RESUMEN

BACKGROUND: Following resection and standard adjuvant radio- and chemotherapy, approved maintenance therapies for glioblastoma are lacking. Intracavitary radioimmunotherapy (iRIT) with 177Lu-labeled 6A10-Fab fragments targeting tumor-associated carbonic anhydrase XII and injected into the resection cavity offers a novel and promising strategy for improved tumor control. METHODS: Three glioblastoma patients underwent tumor resection followed by standard radio- and chemotherapy. These patients with stable disease following completion of standard therapy underwent iRIT on compassionate grounds. After surgical implantation of a subcutaneous injection reservoir with a catheter into the resection cavity, a leakage test with [99mTc]Tc-DTPA was performed to rule out leakage into other cerebral compartments. IRIT comprised three consecutive applications over three months for each patient, with 25%, 50%, 25% of the total activity injected. A dosimetry protocol was included with blood sampling and SPECT/CT of the abdomen to calculate doses for the bone marrow and kidneys as potential organs at risk. RESULTS: All three patients presented without relevant leakage after application of [99mTc]Tc-DTPA. Two patients underwent three full cycles of iRIT (592 MBq and 1228 MBq total activity). One patient showed histologically proven tumor progression after the second cycle (526 MBq total activity). No relevant therapy-associated toxicities or adverse events were observed. Dosimetry did not reveal absorbed doses above upper dose limits for organs at risk. CONCLUSIONS: In first individual cases, iRIT with [177Lu]Lu-6A10-Fab appears to be feasible and safe, without therapy-related side effects. A confirmatory multicenter phase-I-trial was recently opened and is currently recruiting.

RESUMEN

A series of 6-ureido/amidocoumarins (5a-p and 7a-c) has been designed and synthesised to develop potent and isoform- selective carbonic anhydrase hCA XI and XII inhibitors. All coumarin derivatives were investigated for their CA inhibitory effect against hCA I, II, IX, and XII. Interestingly, target coumarins potently inhibited both tumour-related isoforms hCA IX (KIs: 14.7-82.4 nM) and hCA XII (KIs: 5.9-95.1 nM), whereas the cytosolic off-target hCA I and II isoforms have not inhibited by all tested coumarins up to 100 µM. These findings granted the target coumarins an excellent selectivity profile towards both hCA IX and hCA XII isoforms, supporting their development as promising anticancer candidates. Moreover, all target molecules were evaluated for their anticancer activities against HCT-116 and MCF-7 cancer cells. The 3,5-bis-trifluoromethylphenyl ureidocoumarin 5i, exerted the best anticancer activity. Overall, ureidocoumarins, particularly compound 5i, could serve as a promising prototype for the development of potent anticancer CAIs.

Asunto(s)

Anhidrasas Carbónicas , Humanos , Anhidrasas Carbónicas/metabolismo , Relación Estructura-Actividad , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasa Carbónica IX , Antígenos de Neoplasias , Células MCF-7 , Cumarinas/farmacología , Estructura Molecular

RESUMEN

The overexpression and activity of carbonic anhydrase (CA, EC 4.2.1.1) isoforms CA IX and CA XII promote the accumulation of exceeding protons and acidosis in the extracellular tumor environment. Sulfonamides are effective inhibitors of most families of CAs. In this study, using scaffold-hopping, indoline-5-sulfonamide analogs 4a-u of the CA IX-selective inhibitor 3 were designed and synthesized to evaluate their biological properties. 1-Acylated indoline-5-sulfonamides demonstrated inhibitory activity against tumor-associated CA IX and XII with KI values up to 132.8 nM and 41.3 nM. Compound 4f, as one of the most potent inhibitors of CA IX and XII, exhibits hypoxic selectivity, suppressing the growth of MCF7 cells at 12.9 µM, and causes partial inhibition of hypoxia-induced CA IX expression in A431 skin cancer cells. 4e and 4f reverse chemoresistance to doxorubicin of K562/4 with overexpression of P-gp.

