabolic reprogramming and pH regulation. Carbonic anhydrase IX (CAIX) is a membrane-bound, hypoxia-inducible enzyme that is highly expressed in solid tumors and functions as a critical component of the pH regulatory machinery required by hypoxic cancer cells for survival and metastasis. We have recently developed and validated novel small molecule inhibitors of CAIX and have demonstrated that these inhibitors are effective at reducing breast tumor growth and metastasis. The lead compound, SLC-0111, is currently being assessed in a Phase 1 clinical trial (NCT02215850). In the present study, we have evaluated the efficacy of targeting CAIX in PDAC using genetic and pharmacologic strategies. CAIX expression was found to be induced by hypoxia in a panel of human PDAC cell lines and was also upregulated in cell line-derived xenografts, patient-derived xenografts as well as the KRASG12D/p53-/- KPCY transgenic mouse model of PDAC (Rhim, AD et al. Cell, 2012:148;349-361). shRNA-mediated silencing of CAIX expression in PK-8 PDAC cells significantly reduced hypoxia-mediated cell proliferation (P&amp;lt;0.05) and invasion (P&amp;lt;0.01) in vitro, and dramatically delayed tumor growth (P&amp;lt;0.001) in vivo, resulting in increased median survival of the mice from 36 days to 59 days (P&amp;lt;0.01), similar to that observed following treatment with gemcitabine (36 days to 58 days, P&amp;lt;0.01). Interestingly, gemcitabine treatment in this model increased the fraction of CAIX-positive tumor cells, and silencing of CAIX expression in combination with gemcitabine treatment further delayed tumor growth (P&amp;lt;0.01) and increased median survival to 86 days (P&amp;lt;0.01). Similarly, sequential administration of gemcitabine and the CAIX inhibitor, SLC-0111, significantly delayed tumor growth (P&amp;lt;0.01) and enhanced survival (P&amp;lt;0.01) in PK-8 and PK-1 xenografts, compared to gemcitabine alone. Preliminary data indicate that treatment with SLC-0111, alone and in combination with gemcitabine inhibits the growth of spontaneous PDAC tumors in the KPCY transgenic model of PDAC, as assessed by ultrasound imaging. These results demonstrate that CAIX plays an important role in PDAC progression and is a potential therapeutic target for pancreatic cancer. A focused Phase 1b clinical trial of gemcitabine and SLC-0111 in patients with metastatic PDAC is planned, based on the outcome of the Phase 1 trial. Citation Format: Paul C. McDonald, Shawn C. Chafe, Marylou Vallejo, David Schaeffer, Don Yapp, Claudiu T. Supuran, Ben Stanger, Mac Parmar, Daniel Renouf, Shoukat Dedhar. Targeting carbonic anhydrase IX in multiple pancreatic ductal adenocarcinoma models results in tumor growth inhibition and increased survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 754.

Overexpression of Carbonic Anhydrase IX is a Dismal Prognostic Marker in Breast Carcinoma in Egyptian Patients.

Carbonic anhydrase IX (CAIX) is an enzyme whose expression is very limited in normal tissues and it is highly expressed in various cancers. Therefore, inhibition of CAIX is considered as a promising therapeutic target for the treatment of solid tumors where hypoxic environment has developed. The aim of the current work is to evaluate the immunohistochemical (IHC) expression of CAIX in breast cancer (BC) of Egyptian patients and to investigate the associations of CAIX expression with the standard clinicopathologic features, IHC subtypes of BC, and overall survival. This retrospective study was conducted on 56 archival cases of Egyptian BC patients. Fifty-one of 56 cases (91.1%) showed positive expression of CAIX with cytoplasmic localization, whereas 5 cases (8.9%) showed negative expression. CAIX IHC overexpression is significantly associated with advanced stage and presence of coagulative tumor cell necrosis (P=0.03 and 0.02, respectively). Multivariate analysis revealed Ki67 labeling index and CAIX H-score grouping (P=0.03 and 0.02, respectively) as independent prognostic factors affecting BC patients' overall survival. We concluded that CAIX could play a role in the progression of the studied BC cases. CAIX is a good candidate for target therapy.

