Carbonic Anhydrase IX Inhibitors Research Articles

Abstract 3780: On the role of experimental microenvironmental conditions in targeted inhibition of the pH-regulating carbonic anhydrase IX in colorectal carcinoma cells

Abstract Tumor hypoxia, causing metabolic changes towards enhanced glycolysis and lactic acid production, is a common microenvironmental feature of solid tumors, and is recognized as a main mechanism both of therapeutic resistance and metastasis. The hypoxia-inducible factor-1α conducts transcription of a number of genes involved in adaption to the hypoxic condition. Among these is the tumor-specific carbonic anhydrase IX (CAIX), a membrane-bound protein that regulates extracellular (pHe) and intracellular (pHi) pH by catalyzing the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3-) and protons, enabling tumor cell survival in the acidic microenvironment. To improve therapeutic outcome and reduce metastatic risk, targeted inhibition of CAIX is an attractive strategy. Here we report on effects of a CAIX inhibitor in a panel of colorectal carcinoma (CRC) cell lines, giving specific emphasis to the microenvironmental conditions. Five CRC cell lines were incubated for 24 hours under normoxic (21% O2) or hypoxic (0.2% O2) condition, either in the presence (22 mM HCO3-, 5% CO2) or absence (0 mM HCO3-, 0% CO2) of HCO3- in the culture medium. The cells were treated with 4-(3′-(3”,5”-dimethylphenyl)ureido)phenyl sulfamate (termed S4), considered to be a CAIX-specific inhibitor, and clonogenicity, pHe, and pHi (measured as cells labeled with a pH-sensitive dye) were assessed. In CO2/HCO3--buffered medium under normoxia, differential inhibition of clonogenicity following S4 treatment was observed among the five cell lines, correlating with individual CAIX levels: strong suppression in cell lines with high CAIX expression, weaker S4 effect in cell lines with low expression, and no effect in the CAIX-negative cell line. When incubating with this medium under hypoxia, despite significant induction of CAIX, no additional effect of S4 on clonogenicity was observed. Of note, when changing the cell culture medium to CO2/HCO3--free condition, S4 strongly inhibited clonogenicity of hypoxic HT29 cells (high CAIX expression) but not of the normoxic counterpart, while HCT116 cells (low CAIX expression) showed no clonogenic response to S4 with either oxygenation status. In all of the five cell lines, hypoxia caused significant acidification (decrease in pHe) of culture media. In parallel with the clonogenic response, S4 treatment significantly counteracted the hypoxia-induced pHe decline in HT29 cultures incubated in the CO2/HCO3--free condition. pHi was not altered with any of the tested conditions. This set of data demonstrated that the CO2/HCO3- buffering system determined CRC cell response to targeted CAIX inhibition. To enable reliable assessment of experimental S4 effects, strict control of the microenvironmental conditions was required. We believe this information may be of note for future evaluation of this specific therapeutic approach. Citation Format: Helga Helseth Hektoen, Kjersti Flatmark, Kirsti Solberg Landsverk, Marie Gron Saelen, Kathrine Roe, Anne Hansen Ree. On the role of experimental microenvironmental conditions in targeted inhibition of the pH-regulating carbonic anhydrase IX in colorectal carcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3780. doi:10.1158/1538-7445.AM2014-3780

Receptor Chemoprint Derived Pharmacophore Model for Development of CAIX Inhibitors

