A spinal muscular atrophy reporter system for in vivo drug discovery in drosophila melanogaster

Resumen

Background Spinal Muscular Atrophy is a rare and fatal neuromuscular disorder caused by the loss or reduction in the Survival Motor Neuron (SMN) protein levels. The affected individuals have mutated SMN1 gene and unaffected human-specific SMN2 copy, which is only partially translated into a functional SMN protein. Pharmacological activation of SMN2 exon 7 inclusion by small molecules or modified antisense oligonucleotides is a promising approach to treat SMA. Getting new, potentially therapeutic, compounds to clinical trials is a long and expensive process. Drug repositioning approach can reduce the transition time from screen to human tests, especially when undertaken in a whole organism like Drosophila melanogaster. Results Here we describe a reporter system, based on the SMN2 minigene (Zhang, Lorson et al. 2001), informative of exon 7 splicing modulation in Drosophila motor neurons. The inclusion of exon 7 results in translation of functional luciferase and luminescence detection. The higher the inclusion rate the stronger the signal, which has been confirmed by feeding the flies with compounds known to promote splicing of a full-length transcript. The highly conserved Drosophila blood-brain barrier makes the screening selective to compounds with the ability to target central nervous system. This screening model was used to test 1100 drugs from Prestwick Chemical Library, with a 2.5% hit rate. The 11 most promising drugs were tested in SMA patient-derived fibroblasts were 3 of them significantly increased the SMN protein levels. The best hit (GT5) was further validated and cells were treated with the maximal non-toxic GT5 concentration (50 µM) for 72 h. The drug significantly increased the full-length SMN2 transcript and SMN protein levels in a dose-dependent manner. The SMN immunofluorescence images from SMA patient-derived fibroblasts, treated with GT5, clearly confirm the increase of SMN protein level, compared to solvent treated cells. Conclusions We developed a Drosophila-based SMN2 spliceosensor system capable of detecting compounds that affect the minigene in fly motor neurons. One of the selected hits, named GT5, proved to significantly increase the SMN protein levels in SMA-patient derived fibroblasts.