RESUMEN
Disturbances of the endothelin axis have been described in tumor angiogenesis and in highly vascularized tumors, such as thyroid carcinoma. Consequently, the endothelin (ET) receptor offers a molecular target for the visualization of the endothelin system in vivo by positron emission tomography (PET). We therefore endeavoured to develop a subtype-selective ETA receptor (ETAR) radioligand by introduction of a glycosyl moiety as a hydrophilic building block into the lead compound PD156707. Employing click chemistry we synthesized the triazolyl conjugated fluoroglucosyl derivative 1 that had high selectivity for ETAR (4.5 nM) over ETBR (1.2 µM). The radiosynthesis of the glycoconjugate [(18)F]1 was achieved by concomitant (18)F-labeling and glycosylation, providing [(18)F]1 in high radiochemical yields (20-25%, not corrected for decay, 70 min) and a specific activity of 41-138 GBq/µmol. Binding properties of [(18)F]1 were evaluated in vitro, and its biodistribution was measured in K1 thyroid carcinoma xenograft nude mice ex vivo and by molecular imaging. Although the very substantial excretion via hepatobiliary clearance was not decisively influenced by glycosylation, the (18)F-glycoconjugate was more stable in blood during PET recordings than was the previously described (18)F-fluoroethoxy analog. Small-animal PET imaging showed displacable binding of [(18)F]1 at ETAR in K1 tumors. The simple and efficient (18)F-radiosynthesis together with the excellent stability make the (18)F-labeled glycoconjugate [(18)F]1 a promising molecular tool for preclinical PET imaging studies of ETAR expression in thyroid carcinoma and other conditions with marked angiogenesis.
RESUMEN
Endothelin (ET) receptor dysregulation has been described in a number of pathophysiological processes, including cardiovascular disorders, renal failure, and cancer. The aim of this study was to evaluate the expression of the ET-A receptor (ET(A)R) in murine models of thyroid carcinoma using optical imaging methods. A recently developed near-infrared fluorescent tracer was first assessed in isolated artery preparations for its functional performance in comparison with known ET(A)R antagonists BQ123 and PD156707. Before evaluation of the tracer in vivo, different thyroid carcinoma cell lines were characterized with respect to their ET receptor expression by RT-PCR and autoradiography. In vivo, sc and orthotopic papillary thyroid tumor xenografts were clearly visualized by fluorescence reflectance imaging and fluorescence-mediated tomography up to 48 h after injection of the tracer. Binding specificity of the probe was demonstrated by predosing with PD156707 as a competing inhibitor. In conclusion, optical imaging with a fluorescent ET(A)R tracer allows the noninvasive imaging of tumor-associated ET(A)R expression in vivo. In the future, this technique may help surgeons to evaluate lesion dimensions in intraoperative settings (e.g. thyroidectomy).
Asunto(s)
Diagnóstico por Imagen/métodos , Receptor de Endotelina A/metabolismo , Neoplasias de la Tiroides/metabolismo , Tomografía/métodos , Animales , Autorradiografía/métodos , Línea Celular Tumoral , Dioxoles/metabolismo , Dioxoles/farmacología , Antagonistas de los Receptores de la Endotelina A , Femenino , Fluorescencia , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias/métodos , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Receptor de Endotelina A/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Trasplante HeterólogoRESUMEN
UNLABELLED: Hepatitis C virus (HCV) is a leading cause of chronic liver disease worldwide and establishes a persistent infection in more than 60% of infected individuals. This high frequency of persistent infection indicates that HCV has evolved efficient strategies to interfere with the adaptive and innate immune response and to occupy and use host cell infrastructure. The present study provides evidence that c-Src, a member of the Src family kinases that participates in many signal transduction pathways, represents an essential host factor exploited for viral replication. c-Src directly interacts with the viral RNA-dependent RNA polymerase (NS5B) via its SH3 domain and with the nonstructural phosphoprotein NS5A via its SH2 domain. Both interactions are required to maintain the protein-protein interaction of NS5A and NS5B, which has been previously demonstrated to be essential for viral replication. Accordingly, HCV genome replication and production of the viral proteins was strongly reduced upon small interfering RNA-mediated knockdown of c-Src or in the presence of the tyrosine kinase inhibitor herbimycin A. This effect could not be rescued by supplementation of the two other ubiquitously expressed Src family kinases Fyn or Yes. CONCLUSION: Our data suggest that c-Src participates in the formation of an NS5A/NS5B protein complex that is required for efficient replication of HCV.