Targeting carbonic anhydrase IX improves the anti-cancer efficacy of mTOR inhibitors.

The inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) by chemical inhibitors, such as rapamycin, has demonstrated anti-cancer activity in preclinical and clinical trials. Their efficacy is, however, limited and tumors eventually relapse through resistance formation. In this study, using two different cancer mouse models, we identify tumor hypoxia as a novel mechanism of resistance of cancer cells against mTORC1 inhibitors. Indeed, we show that the activity of mTORC1 is mainly restricted to the non-hypoxic tumor compartment, as evidenced by a mutually exclusive staining pattern of the mTORC1 activity marker pS6 and the hypoxia marker pimonidazole. Consequently, whereas rapamycin reduces cancer cell proliferation in non-hypoxic regions, it has no effect in hypoxic areas, suggesting that cancer cells proliferate independently of mTORC1 under hypoxia. Targeting the hypoxic tumor compartment by knockdown of carbonic anhydrase IX (CAIX) using short hairpin RNA or by chemical inhibition of CAIX with acetazolamide potentiates the anti-cancer activity of rapamycin. Taken together, these data emphasize that hypoxia impairs the anti-cancer efficacy of rapalogs. Therapeutic strategies targeting the hypoxic tumor compartment, such as the inhibition of CAIX, potentiate the efficacy of rapamycin and warrant further clinical evaluation.

PET Imaging of Carbonic Anhydrase IX Expression of HT-29 Tumor Xenograft Mice with (68)Ga-Labeled Benzenesulfonamides.

Carbonic anhydrase IX (CA-IX) is a HIF-1-inducible enzyme that is overexpressed in many cancer subtypes to promote survival and invasion in hypoxic niches. Pharmacologic inhibition of CA-IX is achievable through sulfonamide-based inhibitors and has been shown to reduce primary growth of cancers and distant metastasis in preclinical models. We explored a multivalent approach for targeting CA-IX in vivo, noninvasively, with positron emission tomography. Three (68)Ga-polyaminocarboxylate chelator complex-conjugated tracers containing one, two, or three 4-(2-aminoethyl)benzenesulfonamide moieties were synthesized and evaluated for protein binding and imaging properties. Binding affinity to CA-I, -II, -IX, and -XII were determined using a stopped-flow CA catalyzed CO2 hydration assay. Biodistribution and PET/CT imaging were performed using immunocompromised mice bearing CA-IX expressing HT-29 colorectal tumors. Compounds demonstrated good binding affinity to CA-IX (Ki: 7.7-25.4 nM). (68)Ga-labeled sulfonamides were obtained in 64-91% decay-corrected average radiochemical yields with 50-536 GBq/μmol specific activity and >97% average radiochemical purity. All three tracers allowed for the visualization of tumor xenografts at 1 h postinjection, with the monomer displaying the highest contrast. Tumor uptake of the monomer was blockable in the presence of acetazolamide, confirming target specificity. The monomer was excreted predominantly through the kidneys, while the dimer and trimer were cleared by both renal and hepatobiliary pathways. According to biodistribution analysis, tumor uptake (%ID/g) of the monomeric, dimeric, and trimeric tracers were 0.81 ± 0.15, 1.93 ± 0.26, and 2.30 ± 0.53 at 1 h postinjection. This corresponded to tumor-to-muscle ratios of 5.02 ± 0.22, 4.07 ± 0.87, and 4.18 ± 0.84, respectively. Our data suggest that (68)Ga-polyaminocarboxylate chelator-conjugated sulfonamides can be used to noninvasively image CA-IX. These CA-IX targeting PET tracers may be used to identify patients who can benefit from treatments targeting this protein or serve as surrogate imaging agents for tumor hypoxia.

