RESUMEN
DNA points accumulation in nanoscale topography (DNA-PAINT) is a relatively easy-to-implement super-resolution technique. However, image acquisition is slow compared to most other approaches. Here, we overcome this limitation by designing optimized DNA sequences and buffer conditions. We demonstrate our approach in vitro with DNA origami and in situ using cell samples, and achieve an order of magnitude faster imaging speeds without compromising image quality or spatial resolution. This improvement now makes DNA-PAINT applicable to high-throughput studies.
Asunto(s)
ADN/química , Microscopía Fluorescente/métodos , Nanotecnología/métodos , Animales , Secuencia de Bases , Tampones (Química) , Células COS , Chlorocebus aethiops , Células HeLa , HumanosRESUMEN
Variant rs351855-G/A is a commonly occurring single-nucleotide polymorphism of coding regions in exon 9 of the fibroblast growth factor receptor FGFR4 (CD334) gene (c.1162G>A). It results in an amino-acid change at codon 388 from glycine to arginine (p.Gly388Arg) in the transmembrane domain of the receptor. Despite compelling genetic evidence for the association of this common variant with cancers of the bone, breast, colon, prostate, skin, lung, head and neck, as well as soft-tissue sarcomas and non-Hodgkin lymphoma, the underlying biological mechanism has remained elusive. Here we show that substitution of the conserved glycine 388 residue to a charged arginine residue alters the transmembrane spanning segment and exposes a membrane-proximal cytoplasmic signal transducer and activator of transcription 3 (STAT3) binding site Y(390)-(P)XXQ(393). We demonstrate that such membrane-proximal STAT3 binding motifs in the germline of type I membrane receptors enhance STAT3 tyrosine phosphorylation by recruiting STAT3 proteins to the inner cell membrane. Remarkably, such germline variants frequently co-localize with somatic mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Using Fgfr4 single nucleotide polymorphism knock-in mice and transgenic mouse models for breast and lung cancers, we validate the enhanced STAT3 signalling induced by the FGFR4 Arg388-variant in vivo. Thus, our findings elucidate the molecular mechanism behind the genetic association of rs351855 with accelerated cancer progression and suggest that germline variants of cell-surface molecules that recruit STAT3 to the inner cell membrane are a significant risk for cancer prognosis and disease progression.
Asunto(s)
Membrana Celular/metabolismo , Mutación de Línea Germinal , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Factor de Transcripción STAT3/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Exones/genética , Femenino , Técnicas de Sustitución del Gen , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Fosforilación , Fosfotirosina/metabolismo , Polimorfismo de Nucleótido Simple/genética , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/química , Transducción de SeñalRESUMEN
Although naturally occurring low-molecular-weight compounds have many known roles within the cell, these do not usually involve the direct inhibition of protein-protein interactions. Based on the results of high-throughput screening of a library of bioactive compounds and neurotransmitters, we report here that the four nucleoside triphosphates ATP, GTP, CTP and UTP inhibit the SH2 domain of the tumor-related transcription factor STAT5b. ATP and GTP are the most active nucleoside triphosphates and show specificity for STAT5b over STAT5a, STAT3, STAT6 and the p53-binding protein HDM2. As the inhibition constant of ATP against STAT5b is significantly lower than published values for the intracellular ATP concentration, our data suggest that ATP might inhibit the protein-protein interactions of STAT5b in living cells.
Asunto(s)
Adenosina Trifosfato/farmacología , Factor de Transcripción STAT5/antagonistas & inhibidores , Guanosina Trifosfato/farmacología , Humanos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Bibliotecas de Moléculas Pequeñas , Dominios Homologos srcRESUMEN
[This corrects the article DOI: 10.18632/oncotarget.26817.].
RESUMEN
The modulation of biological signal transduction pathways by masking phosphorylated amino acid residues represents a viable route toward pharmacologic protein regulation. Binding of phosphorylated amino acid residues has been achieved with synthetic metal-chelate receptors. The affinity and selectivity of such receptors can be enhanced if combined with a second binding site. We demonstrate this principle with a series of synthetic ditopic metal-chelate receptors, which were synthesized and investigated for their binding affinity to phosphorylated short peptides under conditions of physiological pH. The compounds showing highest affinity were subsequently used to inhibit the interaction of the human STAT1 protein to a peptide derived from the interferon-gamma receptor, and between the checkpoint kinase Chk2 and its preferred binding motif. Two of the investigated ditopic synthetic receptors show a significant increase in inhibition activity. The results show that regulation of protein function by binding to phosphorylated amino acids is possible. The introduction of additional binding sites into the synthetic receptors increases their affinity, but the flexibility of the structures investigated so far prohibited stringent amino acid sequence selectivity in peptide binding.