RESUMEN

Puerarin is a flavonoid molecule that widely exists in various plants. Puerarin has been reported to exhibit anti-tumor effects in various cancers. However, its exact underlying pharmacological mechanism is unclear. This study evaluated the anticancer effect of puerarin combined with oxaliplatin (OXA) in vitro and in vivo. Our results indicated that puerarin can reverse platinum-based anti-cancer drug resistance, and enhance the OXA's anticancer effects on breast cancer. Furthermore, puerarin can inhibit migration and reverse the epithelial-mesenchymal transition (EMT) induced by low-dose OXA. Further studies showed that the carbonic anhydrase (CA) XII is a potential target of puerarin. In conclusion, puerarin is expected to become an adjuvant chemotherapy drug and potentially become one of the medicated foods for breast cancer patients.

RESUMEN

Neuroblastoma is a rare disease. Rare are also the possibilities to test new therapeutic options for neuroblastoma in clinical trials. Despite the constant need to improve therapy and outcomes for patients with advanced neuroblastoma, clinical trials currently only allow for testing few substances in even fewer patients. This increases the need to improve and advance preclinical models for neuroblastoma to preselect favorable candidates for novel therapeutics. Here we propose the use of a new patient-derived 3D slice-culture perfusion-based 3D model in combination with rapid treatment evaluation using isothermal microcalorimetry exemplified with treatment with the novel carbonic anhydrase IX and XII (CAIX/CAXII) inhibitor SLC-0111. Patient samples showed a CAIX expression of 18% and a CAXII expression of 30%. Corresponding with their respective CAIX expression patterns, the viability of SH-EP cells was significantly reduced upon treatment with SLC-0111, while LAN1 cells were not affected. The inhibitory effect on SH-SY5Y cells was dependent on the induction of CAIX expression under hypoxia. These findings corresponded to thermogenesis of the cells. Patient-derived organotypic slice cultures were treated with SLC-0111, which was highly effective despite heterogeneity of CAIX/CAXII expression. Thermogenesis, in congruence with the findings of the histological observations, was significantly reduced in SLC-0111-treated samples. In order to extend the evaluation time, we established a perfusion-based approach for neuroblastoma tissue in a 3D perfusion-based bioreactor system. Using this system, excellent tissue quality with intact tumor cells and stromal structure in neuroblastoma tumors can be maintained for 7 days. The system was successfully used for consecutive drug response monitoring with isothermal microcalorimetry. The described approach for drug testing, relying on an advanced 3D culture system combined with a rapid and highly sensitive metabolic assessment, can facilitate development of personalized treatment strategies for neuroblastoma.

Asunto(s)

Inhibidores de Anhidrasa Carbónica , Neuroblastoma , Antígenos de Neoplasias/metabolismo , Reactores Biológicos , Anhidrasa Carbónica IX/metabolismo , Inhibidores de Anhidrasa Carbónica/química , Humanos , Neuroblastoma/tratamiento farmacológico , Perfusión , Compuestos de Fenilurea , Sulfonamidas

RESUMEN

Human carbonic anhydrases IX (hCA IX) and XII (hCA XII) are two proteins associated with tumor formation and development. These enzymes have been largely investigated both from a biochemical and a functional point of view. However, limited data are currently available on the characterization of their post-translational modifications (PTMs) and the functional implication of these structural changes in the tumor environment. In this review, we summarize existing literature data on PTMs of hCA IX and hCA XII, such as disulphide bond formation, phosphorylation, O-/N-linked glycosylation, acetylation and ubiquitination, highlighting, when possible, their specific role in cancer pathological processes.

Asunto(s)

Anhidrasas Carbónicas , Neoplasias , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/metabolismo , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Humanos , Procesamiento Proteico-Postraduccional , Relación Estructura-Actividad

RESUMEN

Amino acids with small side chains and motifs of small residues in a distance of four are rather abundant in human single-span transmembrane helices. While interaction of such helices appears to be common, the role of the small residues in mediating and/or stabilizing transmembrane helix oligomers remains mostly elusive. Yet, the mere existence of (small)xxx(small) motifs in transmembrane helices is frequently used to model dimeric TM helix structures. The single transmembrane helix of the human carbonic anhydrases XII contains a large number of amino acids with small side chains, and critical involvement of these small amino acids in dimerization of the transmembrane domain has been suggested. Using the GALLEX assay, we show here that the transmembrane domain indeed forms a strong transmembrane helix oligomer within a biological membrane. However, single or multiple mutations of small residue(s) to isoleucine almost always increased, rather than decreased, the interaction propensities. Reduction of helix flexibility and of protein-lipid contacts caused by a reduced lipid accessible surface area likely results in stabilization of helix-helix interactions within the membrane.