Background: Carbonic anhydrase IX (CAIX) is an attractive target for anticancer therapy because it is selectively overexpressed in tumor cells. Various CAs’ inhibitors (sulfonamides/sulfaumates and coumarins) are reported as promising anti-cancer agents, showed appreciable affinity and selectivity. Novel chemical scaffolds with improved pharmacological properties are essential for the development of safe and potent CAIX inhibitors. Materials and methods: Crystal structure of CAIX with its inhibitors revealed critical residues of CAIX that interacts to inhibitor(s). These information was used to design a receptor-chemoprint based pharmacophore model. In silico pharmacokinetic assays were carried for novel hits with both ADMET and TOPKAT tools of Discovery Studio 3.5. Results: Pharmacophore model consists of one hydrogen bond donor, three hydrogen bond acceptors, and two hydrophobic moieties which are defined as essential feature for CAIX inhibitors. Virtual screening of ZINC chemical databases leads to identification of 1242 hits, having pharmacophore fit score ≥ 0.95. These hits were subsequently subjected to molecular docking analysis but they are limited to 321 only. Conclusion: Based on consensus scoring values, critical interactions with active site residues, and predicted activity values, five compounds (ZINC03363328, ZINC08828920, ZINC12941947, ZINC03622539 and ZINC16650541) are proposed as possible lead for CAIX inhibitor. Present study suggests that a conceptual adjustment of these hits may lead to rational design of novel and potent CAIX inhibitor.

Abstract 5065: Orally active nontoxic dual MMP2 and CAIX inhibitors as anticancer therapy.

Abstract Carbonic anhydrases IX and XII, (CAIX and CAXII) are critical to maintain a low pH in the tumor cell's microenvironment, and MMP-2 is critical for breaching the basement membrane that opens the way to the dissemination of metastases. The expression of carbonic anhydrase IX (CAIX), a marker for hypoxic tumors, is correlated with poor prognosis. In this presentation, we wish to report that carbamoylphosphonates (CPOs) have been identified as orally bioavailable dual inhibitors of matrix metalloproteinase-2 (MMP-2) on the one hand, and carbonic anhydrases (CAs) on the other hand, especially the IX and XII isoforms, well known as cancer promoting factors. We have also demonstrated that the ionized CPO acids are unable to cross the cell membrane and thus are limited to interact with enzymes present in the extracellular domains of as are both CA isozymes CAIX and CAXII and MMP-2. Finally, exposing cancer cells to CPOs in hypoxic conditions resulted in the dose dependent release of lactate dehydrogenase, confirming the direct interaction of the CPOs with the cancer related isozymes CAIX and XII and thereby promoting cellular damage. Thus, CPOs can be regarded as novel orally active nontoxic drug candidates for CAIX expressing tumor targeted chemotherapy acting by two synergistic mechanisms, namely, inhibiting CAs and MMPs simultaneously. Citation Format: Eli Breuer. Orally active nontoxic dual MMP2 and CAIX inhibitors as anticancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5065. doi:10.1158/1538-7445.AM2013-5065

Synthesis and SAR of Novel Re/99mTc-Labeled Benzenesulfonamide Carbonic Anhydrase IX Inhibitors for Molecular Imaging of Tumor Hypoxia

Carbonic anhydrase IX (CA-IX) is upregulated in cancer in response to the hypoxic tumor microenvironment, making it an attractive molecular target for the detection of hypoxic solid tumors. A series of small molecule benzenesulfonamide based CA-IX inhibitors containing novel tridentate chelates complexed with the M(CO)(3) core (M = Re or (99m)Tc) were designed and synthesized. The in vitro binding affinity of the benzenesulfonamide rhenium complexes yielded IC(50) values ranging from 3 to 116 nM in hypoxic CA-IX expressing HeLa cells. One of the most potent compounds, 3d (IC(50) = 9 nM), was radiolabeled with technetium tricarbonyl ({(99m)Tc(CO)(3)}(+)) to afford the {(99m)Tc(CO)(3)}(+) complex in excellent yield and high purity. (99m)Tc(CO)(3)-3d bound specifically to CA-IX expressing hypoxic HeLa cells. This effort led to the identification of a diverse series of promising high affinity {(99m)Tc(CO)(3)}(+) radiolabeled CA-IX inhibitors with the potential to significantly impact diagnosis, staging, and treatment selection of hypoxic solid tumors.

