Toward drug treatment of spinal muscular atrophy: Mechanism of action

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 DESCRIPTION (provided by applicant): Low levels of the SMN protein produced from the SMN2 gene are the cause of spinal muscular atrophy (SMA), an inherited disease of motor neuron degeneration. We successfully implemented a high throughput chemical diversity screen for compounds that increase cellular levels of this protein. Following a synthetic chemistry program, we identified two unique chemical scaffolds called LDN-75 and LDN-76 that increase SMN expression in cell culture. Critical validation studies in a severe murine model of SMA demonstrated these compounds increase SMN protein in brain, mean survival, and gross motor function. This grant proposal seeks to determine the mechanism of action of these active pre-clinical lead candidates. Our initial characterization reveals that the two series of compounds act through distinct mechanisms. Quantitative real-time PCR demonstrates that compounds from the LDN-76 series activate transcription with a proportional increase in inclusion of SMN2 exon 7. While the LDN-75 series had no detectable effect on SMN2 mRNA levels or processing, these compounds stabilize the SMN protein. Consistent with these results, we find that compounds from the two series show synergistic increases in SMN protein, further evidence that these act through distinct mechanisms. Aim 1 will determine whether LDN-75 compounds alter the ubiquitination or proteasome mediated destruction of SMN protein. It has been reported that protein kinase A (PKA) activation stimulated SMN incorporation into a stable complex. We will explore the possibility that LDN-75 regulates phosphorylation of SMN and increases its stability. In Aim 2, we will investigate the role of the PI3K/AKT/Wnt signaling pathway as a target for LDN-76. We hypothesize that transcription of SMN is modified by the beta- catenin pathway and that this is the downstream target for the LDN-76 based compounds. Aim 3 describes an affinity based approach to capture protein targets of our lead compounds. Mass sequencing will be performed for target identification. Genetic and biochemical experiments will be used to validate the candidate factors for effects on SMN expression and interaction with the lead compounds. There is no FDA approved treatment for SMA. While several new therapeutic designs are in development, there are no published data that any are clinically effective or will be absent of prohibitive side effects. The results generated in this R21 will supply invaluable insights into potential safety and toxicity concerns for our active lead compounds and potentially provide insights into chemical refinement of our leads.