RESUMEN

Intrinsic or acquired resistance to chemotherapy is a major hurdle in the treatment of cancer. One of the key mechanisms of resistance is the overexpression of the drug efflux transporter P-glycoprotein (Pgp). Pgp overexpression renders a large number of mechanistically unrelated chemotherapies ineffective. Targeting Pgp inhibition directly to overcome drug resistance, although conceptually and mechanistically attractive, has not translated to the clinic, in part because Pgp also has a critical protective function in many healthy tissues. It was recently discovered that carbonic anhydrase XII (CA XII), an enzyme associated with pH regulation in cancer, is co-expressed and co-located with Pgp in drug resistant cancer cells. CA XII is also upregulated by hypoxia, which is another microenvironmental factor that contributes to drug resistance. Here, we review findings that demonstrate modulation of CA XII may offer a promising new approach towards overcoming the longstanding hurdle of drug resistance and therapy failure against solid cancers. This review covers the use of CA XII inhibitors, both small molecule and antibody, in combination with chemotherapeutics that are substrates for Pgp. This combination therapy approach restores the efficacy of chemotherapy in resistant cells and offers a potential new therapeutic window to re-examine the targeting of Pgp as a safe, effective, and novel anticancer strategy.

RESUMEN

Monoclonal and recombinant antibodies are widely used for the diagnostics and therapy of cancer. They are generated to interact with cell surface proteins which are usually involved in the development and progression of cancer. Carbonic anhydrase XII (CA XII) contributes to the survival of tumors under hypoxic conditions thus is considered a candidate target for antibody-based therapy. In this study, we have generated a novel collection of monoclonal antibodies (MAbs) against the recombinant extracellular domain of CA XII produced in HEK-293 cells. Eighteen out of 24 MAbs were reactive with cellular CA XII on the surface of live kidney and lung cancer cells as determined by flow cytometry. One MAb 14D6 also inhibited the enzymatic activity of recombinant CA XII as measured by the stopped-flow assay. MAb 14D6 showed the migrastatic effect on human lung carcinoma A549 and renal carcinoma A498 cell lines in a 'wound healing' assay. It did not reduce the growth of multicellular lung and renal cancer spheroids but reduced the cell viability by the ATP Bioluminescence assay. Epitope mapping revealed the surface-exposed amino acid sequence (35-FGPDGENS-42) close to the catalytic center of CA XII recognized by the MAb 14D6. The variable regions of the heavy and light chains of MAb 14D6 were sequenced and their complementarity-determining regions were defined. The obtained variable sequences were used to generate recombinant antibodies in two formats: single-chain fragment variable (scFv) expressed in E. coli and scFv fused to human IgG1 Fc fragment (scFv-Fc) expressed in Chinese Hamster Ovary (CHO) cells. Both recombinant antibodies maintained the same specificity for CA XII as the parental MAb 14D6. The novel antibodies may represent promising tools for CA XII-related cancer research and immunotherapy.

Asunto(s)

Anhidrasas Carbónicas/inmunología , Anticuerpos de Cadena Única/inmunología , Células A549 , Animales , Células CHO , Anhidrasas Carbónicas/química , Células Cultivadas , Cricetinae , Cricetulus , Mapeo Epitopo , Células HEK293 , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética

