ABSTRACT
Dynamic combinatorial chemistry (DCC) has proven its potential in drug discovery speeding the identification of modulators of biological targets. However, the exchange chemistries typically take place under specific reaction conditions, with limited tools capable of operating under physiological parameters. Here we report a catalyzed protein-directed DCC working at low temperatures that allows the calcium sensor NCS-1 to find the best ligands in situ. Ultrafast NMR identifies the reaction intermediates of the acylhydrazone exchange, tracing the molecular assemblies and getting a real-time insight into the essence of DCC processes at physiological pH. Additionally, NMR, X-ray crystallography and computational methods are employed to elucidate structural and mechanistic aspects of the molecular recognition event. The DCC approach leads us to the identification of a compound stabilizing the NCS-1/Ric8a complex and whose therapeutic potential is proven in a Drosophila model of disease with synaptic alterations.
Subject(s)
Calcium/metabolism , Gene Library , Neuronal Calcium-Sensor Proteins/metabolism , Animals , Catalysis , Cells, Cultured , Combinatorial Chemistry Techniques , Drosophila/physiology , Magnetic Resonance Imaging , Male , Membranes, Artificial , Mice , Neuronal Calcium-Sensor Proteins/genetics , Neurons/metabolism , Palmitoyl-CoA Hydrolase , Permeability , Protein Conformation , ProteinsABSTRACT
Genetic lesions in glioblastoma (GB) include constitutive activation of PI3K and EGFR pathways to drive cellular proliferation and tumor malignancy. An RNAi genetic screen, performed in Drosophila melanogaster to discover new modulators of GB development, identified a member of the secretory pathway: kish/TMEM167A. Downregulation of kish/TMEM167A impaired fly and human glioma formation and growth, with no effect on normal glia. Glioma cells increased the number of recycling endosomes, and reduced the number of lysosomes. In addition, EGFR vesicular localization was primed toward recycling in glioma cells. kish/TMEM167A downregulation in gliomas restored endosomal system to a physiological state and altered lysosomal function, fueling EGFR toward degradation by the proteasome. These endosomal effects mirrored the endo/lysosomal response of glioma cells to Brefeldin A (BFA), but not the Golgi disruption and the ER collapse, which are associated with the undesirable toxicity of BFA in other cancers. Our results suggest that glioma growth depends on modifications of the vesicle transport system, reliant on kish/TMEM167A. Noncanonical genes in GB could be a key for future therapeutic strategies targeting EGFR-dependent gliomas.
Subject(s)
Brain Neoplasms/metabolism , Drosophila Proteins/metabolism , Gene Expression Regulation, Neoplastic/physiology , Glioma/metabolism , Protein Transport/genetics , Animals , Animals, Genetically Modified , Brain/cytology , Brain Neoplasms/pathology , Cell Line, Tumor , Disease Models, Animal , Drosophila Proteins/genetics , Drosophila melanogaster , Enzyme Inhibitors/pharmacology , ErbB Receptors/genetics , ErbB Receptors/metabolism , Female , Glioma/pathology , Heterografts , Humans , Leupeptins/pharmacology , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Male , Mice , RNA Interference/physiologyABSTRACT
The appendages of arthropods and vertebrates are not homologous structures, although the underlying genetic mechanisms that pattern them are highly conserved. Members of the Sp family of transcription factors are expressed in the developing limbs and their function is required for limb growth in both insects and chordates. Despite the fundamental and conserved role that these transcription factors play during appendage development, their target genes and the mechanisms by which they participate in control limb growth are mostly unknown. We analyzed here the individual contributions of two Drosophila Sp members, buttonhead (btd) and Sp1, during leg development. We show that Sp1 plays a more prominent role controlling leg growth than does btd We identified a regulatory function of Sp1 in Notch signaling, and performed a genome-wide transcriptome analysis to identify other potential Sp1 target genes contributing to leg growth. Our data suggest a mechanism by which the Sp factors control appendage growth through the Notch signaling.
Subject(s)
Drosophila Proteins/metabolism , Receptors, Notch/metabolism , Sp1 Transcription Factor/metabolism , Animals , Chromatin Immunoprecipitation , Drosophila , Drosophila Proteins/genetics , Evolution, Molecular , Female , Gene Expression Regulation, Developmental , Male , Receptors, Notch/genetics , Signal Transduction/genetics , Signal Transduction/physiology , Sp1 Transcription Factor/geneticsABSTRACT
La aparición de nuevas tecnologías trae añadida nuevas formas de abordaje quirúrgico. En el Hospital Universitario «Río Hortega» de Valladolid, desde el año 1988 se han tratado aproximadamente 780 pacientes con cirugía percutánea. Cirugía con abordaje percutáneo se definiría como el acto quirúrgico sobre la estructura, en este caso, renal y ureteral cuya vía de acceso o de entrada al órgano se efectúa a través de la piel, concretamente una nefrostomía. En nuestra Unidad, un 77% de los casos de cirugía percutánea es aplicada en el tratamiento de litiasis renales-ureterales cuando otros tratamientos fallan o no son factibles, por ejemplo litotricias fallidas, cálculos ureterales de mal abordaje, cálculos renales coraliformes, etc. También se aplica en tratamientos tumorales, endopielotomías, etc. A priori esta cirugía es de elección preferente por ser mínimamente invasiva. Por otra parte, la implantación del programa informático GACELA permite estandarizar los cuidados enfermeros, lo que facilita la creación de un plan de cuidados específico para este tipo de pacientes, ya que los diagnósticos y acciones cuidadoras descritas son comunes en un alto porcentaje de casos tratados (próximo al 100%).La taxonomía empleada en la descripción de diagnósticos enfermeros corresponde a la clasificación NANDA y las acciones se encuentran en la base de datos del programa GACELA (AU)
In the University Hospital «Río Hortega» in Valladolid, since 1988 approximately 780 patients have been treated with percutaneous surgery. Surgery with percutaneous approach would be defined as the surgical act on the structure, in this case, renal and uretheral, which access tract to the organ is carried out through the skin, precisely a nephrostomy. In our Unit 77% of the cases of percutaneous surgery it is applied in the treatment of renal lithiasis-uretheral when other treatments fail or are not feasible, for example unsuccessful lithotripsy, ureteral calculus of difficult approach, coralliform kidney calculis, etc. It is also applied in tumoral treatments, endopyelotomy, etc. A priori this surgery is of preferential choice because it is minimally invasive. On the other hand, the introduction of the GAZELLE computer program allows standardizing the Nursing Cares, what facilitates the creation of a specific cares plan for this type of patients, since the diagnoses and care actions described are common in a high percentage of cases treated (close to 100%). The taxonomy used in the description of nursing diagnoses corresponds to the classification NANDA and the actions are found in the database of the GAZELLE program (AU)
