RESUMEN
Polyglutamine expansion in certain proteins causes neurodegeneration in inherited disorders such as Huntington disease and X-linked spinobulbar muscular atrophy. Polyglutamine tracts promote protein aggregation in vitro and in vivo with a strict length-dependence that strongly implicates alternative protein folding and/or aggregation as a proximal cause of cellular toxicity and neurodegeneration. We used an intracellular polyglutamine protein aggregation assay based on fluorescence resonance energy transfer (FRET) to identify inhibitors of androgen receptor (AR) aggregation in three libraries of biologically active small molecules: the Annotated Compound Library, the NINDS Custom Collection and a kinase inhibitor collection. In the primary screen 10 compounds reduced AR aggregation. While 10/10 also reduced huntingtin (Htt) exon 1 aggregation, only 2/10 reduced aggregation of pure polyglutamine peptides. In a PC-12 model 9/10 compounds reduced aggregation. Five out of nine compounds tested in an Htt exon 1 assay of neurodegeneration in Drosophila partially rescued the phenotype. Three of the five compounds effective in flies are FDA-approved drugs. These compounds provide new leads for therapeutic development for the polyglutamine diseases based on their efficacy in mammalian cells and a Drosophila model. The high predictive value of the primary screen suggests that the FRET-based screening assay may be useful for further primary and secondary screens for genes or small molecules that inhibit polyglutamine protein aggregation.
Asunto(s)
Antagonistas de Receptores Androgénicos , Drogas en Investigación/farmacología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Péptidos/antagonistas & inhibidores , Animales , Bioensayo/métodos , Línea Celular , Relación Dosis-Respuesta a Droga , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Drogas en Investigación/química , Drogas en Investigación/uso terapéutico , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , Proteína Huntingtina , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/metabolismo , Estructura Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Células PC12 , Péptidos/química , Péptidos/metabolismo , Pliegue de Proteína , Ratas , Receptores Androgénicos/química , Receptores Androgénicos/metabolismo , Relación Estructura-Actividad , Expansión de Repetición de Trinucleótido/efectos de los fármacosRESUMEN
Many neurodegenerative diseases, including tauopathies, Parkinson's disease, amyotrophic lateral sclerosis, and the polyglutamine diseases, are characterized by intracellular aggregation of pathogenic proteins. It is difficult to study modifiers of this process in intact cells in a high-throughput and quantitative manner, although this could facilitate molecular insights into disease pathogenesis. Here we introduce a high-throughput assay to measure intracellular polyglutamine protein aggregation using fluorescence resonance energy transfer (FRET). We screened over 2800 biologically active small molecules for inhibitory activity and have characterized one lead compound in detail. Y-27632, an inhibitor of the Rho-associated kinase p160ROCK, diminished polyglutamine protein aggregation (EC(50) congruent with 5 microM) and reduced neurodegeneration in a Drosophila model of polyglutamine disease. This establishes a novel high-throughput approach to study protein misfolding and aggregation associated with neurodegenerative diseases and implicates a signaling pathway of previously unrecognized importance in polyglutamine protein processing.
Asunto(s)
Amidas/farmacología , Bioensayo/métodos , Inhibidores Enzimáticos/farmacología , Transferencia Resonante de Energía de Fluorescencia/métodos , Péptidos/antagonistas & inhibidores , Péptidos/análisis , Piridinas/farmacología , Amidas/uso terapéutico , Animales , Animales Modificados Genéticamente , Células COS , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Drosophila melanogaster , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/uso terapéutico , Humanos , Proteína Huntingtina , Cuerpos de Inclusión/química , Cuerpos de Inclusión/efectos de los fármacos , Cuerpos de Inclusión/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Péptidos/metabolismo , Células Fotorreceptoras de Invertebrados/efectos de los fármacos , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/patología , Pliegue de Proteína , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Expansión de Repetición de Trinucleótido/efectos de los fármacos , Expansión de Repetición de Trinucleótido/genética , Quinasas Asociadas a rhoRESUMEN
Huntington's disease (HD) is one of a number of familial polyglutamine (polyQ) repeat diseases. These neurodegenerative disorders are caused by expression of otherwise unrelated proteins that contain an expansion of a polyQ tract, rendering them toxic to specific subsets of vulnerable neurons. These expanded repeats have an inherent propensity to aggregate; insoluble neuronal nuclear and cytoplasmic polyQ aggregates or inclusions are hallmarks of the disorders [1,2]. In HD, inclusions in diseased brains often precede onset of symptoms, and have been proposed to be involved in pathogenicity [3-5]. Various strategies to block the process of aggregation have been developed in an effort to create drugs that decrease neurotoxicity. A discussion of the effect of antibodies, caspase inhibitors, chemical inhibitors, heat-shock proteins, suppressor peptides and transglutaminase inhibitors upon aggregation and disease is presented.