RESUMEN

6A10 is a CA XII inhibitory monoclonal antibody, which was demonstrated to reduce the growth of cancer cells in vitro and in a xenograft model of lung cancer. It was also shown to enhance chemosensitivity of multiresistant cancer cell lines and to significantly reduce the number of lung metastases in combination with doxorubicin in mice carrying human triple-negative breast cancer xenografts. Starting from these data, we report here on the development of the 6A10 antigen-binding fragment (Fab), termed Fab6A10, and its functional, biochemical, and structural characterization. In vitro binding and inhibition assays demonstrated that Fab6A10 selectively binds and inhibits CA XII, whereas immunohistochemistry experiments highlighted its capability to stain malignant glioma cells in contrast to the surrounding brain tissue. Finally, the crystallographic structure of CA XII/Fab6A10 complex provided insights into the inhibition mechanism of Fab6A10, showing that upon binding, it obstructs the substrate access to the enzyme active site and interacts with CA XII His64 freezing it in its out conformation. Altogether, these data indicate Fab6A10 as a new promising therapeutic tool against cancer.

Asunto(s)

Anticuerpos Monoclonales/química , Complejo Antígeno-Anticuerpo/química , Anhidrasas Carbónicas/química , Fragmentos Fab de Inmunoglobulinas/química , Afinidad de Anticuerpos , Sitios de Unión , Catálisis , Humanos , Fragmentos Fab de Inmunoglobulinas/farmacología , Inmunohistoquímica , Modelos Moleculares , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad

RESUMEN

Extensive growth of cancer in humans is a major cause of death. Numerous studies are being conducted to improve the early diagnosis, prevention, and treatment of cancer. Recent technological advancements in medical science and research indicate molecular target therapy holds much promise in cancer treatment. In the past, therapeutic and diagnostic targeting of non-glycolytic and glycolytic enzymes in cancer have been successful, and discoveries of biomarker enzymes in cancer hold promise for therapeutic treatments. In this review, we discuss the roles of several cancer-associated enzymes that could potentially act as therapeutic targets, and place special focus on non-glycolytic and glycolytic enzymes. This review indicates that the targeting of metabolic signaling offers a promising means of developing novel anti-cancer therapies.

Asunto(s)

Biomarcadores de Tumor/antagonistas & inhibidores , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Animales , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Glucólisis/efectos de los fármacos , Humanos , Neoplasias/etiología , Neoplasias/prevención & control

RESUMEN

Carbonic anhydrases (CAs) catalyze the reversible hydration of carbon dioxide to produce bicarbonate and a proton. Multiple CA isoforms are implicated in a range of diseases, including cancer. In solid tumors, continuously dividing cells create hypoxic conditions that eventually lead to an acidic microenvironment. Hypoxic tumor cells have different mechanisms in place to regulate and adjust the surrounding microenvironment for survival. These mechanisms include expression of CA isoform IX (CA IX) and XII (CA XII). These enzymes help maintain a physiological intracellular pH while simultaneously contributing to an acidic extracellular pH, leading to tumor cell survival. Expression of CA IX and CA XII has also been shown to promote tumor cell invasion and metastasis. This review discusses the characteristics of CA IX and CA XII, their mechanism of action, and validates their prospective use as anticancer targets. We discuss the current status of small inhibitors that target these isoforms, both classical and non-classical, and their future design in order to obtain isoform-specificity for CA IX and CA XII. Biologics, such as monoclonal antibodies, monoclonal-radionuclide conjugated chimeric antibodies, and antibody-small molecule conjugates are also discussed.

Asunto(s)

Inhibidores de Anhidrasa Carbónica/química , Anhidrasas Carbónicas/química , Neoplasias/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/química , Inhibidores de Anhidrasa Carbónica/farmacología , Inhibidores de Anhidrasa Carbónica/uso terapéutico , Anhidrasas Carbónicas/metabolismo , Dominio Catalítico/efectos de los fármacos , Hipoxia de la Célula , Movimiento Celular/efectos de los fármacos , Ensayos Clínicos como Asunto , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Modelos Moleculares , Neoplasias/enzimología , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéutico

RESUMEN

The pH of the tumor microenvironment drives the metastatic phenotype and chemotherapeutic resistance of tumors. Understanding the mechanisms underlying this pH-dependent phenomenon will lead to improved drug delivery and allow the identification of new therapeutic targets. This includes an understanding of the role pH plays in primary tumor cells, and the regulatory factors that permit cancer cells to thrive. Over the last decade, carbonic anhydrases (CAs) have been shown to be important mediators of tumor cell pH by modulating the bicarbonate and proton concentrations for cell survival and proliferation. This has prompted an effort to inhibit specific CA isoforms, as an anti-cancer therapeutic strategy. Of the 12 active CA isoforms, two, CA IX and XII, have been considered anti-cancer targets. However, other CA isoforms also show similar activity and tissue distribution in cancers and have not been considered as therapeutic targets for cancer treatment. In this review, we consider all the CA isoforms and their possible role in tumors and their potential as targets for cancer therapy.