Inhibition of hypoxia-inducible carbonic anhydrase-IX enhances hexokinase II inhibitor-induced hepatocellular carcinoma cell apoptosis

The hypoxic condition within large or infiltrative hypovascular tumors produces intracellular acidification, which could activate many signaling pathways and augment cancer cell growth and invasion. Carbonic anhydrase-IX (CA-IX) is an enzyme lowering pH. This study is to examine whether hypoxia induces CA-IX in hepatocellular carcinoma (HCC) cells, and to evaluate its clinical implication in HCC patients. Human HCC cell lines (Huh-7 and HepG2 cells) were used, and cell growth was assessed using MTS assay. CA-IX expression and apoptotic/kinase signaling were evaluated using immunoblotting. The cells were transfected with CA-IX-specific siRNA, or treated with its inhibitor 4-(2-aminoethyl) benzenesulfonamide (CAI#1), and/or the hexokinase II inhibitor, 3-bromopyruvate (3-BP). A clinic pathological analysis of 69 patients who underwent an HCC resection was performed using a tissue array. Incubation of HCC cells under hypoxia (1% O₂, 5% CO₂, 94% N₂) for 36 h significantly increased CA-IX expression level. CAI#1 (400 μmol/L) or CA-IX siRNA (100 μmol/L) did not influence HCC cell growth and induce apoptosis. However, CAI#1 or CA-IX siRNA at these concentrations enhanced the apoptosis induced by 3-BP (100 μmol/L). This enhancement was attributed to increased ER stress and JNK activation, as compared with 3-BP alone. Furthermore, a clinic pathological analysis of 69 HCC patients revealed that tumor CA-IX intensity was inversely related to E-cadherin intensity. Inhibition of hypoxia-induced CA-IX enhances hexokinase II inhibitor-induced HCC apoptosis. Furthermore, CA-IX expression profiles may have prognostic implications in HCC patients. Thus, the inhibition of CA-IX, in combination with a hexokinase II inhibitor, may be therapeutically useful in patients with HCCs that are aggressively growing in a hypoxic environment.

Carbonic Anhydrase IX Overexpression is Associated with Diminished Prognosis in Esophageal Cancer and Correlates with Her-2 Expression

Carbonic anhydrase IX (CAIX), a transmembrane glycoprotein, seems to play a key role in the adaption of tumor cells to hypoxia. This study was designed to investigate the clinical role of CAIX and its association with Her-2 in a large cohort of adeno- (AC) and squamous cell carcinomas (SCC) of the esophagus and their metastases. Expression of CAIX and Her-2 was investigated immunohistochemically in formalin fixed, paraffin-embedded tissue from 330 esophageal cancers (182 ACS, 148 SCCs). Corresponding lymph node metastases in 137 cases, distant metastases in 34 cases, and local recurrences in 14 cases were analyzed for CAIX expression. A total of 147 cases (44.5%) showed strong CAIX expression (AC: 46.7%; ACC: 41.9%). CAIX status of the primary tumor influenced CAIX expression in corresponding lymph node metastases (P<0.001, linear regression). High CAIX-expression was an independent prognostic factor for shorter overall and disease-free survival (P≤0.05, Cox regression). Twenty-nine ACs (15.9%) and 6 SCCs (4.1%) showed Her-2 overexpression. In AC, a significant positive correlation between the Her-2 status and CAIX expression was found (P=0.009, chi-square test). High CAIX expression is associated with shorter survival in esophageal cancer, and the hypoxic phenotype seems to be preserved at least during formation of lymph node metastases. Inhibition of CAIX might reduce the ability of tumor cells to establish disseminated disease. In Her-2 overexpressing ACs, blocking of this tyrosine kinase, e.g., by monoclonal antibodies, might induce this effect.