Asunto(s)
Metales/metabolismo , Péptidos , Quinasa de Punto de Control 2 , Quelantes/metabolismo , Humanos , Estructura Molecular , Péptidos/química , Péptidos/metabolismo , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Factor de Transcripción STAT1/química , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/fisiologíaRESUMEN
In this Letter, we report the natural products salvianolic acid A, salvianolic acid B, and caftaric acid as inhibitors of the protein-protein interactions mediated by the SH2 domains of the Src-family kinases Src and Lck, two established disease targets. Moreover, we propose a binding mode for the inhibitors based on molecular modeling, which will facilitate chemical optimization efforts of these important lead structures for drug discovery.
Asunto(s)
Productos Biológicos/farmacología , Dominios Homologos src/efectos de los fármacos , Familia-src Quinasas/antagonistas & inhibidores , Benzofuranos/farmacología , Ácidos Cafeicos/farmacología , Descubrimiento de Drogas , Humanos , Lactatos/farmacología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Modelos Moleculares , Fenoles/farmacología , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas pp60(c-src)/antagonistas & inhibidoresRESUMEN
The lung cancer stem cell (LuCSC) model comprises an attractive framework to explore acquired drug resistance in non-small cell lung cancer (NSCLC) treatment. Here, we used NSCLC cell line model to translate cellular heterogeneity into tractable populations to understand the origin of lung cancers and drug resistance. The epithelial LuCSCs, presumably arising from alveolar bipotent stem/progenitor cells, were lineage naïve, noninvasive, and prone to creating aggressive progeny expressing AT2/AT1 markers. LuCSC-holoclones were able to initiate rimmed niches, where their specialization created pseudo-alveoli structures. Mechanistically, LuCSC transitioning from self-renewal (ß-catenin and Nanog signaling) to malignant lineage differentiation is regulated by EGFR activation and the inverse inhibition of tumor suppressor MIG6. We further identified the functional roles of endogenous EGFR signaling in mediating progeny invasiveness and their ligands in LuCSC differentiation. Importantly, drug screening demonstrated that EGFR driving progeny were strongly responsive to TKIs; however, the LuCSCs were exclusively resistant but sensitive to AMPK agonist Metformin, antibiotic Salinomycin and to a lesser degree Carboplatin. Our data reveals previously an unknown mechanism of NSCLC resistance to EGFR-TKIs, which is associated with LuCSCs bearing a silenced EGFR and inversely expressed MIG6 suppressor gene. Taken altogether, successful NSCLC treatment requires development of a novel combination of drugs, efficiently targeting both LuCSCs and heterogeneous progeny.
RESUMEN
CONTEXT: The effect of a lifestyle intervention to reduce liver fat content in nonalcoholic fatty liver disease in humans is influenced by genetics. We hypothesized that the amino acid exchange in human Gly388Arg (mouse homolog: Gly385Arg) in fibroblast growth factor receptor 4 (FGFR4), which regulates bile acid, lipid, and glucose metabolism, could determine hepatic lipid accumulation and insulin sensitivity. Mechanisms of this substitution were studied in mice under normal chow and high-fat diets. DESIGN: In humans, the Gly388Arg polymorphism was studied for its relationship with changes in liver fat content and insulin sensitivity during 9 months of a lifestyle intervention. We also studied a knock-in mouse strain with an Arg385 allele introduced into the murine FGFR4 gene under normal chow and high-fat diets. RESULTS: In humans, the FGFR4 Arg388 allele was not associated with liver fat content or insulin sensitivity in subjects who were overweight and obese before lifestyle intervention. However, it was associated with less decrease in liver fat content and less increase in insulin sensitivity during the intervention. In mice receiving normal chow, the FGFR4 Arg385 allele was associated with elevated hepatic triglyceride content, altered hepatic lipid composition, and increased hepatic expression of genes inducing de novo lipogenesis and glycolysis. Body fat mass and distribution, glucose tolerance, and insulin sensitivity were unaltered. The FGFR4 Arg385 allele had no effect on glucose or lipid metabolism under the high-fat diet. CONCLUSION: Our data indicate that the FGFR4 Arg388(385) allele affects hepatic lipid and glucose metabolism specifically during healthy caloric intake.