RESUMEN

INTRODUCTION: Due to their infiltrative growth behavior, gliomas have, even after surgical resection, a high recurrence tendency. The approach of intracavitary radioimmunotherapy (RIT) is aimed at inhibiting tumor re-growth by directly administering drugs into the resection cavity (RC). Direct application of the radioconjugate into the RC has the advantage of bypassing the blood-brain barrier, which allows the administration of higher radiation doses than systemic application. Carbonic anhydrase XII (CA XII) is highly expressed on glioma cells while being absent from normal brain and thus an attractive target molecule for RIT. We evaluated a CA XII-specific 6A10 Fab (fragment antigen binding) labelled with 177Lu as an agent for RIT. METHODS: 6A10 Fab fragment was modified and radiolabelled with 177Lu and characterized by MALDI-TOF, flow cytometry and radio-TLC. In vitro stability was determined under physiological conditions. Biodistribution studies, autoradiography tumor examinations and planar scintigraphy imaging were performed on SCID-mice bearing human glioma xenografts. RESULTS: The in vitro CA XII binding capacity of the modified Fab was confirmed. Radiochemical purity was determined to be >90% after 72 h of incubation under physiological conditions. Autoradiography experiments proved the specific binding of the Fab to CA XII on tumor cells. Biodistribution studies revealed a tumor uptake of 3.0%ID/g after 6 h and no detectable brain uptake. The tumor-to-contralateral ratio of 10/1 was confirmed by quantitative planar scintigraphy. CONCLUSION: The radiochemical stability in combination with a successful in vivo tumor uptake shows the potential suitability for future RIT applications with the 6A10 Fab.

Asunto(s)

Anhidrasas Carbónicas/metabolismo , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Lutecio , Ácido Pentético/química , Radioinmunoterapia/métodos , Radioisótopos , Células A549 , Animales , Femenino , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Inmunoconjugados/uso terapéutico , Fragmentos Fab de Inmunoglobulinas/metabolismo , Ratones , Radioquímica , Distribución Tisular

RESUMEN

Carbonic anhydrase XII (CAXII) is a membrane enzyme that maintains pH homeostasis and sustains optimum P-glycoprotein (Pgp) efflux activity in cancer cells. Here, we investigated a panel of eight CAXII inhibitors (compounds 1-8), for their potential to reverse Pgp mediated tumor cell chemoresistance. Inhibitors (5 nM) were screened in human and murine cancer cells (colon, lung, breast, bone) with different expression levels of CAXII and Pgp. We identified three CAXII inhibitors (compounds 1, 2 and 4) that significantly (≥ 2 fold) increased the intracellular retention of the Pgp-substrate and chemotherapeutic doxorubicin, and restored its cytotoxic activity. The inhibitors lowered intracellular pH to indirectly impair Pgp activity. Ca12-knockout assays confirmed that the chemosensitizing property of the compounds was dependent on active CAXII. Furthermore, in a preclinical model of drug-resistant breast tumors compound 1 (1900 ng/kg) restored the efficacy of doxorubicin to the same extent as the direct Pgp inhibitor tariquidar. The expression of carbonic anhydrase IX had no effect on the intracellular doxorubicin accumulation. Our work provides strong evidence that CAXII inhibitors are effective chemosensitizer agents in CAXII-positive and Pgp-positive cancer cells. The use of CAXII inhibitors may represent a turning point in combinatorial chemotherapeutic schemes to treat multidrug-resistant tumors.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/fisiología , Neoplasias Experimentales/tratamiento farmacológico , Animales , Línea Celular Tumoral , Doxorrubicina/farmacocinética , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/enzimología

