RESUMEN
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.
Asunto(s)
Calcio/metabolismo , Biblioteca de Genes , Proteínas Sensoras del Calcio Neuronal/metabolismo , Animales , Catálisis , Células Cultivadas , Técnicas Químicas Combinatorias , Drosophila/fisiología , Imagen por Resonancia Magnética , Masculino , Membranas Artificiales , Ratones , Proteínas Sensoras del Calcio Neuronal/genética , Neuronas/metabolismo , Palmitoil-CoA Hidrolasa , Permeabilidad , Conformación Proteica , ProteínasRESUMEN
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.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Proteínas de Drosophila/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Glioma/metabolismo , Transporte de Proteínas/genética , Animales , Animales Modificados Genéticamente , Encéfalo/citología , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Proteínas de Drosophila/genética , Drosophila melanogaster , Inhibidores Enzimáticos/farmacología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Glioma/patología , Xenoinjertos , Humanos , Leupeptinas/farmacología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Interferencia de ARN/fisiologíaRESUMEN
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.