Design and synthesis of a novel class of carbonic anhydrase-IX inhibitor 1-(3-(phenyl/4-fluorophenyl)-7-imino-3H-[1,2,3]triazolo[4,5d]pyrimidin 6(7H)yl)urea

Carbonic anhydrase IX (CAIX) is a promising target in cancer therapy especially in the case of hypoxia-induced tumors. The selective inhibition of CA isozymes is a challenging task in drug design and discovery process. Here, we performed fluorescence-binding studies and inhibition assay combined with molecular docking and molecular dynamics (MD) simulation analyses to determine the binding affinity of two synthesized triazolo-pyrimidine urea derived (TPUI and TPUII) compounds with CAIX and CAII. Fluorescence binding results are showing that molecule TPUI has an excellent binding-affinity for CAIX (kD=0.048μM). The TPUII also exhibits an appreciable binding affinity (kD=7.52μM) for CAIX. TPUI selectively inhibits CAIX as compared to TPUII in the 4-NPA assay. Docking studies show that TPUI is spatially well-fitted in the active site cavity of CAIX, and is involve in H-bond interactions with His94, His96, His119, Thr199 and Thr200. MD simulation studies revealed that TPUI efficiently binds to CAIX and essential active site residual interaction is consistent during the entire simulation of 40ns. These studies suggest that TPUI appeared as novel class of CAIX inhibitor, and may be used as a lead molecule for the development of potent and selective CAIX inhibitor for the hypoxia-induced cancer therapy.

Sulfamate inhibitor S4 influences carbonic anhydrase IX ectodomain shedding in colorectal carcinoma cells

Carbonic anhydrase IX (CAIX) is a pivotal pH regulator under hypoxia, which by its tumor-specific expression represents an attractive target for cancer therapy. Here, we report on effects of the sulfamate CAIX inhibitor S4 (4-(3′-(3″,5″-dimethylphenyl)ureido)phenyl sulfamate) in colorectal carcinoma cell lines. S4 was administered under experimental hypoxia or normoxia to HT29, KM20L2 and HCT116 cells. Effects on survival, proliferation, pH, lactate extrusion and CAIX protein expression were evaluated. S4 treatment resulted in attenuated hypoxia-induced extracellular acidification and reduced clonogenic survival under hypoxia in HT29 cells. The pH effects were present only in a -free buffer system and were accompanied by decreased lactate extrusion. The main finding of this work was that S4 treatment caused alterations in CAIX ectodomain shedding. This merits further investigation to understand how sulfamates influence CAIX activity and how such drugs may be of use in cancer treatment.

Evaluation of carbonic anhydrase IX as a therapeutic target for inhibition of breast cancer invasion and metastasis using a series ofin vitrobreast cancer models

Triple negative, resistant or metastatic disease are major factors in breast cancer mortality, warranting novel approaches. Carbonic anhydrase IX (CAIX) is implicated in survival, migration and invasion of breast cancer cells and inhibition provides an innovative therapeutic strategy. The efficacy of 5 novel ureido-substituted sulfamate CAIX inhibitors were assessed in increasingly complex breast cancer models, including cell lines in normoxia and hypoxia, 3D spheroids and an ex-vivo explant model utilizing fresh biopsy tissue from different breast cancer subtypes. CAIX expression was evaluated in a tissue microarray (TMA) of 92 paired lymph node and primary breast cancers and 2 inhibitors were appraised in vivo using MDA-MB-231 xenografts. FC11409B, FC9398A, FC9403, FC9396A and S4 decreased cell proliferation and migration and inhibited 3D spheroid invasion. S4, FC9398A and FC9403A inhibited or prevented invasion into collagen. FC9403A significantly reversed established invasion whilst FC9398A and DTP348 reduced xenograft growth. TMA analysis showed increased CAIX expression in triple negative cancers. These data establish CAIX inhibition as a relevant therapeutic goal in breast cancer, targeting the migratory, invasive, and metastatic potential of this disease. The use of biopsy tissue suggests efficacy against breast cancer subtypes, and should provide a useful tool in drug testing against invasive cancers.