RESUMEN

PURPOSE: Hypoxia is commonly observed in regions of primary tumors and metastases, and is associated with resistance to treatment, more aggressive tumor phenotypes and poor prognosis. Reliable and validated imaging biomarkers of hypoxia are needed for pre-clinical studies and clinical use. Expression of cell-surface carbonic anhydrases IX and XII (CAIX and CAXII) in tumor cells has been associated with tumor hypoxia. CAIX and CAXII specific antibodies conjugated to fluorescent dye were evaluated for the non-invasive detection of hypoxia in vivo. PROCEDURES: Human breast cancer cell lines (MCF10A, DCIS, MCF7, ZR-75.1 and MDA-mb231) were characterized for CAIX and CAXII expression by real-time RT-PCR and immunocytochemistry (ICC) under normoxic and hypoxic conditions. Immunohistochemical (IHC) staining of CAIX, CAXII and the commercially available exogenous hypoxia marker, pimonidazole, was performed using sections of ZR-75.1 and MDA-mb-231 orthotopic breast cancer xenograft tumors from nude mice. In vivo fluorescence imaging of ZR-75.1 tumors in animals housed at varied levels of oxygen was used to quantify the relative uptake of the CAIX and CAXII agents and a commercially available sulfonamide-based agent. Corresponding tumor sections were IHC stained for CAIX, CAXII and pimonidazole. RESULTS: CAIX mRNA expression was significantly higher (p < 0.05) in hypoxia for all cell lines, which was in agreement with protein expression by ICC. CAXII expression was mixed, with a modest hypoxia-related increase in two cell lines (p < 0.05) and no change in others. Quantified IHC staining of ZR-75.1 and MDA-mb-231 tumor sections showed that CAIX and CAXII expression was elevated in regions with pimonidazole staining, but CAXII levels were lower than CAIX. Tumor uptake of the CAIX targeted agent, and IHC staining of CAIX and pimonidazole in corresponding tumor sections were correlated, and co-registered, and shown to be significantly elevated by level of oxygenation (p < 0.001): hypoxia > normoxia > hyperoxia. However, the CAXII and sulfonamide agents were not significantly correlated with hypoxia. CONCLUSION: These studies suggest that the fluorescently labeled CAIX-specific agent is a more robust indicator of hypoxia in vivo compared to the CAXII-specific agent or the agent specific to the CA active site.

Asunto(s)

Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/enzimología , Anhidrasa Carbónica IX/metabolismo , Anhidrasas Carbónicas/metabolismo , Imagen Molecular/métodos , Oxígeno/farmacología , Hipoxia Tumoral/efectos de los fármacos , Hipoxia Tumoral/genética , Análisis de Varianza , Animales , Antígenos de Neoplasias/genética , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Anhidrasa Carbónica IX/genética , Anhidrasas Carbónicas/genética , Línea Celular Tumoral , Femenino , Fluorescencia , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones Desnudos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tomografía , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Anion exchanger 2 (AE2) has a critical role in epithelial cells and is involved in the ionic homeostasis such as Cl(-) uptake and HCO3(-) secretion. However, little is known about the regulatory mechanism of AE2. The main goal of the present study was to investigate potential regulators, such as spinophilin (SPL), inositol-1,4,5-trisphosphate [IP3] receptors binding protein released with IP3 (IRBIT), STE20/SPS1-related proline/alanine-rich kinase (SPAK) kinase, and carbonic anhydrase XII (CA XII). We found that SPL binds to AE2 and markedly increased the Cl(-)/HCO3(-) exchange activity of AE2. Especially SPL 1-480 domain is required for enhancing AE2 activity. For other regulatory components that affect the fidelity of fluid and HCO3(-) secretion, IRBIT and SPAK had no effect on the activity of AE2 and no protein-protein interaction with AE2. It has been proposed that CA activity is closely associated with AE activity. In this study, we provide evidence that the basolateral membrane-associated CA isoform CA XII significantly increased the activity of AE2 and co-localized with AE2 to the plasma membrane. Collectively, SPL and CA XII enhanced the Cl(-)/HCO3(-) exchange activity of AE2. The modulating action of these regulatory proteins could serve as potential therapeutic targets for secretory diseases mediated by AE2.