Asunto(s)
Hepacivirus/fisiología , Familia-src Quinasas/fisiología , Benzoquinonas/farmacología , Línea Celular Tumoral , Hepacivirus/genética , Humanos , Lactamas Macrocíclicas/farmacología , ARN Polimerasa Dependiente del ARN/genética , Rifabutina/análogos & derivados , Proteínas no Estructurales Virales , Replicación Viral , Dominios Homologos src/genéticaRESUMEN
The expression and function of endothelin (ET) receptors are abnormal in cardiovascular diseases, tumor progression, and tumor metastasis. A previously reported promising radioligand for positron emission tomography (PET) based on the non-peptide ET(A) receptor antagonist PD 156707 showed specific binding to target receptors in the myocardium but high accumulation in bile and intestine, probably because of its high lipophilicity. In this study we describe the synthesis of a series of fluorinated derivatives with hydrophilic building blocks. All compounds were evaluated as high affinity ET(A) receptor ligands (16, 17, 23-26, K(i) = 1.4-7.9 nM) with high subtype selectivity over the ET(B) receptor. [(18)F]3-Benzo[1,3]dioxol-5-yl-4-{3-[1-(2-{2-[2-(2-fluoroethoxy)ethoxy]ethoxy}ethyl)-1H-[1,2,3]triazol-4-ylmethoxy]-4,5-dimethoxybenzyl}-5-hydroxy-5-(4-methoxyphenyl)-5H-furan-2-one ([(18)F]17) was synthesized as one of the radioligands of this series that possesses a higher hydrophilicity and an excellent stability in human serum. Improved clearance properties and specific uptake in target organs have been confirmed by biodistribution studies and small animal PET imaging.
Asunto(s)
Furanos/química , Tomografía de Emisión de Positrones/métodos , Radiofármacos/síntesis química , Receptor de Endotelina A/metabolismo , Triazoles/química , Animales , Unión Competitiva , Química Clic/métodos , Antagonistas de los Receptores de la Endotelina A , Radioisótopos de Flúor , Furanos/síntesis química , Humanos , Ligandos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB C , Radiofármacos/química , Ratas , Distribución Tisular , Triazoles/síntesis químicaRESUMEN
WW domain-containing proteins are found in all eukaryotic cells and they are involved in the regulation of a wide variety of cellular functions. We recently identified the neuronal protein KIBRA as novel member of this family of signal transducers. In this report, we describe the identification of protein kinase C (PKC) zeta as a KIBRA-interacting protein. PKCzeta is known to play an important role in synaptic plasticity and memory formation but its specific targets are not well known. Our studies presented here revealed that KIBRA is a novel substrate for PKCzeta and suggest that PKCzeta phosphorylation may regulate the cellular function of KIBRA.
Asunto(s)
Proteína Quinasa C/metabolismo , Proteínas/metabolismo , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Fosfoproteínas , Fosforilación , Especificidad por Sustrato , Técnicas del Sistema de Dos HíbridosRESUMEN
In a yeast two hybrid screen with the human isoform of Dendrin (KIAA0749), a putative modulator of the postsynaptic cytoskeleton, we isolated a cDNA coding for a novel protein, KIBRA, possessing two amino-terminal WW domains, an internal C2-like domain and a carboxy-terminal glutamic acid-rich stretch. Northern blot analysis revealed that the expression of KIBRA mRNA was predominately found in kidney and brain. In vitro interaction studies revealed that the first KIBRA WW domain binds specifically to PPxY motifs. Transient transfection of monkey kidney cells with constructs encoding Myc-tagged KIBRA displayed a cytoplasmic localization and a perinuclear enrichment of the protein.