RESUMEN
We have exploited the existence of a second copy of the human SMN gene (SMN2) to develop a high-throughput screening strategy to identify potential small molecule therapeutics for the genetic disease spinal muscular atrophy (SMA), which is caused by the loss of the SMN1 gene. Our screening process was designed to identify synthetic compounds that increase the total amount of full-length SMN messenger RNA and protein arising from the SMN2 gene, thereby suppressing the deleterious effects of losing SMN1. A cell-based bioassay was generated that detects SMN2 promoter activity, on which greater than 550,000 compounds was tested. This resulted in the identification of 17 distinct compounds with confirmed biological activity on the cellular primary assay, belonging to nine different structural families. Six of the nine scaffolds were chosen on the basis of their drug-like features to be tested for their ability to modulate SMN gene expression in SMA patient-derived fibroblasts. Five of the six compound classes altered SMN mRNA levels or mRNA splicing patterns in SMA patient-derived fibroblasts. Two of the compound classes, a quinazoline compound series and an indole compound, also increased SMN protein levels and nuclear gem/Cajal body numbers in patient-derived cells. In addition, these two distinct scaffolds showed additive effects when used in combination, suggesting that they may act on different molecular targets. The work described here has provided the foundation for a successful medicinal chemistry effort to further advance these compounds as potential small molecule therapeutics for SMA.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Atrofia Muscular Espinal/genética , Proteínas del Tejido Nervioso/genética , Proteínas de Unión al ARN/genética , Secuencia de Bases , Western Blotting , Células Cultivadas , Cuerpos Enrollados/metabolismo , Cartilla de ADN , Relación Dosis-Respuesta a Droga , Humanos , Inmunohistoquímica , Atrofia Muscular Espinal/tratamiento farmacológico , Regiones Promotoras Genéticas , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas del Complejo SMN , Proteína 1 para la Supervivencia de la Neurona Motora , Proteína 2 para la Supervivencia de la Neurona MotoraRESUMEN
Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by mutation of the telomeric copy of the survival motor neuron gene (SMN1). Although a centromeric copy of the survival motor neuron gene (SMN2) is retained in all patients with SMA, it differs from SMN1 at a critical nucleotide such that the majority of SMN2 transcripts lack exon 7 and encode an unstable, truncated protein. Here, we show that valproic acid increases levels of exon 7-containing SMN transcript and SMN protein in type I SMA patient-derived fibroblast cell lines. Valproic acid may increase SMN levels both by activating the SMN promoter and by preventing exon 7 skipping in SMN transcripts. Valproic acid and related compounds warrant further investigation as potential treatment for SMA.