Expression and Activity of Carbonic Anhydrase IX Is Associated With Metabolic Dysfunction in MDA-MB-231 Breast Cancer Cells

The expression of carbonic anhydrase IX (CAIX), a marker for hypoxic tumors, is correlated with poor prognosis in breast cancer patients. We show herein that the MDA-MB-231 cells, a “triple-negative,” basal B line, express exclusively CAIX, while a luminal cell line (T47D) expresses carbonic anhydrase XII (CAXII). CAIX expression in the basal B cells is both density- and hypoxia-dependent and is correlated with carbonic anhydrase activity. Evidence is provided that CAIX contributes to extracellular acidification through studies on pH, lactic acid production, and CAIX inhibition. Together, these studies suggest that CAIX expression and activity is associated with metabolic dysfunction in MDA-MB-231 cells.

ROLE OF CARBONIC ANHYDRASE EXPRESSION IN HEPATOCYTE PROTECTION BY LATE PRECONDITIONING

Liver ischemic preconditioning improves reperfusion damage during hepatic surgery and transplantation. However, the mechanisms involved are still poorly understood. This study investigated the role of carbonic anhydrase IX (CAIX), a transmembrane enzyme that catalyzes bicarbonate production, in the protective effects associated with the late phase of liver preconditioning.Late preconditioning was induced in primary cultured rat hepatocytes by a transient (10 min) hypoxic stress or by 15 min incubation with the adenosine A2A receptors agonist CGS21680 (5μ mol/L) 24 h before the exposure to 90 min hypoxia.Late preconditioning prevented hepatocytes killing and ameliorated intracellular acidosis and Na+ accumulation during hypoxia. Such effects were associated with the expression of CAIX. Blocking the transduction of preconditioning signals by PKC and PI3‐kinase with chelerythrine or wortmannin abolished CAIX expression and the protective action of late preconditioning. The inhibition of CAIX with acetazolamide or the block of bicarbonate influx through the Cl−/HCO−3 exchanger with SITS also reverted the protective effects of late preconditioning on intracellular acidosis and Na+ accumulation.We conclude that the induction of late preconditioning in liver cells involve the expression of CAIX that increases hepatocyte tolerance to ischemia by maintaining intracellular Na+ homeostasis.

Adenosine‐dependent activation of hypoxia‐inducible factor‐1 induces late preconditioning in liver cells†

The cellular mechanisms by which ischemic preconditioning increases liver tolerance to ischemia/reperfusion injury are still poorly understood. This study investigated the role of the hypoxia-inducible factor-1 (HIF-1) in the protection associated with the late phase of liver preconditioning. Late preconditioning was induced in primary cultured rat hepatocytes by a transient (10 minute) hypoxic stress or by 15 minutes incubation with the adenosine A(2A) receptors agonist CGS21680 24 hours before exposure to 90 minutes of hypoxia in a serum-free medium. Late preconditioning induced the nuclear translocation of HIF-1 and the expression of carbonic anhydrase IX (CAIX), a HIF-1-regulated transmembrane enzyme that catalyzes bicarbonate production. Such effects were associated with prevention of hepatocyte killing by hypoxia and the amelioration of intracellular acidosis and Na+ accumulation. The inhibition of PKC-mediated and PI3-kinase-mediated signals with, respectively, chelerythrine and wortmannin abolished HIF-1 activation and blocked both CAIX expression and the protective action of late preconditioning. CAIX expression was also prevented by interfering with the transcriptional activity of HIF-1 using a dominant negative HIF-1beta subunit. The inhibition of CAIX with acetazolamide or the block of bicarbonate influx with disodium-4-acetamido-4'-isothiocyanato-stilben-2,2'-disulfonate also reverted the protective effects of late preconditioning on intracellular acidosis and Na+ accumulation. The stimulation of adenosine A(2A) receptors induced late preconditioning in liver cells through the activation of HIF-1. HIF-1-induced expression of CAIX increases hepatocyte tolerance to ischemia by maintaining intracellular Na+ homeostasis. These observations along with the importance of HIF-1 in regulating cell survival indicates HIF-1 activation as a possible key event in liver protection by late preconditioning.