Asunto(s)

Bicarbonatos/metabolismo , Antiportadores de Cloruro-Bicarbonato/metabolismo , Cloruros/metabolismo , Anhidrasas Carbónicas/metabolismo , Células HeLa , Humanos , Transporte de Proteínas , Transducción de Señal

RESUMEN

Sulfocoumarins behave as interesting inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Here, we report a new series of 7-substituted derivatives which were obtained by the click chemistry approach from 7-propargyloxy-sulfocoumarin and aryl azides incorporating halogens, hydroxy, methoxy and carboxyl moieties in their molecules. The new compounds were screened for the inhibition on four physiologically relevant human CA (hCA) isoforms, the cytosolic hCA I and II and the transmembrane tumor-associated hCA IX and XII. The new compounds did not inhibit the cytosolic isoforms but were low nanomolar inhibitors of the tumor-associated ones hCA IX and XII.

Asunto(s)

Inhibidores de Anhidrasa Carbónica/farmacología , Cumarinas/farmacología , Isoenzimas/antagonistas & inhibidores , Triazoles/química , Cumarinas/química

RESUMEN

Novel Probenecid-based amide derivatives, incorporating different natural amino acids, were synthesized and assayed to test their effect on the human carbonic anhydrase (hCA, EC 4.2.1.1) transmembrane isoforms hCA IX and XII over the ubiquitous isoforms hCA I and II. Most of them presented a complete loss of hCA II inhibition (K(i)s > 10,000 nM) and strong inhibitory activity against hCA IX and XII in the nanomolar range with respect to the parent compound. A residual activity against hCA I was observed for some of them. These biological results have been explained by docking studies within the active sites of the four studied human carbonic anhydrases (with or without the zinc-bound water) and helped us to better comprehend the rationale behind the design of tertiary sulfonamide compounds as potent but atypical inhibitors of specific isoforms of human carbonic anhydrase.

Asunto(s)

Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Probenecid/análogos & derivados , Probenecid/farmacología , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Anhidrasa Carbónica I/antagonistas & inhibidores , Anhidrasa Carbónica I/química , Anhidrasa Carbónica II/antagonistas & inhibidores , Anhidrasa Carbónica II/química , Anhidrasa Carbónica II/metabolismo , Anhidrasa Carbónica IX , Inhibidores de Anhidrasa Carbónica/síntesis química , Anhidrasas Carbónicas/química , Dominio Catalítico , Humanos , Simulación del Acoplamiento Molecular , Probenecid/síntesis química , Relación Estructura-Actividad

RESUMEN

Novel amide derivatives of Probenecid were synthesized and discovered to act as potent and selective inhibitors of the human carbonic anhydrase (hCA, EC 4.2.1.1) transmembrane isoforms hCA IX and XII. The proposed chemical transformation of the carboxylic acid into an amide group led to a complete loss of hCA I and II inhibition (Kis >10,000nM) and enhanced the inhibitory activity against hCA IX and XII, with respect to the parent compound (incorporating a COOH function). These promising biological results have been corroborated by molecular modelling studies within the active sites of the four studied human carbonic anhydrases, which enabled us to rationalize both the isoform selectivity and high activity against the tumor-associated isoforms hCA IX/XII.

Asunto(s)

Antígenos de Neoplasias/química , Anhidrasa Carbónica II/química , Anhidrasa Carbónica I/química , Inhibidores de Anhidrasa Carbónica/síntesis química , Anhidrasas Carbónicas/química , Probenecid/síntesis química , Anhidrasa Carbónica IX , Inhibidores de Anhidrasa Carbónica/química , Dominio Catalítico , Cristalografía por Rayos X , Pruebas de Enzimas , Humanos , Simulación del Acoplamiento Molecular , Probenecid/análogos & derivados , Unión Proteica , Sensibilidad y Especificidad , Relación Estructura-Actividad