A transcriptomic-based drug repositioning approach for the identification of novel muscle-specific therapies for spinal muscular atrophy

Abstract

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder (NMD) caused by depleted survival of motor neuron (SMN) levels and characterised by neuronal degeneration and progressive muscle atrophy. Although three approved SMN-dependent treatments have significantly halted disease progression, they are unfortunately not cures. Thus, additional musclespecific therapies are most likely also required to synergistically ameliorate symptoms in SMA patients. One useful strategy for the discovery of novel SMA muscle-specific therapies is drug repositioning (or repurposing), as using existing approved pharmacological compounds allows for the development of more costeffective treatments compared to traditional drug discovery. We have previously investigated drug repositioning in SMA and demonstrated that prednisolone, a synthetic glucocorticoid (GC), improved muscle health and survival in SMA mice. However, the adverse effects associated with chronic GC use limit prednisolone’s long-term therapeutic potential in SMA. We thus wanted to discover prednisolone-targeted genes and pathways in SMA skeletal muscle and identify commercially available drugs that similarly modulate these effectors.

We initially performed an RNA sequencing, bioinformatics and drug repositioning database pipeline on muscle from symptomatic post-natal day (P)7 prednisolonetreated and untreated Smn-/-;SMN2 SMA mice. These revealed that genes associated with atrophy, metabolism and muscle function pathways were targeted and normalised by prednisolone in SMA skeletal muscle. Furthermore, a total of 223 commercially approved compounds were predicted to similarly target these genes and pathways. We thus selected metformin, a generic antihyperglycaemic biguanide and oxandrolone, an anabolic steroid, for further investigation in SMA, based on their oral bioavailability, safety in infants and previously reported benefits in related conditions.

Metformin was predicted to emulate prednisolone’s activity by upregulating Prkag3 and downregulating Forkhead box O (FoxO) expression. We indeed confirmed that Prkag3 was significantly downregulated in muscle from Smn-/- ;SMN2 and Smn2B/- SMA mice. Furthermore, in vitro experiments in C2C12 myoblast-like cells suggest that the dysregulation of metformin’s molecular targets are SMN-independent and linked to atrophy. However, metformin treatment in both C2C12 cells and Smn2B/- SMA mice (200 mg/kg/day) did not improve disease progression. Furthermore, a higher dose of metformin (400 mg/kg/day) significantly exacerbated disease progression in Smn2B/- SMA mice, which were most likely due to mitochondrial marker dysfunction in the spinal cord. On the other hand, oxandrolone was predicted to upregulate the expression of the androgen receptor (Ar) and its downstream components. However, analyses in both C2C12 cells and muscle from Smn-/-;SMN2 and Smn2B/- SMA mice revealed that most of the predicted oxandrolone targets were in fact not dysregulated. Still, oxandrolone treatment rescued canonical atrophy in C2C12 myotubes and slightly improved survival in Smn2B/- SMA mice (4 mg/kg/day).

Taken together, our in vitro and in vivo experiments revealed that metformin and oxandrolone did not successfully emulate prednisolone’s activity in SMA, suggesting that our in silico approach requires refinement for a better prediction of valid drug candidates. Nevertheless, our discovery of prednisolone-targeted pathways and extensive list of drug candidates supports the usefulness of a transcriptomic-based drug repositioning strategy, and that with alterations, it can be quite beneficial for future therapeutic endeavours in SMA.