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Lipogénesis/genética , Enfermedad del Hígado Graso no Alcohólico/dietoterapia , Obesidad/dietoterapia , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Adulto , Animales , Glucemia/análisis , Glucemia/metabolismo , Dieta Saludable , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Femenino , Estudios de Seguimiento , Técnicas de Sustitución del Gen , Prueba de Tolerancia a la Glucosa , Glucólisis/genética , Humanos , Resistencia a la Insulina/genética , Hígado/química , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad/sangre , Obesidad/etiología , Obesidad/metabolismo , Polimorfismo de Nucleótido Simple , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Triglicéridos/análisis , Triglicéridos/metabolismoAsunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Desinfectantes/farmacología , Neoplasias de la Boca/tratamiento farmacológico , Proteína bcl-X/metabolismo , Antineoplásicos/química , Línea Celular Tumoral , Desinfectantes/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Modelos Moleculares , Estructura Molecular , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Unión Proteica/efectos de los fármacos , Relación Estructura-Actividad , Proteína bcl-X/químicaAsunto(s)
Proteínas Potenciadoras de Unión a CCAAT/antagonistas & inhibidores , Triterpenos/farmacología , ADN/química , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Concentración 50 Inhibidora , Estructura Molecular , Triterpenos Pentacíclicos , Triterpenos/química , Ácido BetulínicoRESUMEN
bZip and bHLHZip protein family members comprise a large fraction of eukaryotic transcription factors and need to bind DNA in order to exert most of their fundamental biological roles. Their binding to DNA requires homo- or heterodimerization via alpha-helical domains, which generally do not contain obvious binding sites for small molecules. We have identified two small molecules, dubbed Mycro1 and Mycro2, which inhibit the protein-protein interactions between the bHLHZip proteins c-Myc and Max. Mycros are the first inhibitors of c-Myc/Max dimerization, which have been demonstrated to inhibit DNA binding of c-Myc with preference over other dimeric transcription factors in vitro. Mycros inhibit c-Myc-dependent proliferation, gene transcription, and oncogenic transformation in the low micromolar concentration range. Our data support the idea that dimeric transcription factors can be druggable even in the absence of obvious small-molecule binding pockets.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , ADN/metabolismo , Proteínas Proto-Oncogénicas c-myc/química , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/antagonistas & inhibidores , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Cartilla de ADN , Dimerización , Inmunoensayo de Polarización Fluorescente , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Signal transducers and activators of transcription (STATs) are a family of latent cytoplasmic transcription factors that transmit signals from the cell membrane to the nucleus. One family member, STAT3, is constitutively activated by aberrant upstream tyrosine kinase activities in a broad spectrum of cancer cell lines and human tumors. Screening of chemical libraries led to the identification of Stattic, a nonpeptidic small molecule shown to selectively inhibit the function of the STAT3 SH2 domain regardless of the STAT3 activation state in vitro. Stattic selectively inhibits activation, dimerization, and nuclear translocation of STAT3 and increases the apoptotic rate of STAT3-dependent breast cancer cell lines. We propose Stattic as a tool for the inhibition of STAT3 in cell lines or animal tumor models displaying constitutive STAT3 activation.