Is carbonic anhydrase IX a validated target for molecular imaging of cancer and hypoxia?

The presence of hypoxia is a general feature of most solid malignancies, and hypoxia is considered as one of major factors for anticancer therapy failure. Carbonic anhydrase IX (CAIX) has been reported to be an endogenous hypoxia marker, CAIX monoclonal antibodies, their segments and inhibitors are developed for CAIX imaging. However, growing evidence indicates that CAIX expression under hypoxia condition may be cancer cell lines or cancer-type dependent. Here we review the current literature on CAIX and discuss the advantage and limitation of CAIX as a target for tumor hypoxia imaging. Accordingly, CAIX would be unreliable as a universal target for cancer and tumor hypoxia visualization.

Carbonic anhydrase IX correlates with survival and is a potential therapeutic target for neuroblastoma

Carbonic anhydrase IX (CAIX) is involved in pathological processes including tumorgenicity, metastases and poor survival in solid tumors. Twenty-two neuroblastoma samples of patients who were surgically treated at the University Medical Center Hamburg-Eppendorf were evaluated immunohistochemically for expression of CAIX. Results were correlated with clinical parameters and outcome. Neuroblastoma Kelly and SH-EP-Tet-21/N cells were examined for CAIX expression and inhibited with specific inhibitors, FC5-207A and FC8-325A. 32% of neuroblastoma tumors expressed CAIX. This was significantly associated with poorer survival. Kelly and SH-EP-Tet-21/N cells showed a major increase of CAIX RNA under hypoxic conditions. Proliferation of Kelly cells was significantly decreased by CAIX inhibitors, FC5-207A and FC8-325A, while proliferation of SH-EP-Tet-21/N cells was only significantly affected by FC8-325A. CAIX is a potent biomarker that predicts survival in neuroblastoma patients. CAIX-targeted therapy in neuroblastoma cell lines is highly effective and strengthens the potential of CAIX as a clinical therapeutic target in a selected patient collective.

Prognostic relevance of carbonic anhydrase IX expression is distinct in various subtypes of breast cancer and its silencing suppresses self-renewal capacity of breast cancer cells.

Carbonic anhydrase IX (CAIX) is a hypoxia-inducible enzyme with extracellular catalytic domain that is overexpressed in a variety of cancers including breast cancer and plays a crucial role in maintaining favourable intracellular pH and reducing extracellular pH. The purpose of the current study was to elucidate the prognostic significance of CAIX in the intrinsic subtypes of breast cancer and to characterise CAIX as a drug target in breast cancer. The prognostic significance of CAIX mRNA expression was interrogated in a cohort of 3,455 breast tumours by using an online tool, Kaplan-Meier plotter. The functional effects of stable CAIX depletion by shRNA in three breast cancer cell lines—MDA-MB-231, MCF7 and SKBR-3, representing basal-like, luminal A and HER2+ subtypes, respectively—were studied by proliferation, invasion, clonal spheroid formation and chemosensitivity assays under normoxia and hypoxia. Finally, the effect of pharmacological CA inhibition alone or in the combination with doxorubicin on self-renewal was assessed by spheroid-forming assay. High CAIX mRNA expression was significantly associated with poor survival in patients with basal-like, luminal B and triple-negative breast cancer, but not luminal A and HER+ subtypes. Silencing of CAIX expression had no significant effect on the cell proliferation or viability upon treatment with doxorubicin in any of the cell lines studied, while it inhibited spheroid formation in hypoxic conditions. Furthermore, pharmacological inhibition of CAs using acetazolamide had a synergistic effect with doxorubicin on decreasing the spheroid-forming efficiency in MDA-MB-231 cells. Inhibition of CAIX reduces the self-renewal capacity of breast cancer cells, and the combination of doxorubicin and CAIX inhibition is an attractive therapeutic strategy in basal-like and triple-negative breast cancer, which warrants further investigations.