Asunto(s)
Óxidos S-Cíclicos/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Óxidos S-Cíclicos/farmacología , Dimerización , Humanos , Fosforilación/efectos de los fármacos , Dominios Homologos srcRESUMEN
Chronic kidney disease (CKD) is a worldwide public health threat that increases risk of death due to cardiovascular complications, including left ventricular hypertrophy (LVH). Novel therapeutic targets are needed to design treatments to alleviate the cardiovascular burden of CKD. Previously, we demonstrated that circulating concentrations of fibroblast growth factor (FGF) 23 rise progressively in CKD and induce LVH through an unknown FGF receptor (FGFR)-dependent mechanism. Here, we report that FGF23 exclusively activates FGFR4 on cardiac myocytes to stimulate phospholipase Cγ/calcineurin/nuclear factor of activated T cell signaling. A specific FGFR4-blocking antibody inhibits FGF23-induced hypertrophy of isolated cardiac myocytes and attenuates LVH in rats with CKD. Mice lacking FGFR4 do not develop LVH in response to elevated FGF23, whereas knockin mice carrying an FGFR4 gain-of-function mutation spontaneously develop LVH. Thus, FGF23 promotes LVH by activating FGFR4, thereby establishing FGFR4 as a pharmacological target for reducing cardiovascular risk in CKD.
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Hipertrofia Ventricular Izquierda/patología , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Calcineurina/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Técnicas de Sustitución del Gen , Glucuronidasa/genética , Glucuronidasa/metabolismo , Células HEK293 , Humanos , Hipertrofia Ventricular Izquierda/metabolismo , Proteínas Klotho , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Factores de Transcripción NFATC/metabolismo , Fosfolipasa C gamma/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/deficiencia , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Transducción de SeñalRESUMEN
The presence of cancer stem cells (CSCs) is linked to preexisting or acquired drug resistance and tumor relapse. Therefore, targeting both differentiated tumor cells and CSCs was suggested as an effective approach for non-small cell lung cancer (NSCLC) treatment. After screening of chemotherapeutic agents, tyrosine kinase inhibitors (TKIs) or monoclonal antibody in combination with the putative stem cell killer Salinomycin (SAL), we found Metformin (METF), which modestly exerted a growth inhibitory effect on monolayer cells and alveospheres/CSCs of 5 NSCLC cell lines regardless of their EGFR, KRAS, EML4/ALK and LKB1 status, interacted synergistically with SAL to effectively promote cell death. Inhibition of EGFR (AKT, ERK1/2) and mTOR (p70 s6k) signaling with the combination of METF and SAL can be augmented beyond that achieved using each agent individually. Phospho-kinase assay further suggested the multiple roles of this combination in reducing oncogenic effects of modules, such as ß-catenin, Src family kinases (Src, Lyn, Yes), Chk-2 and FAK. Remarkably, significant reduction of sphere formation was seen under combinatorial treatment in all investigated NSCLC cell lines. In conclusion, METF in combination with SAL could be a promising treatment option for patients with advanced NSCLC irrespective of their EGFR, KRAS, EML4/ALK and LKB1 status.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Quinasas de la Proteína-Quinasa Activada por el AMP , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Metformina/administración & dosificación , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Células Madre Neoplásicas/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas p21(ras) , Piranos/administración & dosificación , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Transducción de Señal , Proteínas ras/genética , Proteínas ras/metabolismoAsunto(s)
Cromonas/farmacología , Hidrazonas/farmacología , Factor de Transcripción STAT5/antagonistas & inhibidores , Aldehídos/química , Aldehídos/farmacología , Línea Celular Tumoral , Cromonas/química , ADN/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Hidrazonas/química , Ligandos , Estructura Molecular , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Dominios Homologos src/efectos de los fármacosRESUMEN
Phosphorylation-dependent protein binding domains are crucially important for intracellular signaling pathways and thus highly relevant targets in chemical biology. By screening of chemical libraries against 12 structurally diverse phosphorylation-dependent protein binding domains, we have identified fosfosal and dexamethasone-21-phosphate as selective inhibitors of two antitumor targets: the SH2 domain of the transcription factor STAT5b and the substrate-binding domain of the peptidyl-prolyl isomerase Pin1, respectively. Both compounds are phosphate prodrugs with documented clinical use as anti-inflammatory agents in humans and were discovered with a high hit rate from a small subgroup within the screening library. Our study indicates O-phosphorylation of appropriately preselected natural products or natural product derivatives as a generally applicable strategy for the identification of non-reactive and non-peptidic ligands of phosphorylation-dependent protein binding domains. Moreover, our data indicate that it would be advisable to monitor the bioactivities of clinically used prodrugs in their uncleaved state against phosphorylation-dependent protein binding domains.