Drosophila SMN2minigene reporter model identifies moxifloxacin as a candidate therapy for SMA

Abstract

Spinal muscular atrophy is a rare and fatal neuromuscular disorder caused by the loss of alpha motor neurons. The affected individuals have mutated the ubiquitously expressed SMN1 gene resulting in the loss or reduction in the survival motor neuron (SMN) protein levels. However, an almost identical paralog exists in humans: SMN2.Pharmacological activation of SMN2 exon 7 inclusion by small molecules or modified antisense oligonucleotides is a valid approach to treat SMA. Here we describe an in vivo SMN2 minigene reporter system in Drosophila motor neurons that serves as a cost-effective, feasible, and stringent primary screening model for identifying chemicals capable of crossing the conserved Drosophila blood-brain barrier and modulating exon 7 inclusion. The model was used for the screening of 1100 drugs from the Prestwick Chemical Library, resulting in 2.45% hit rate. The most promising candidate drugs were validated in patient-derived fibroblasts where they proved to increase SMN protein levels. Among them, moxifloxacin modulated SMN2 splicing by promoting exon 7 inclusion. The recovery of SMN protein levels was confirmed by increased colocalization of nuclear gems with Cajal Bodies. Thus, a Drosophilabased drug screen allowed the discovery of an FDA-approved small molecule with the potential to become a novel therapy for SMA. K E Y W O R D S drug repurposing, SMA, SMN2, splicing 3022 | KONIECZNY aNd aRTERO | 3035 KONIECZNY aNd aRTEROtogether we have managed to use Drosophila as an in vivo tool to discover compounds with interesting properties on an SMN2 exon 7 reporter construct as evidenced by the ability of moxifloxacin to modulate exon 7 inclusion in the fly motorneurons.

RNA-Targeted Therapies and High-Throughput Screening Methods

“…Another very recent HTS described moxifloxacin as an exon 7 inclusion enhancer using a SMN2 minigene reporter in Drosophila motor neurons. The effects were validated in patient-derived fibroblasts [110].…”

Drug Screening and Drug Repositioning as Promising Therapeutic Approaches for Spinal Muscular Atrophy Treatment

“…On the contrary, DR (also currently referred as drug repurposing, reprofiling, retasking, or therapeutic switching) consists in a strategy to attribute new uses to drugs (generally already FDA approved) that are outside the scope of the original medical indications reducing risks and costs associated with time consuming new drug development programs. Briefly, the two approaches, eventually in combination, exploit the availability of large compound libraries, which include thousands of chemical and natural compounds and/or FDA-approved substances: Prestwick Chemical Library, MicroSource Discovery Systems, ComGenex, National Institute for Neurological Disorders and Stroke, TimTec, IBS, and ChemBridge are just few libraries that have been used to find new SMA therapies during the last years (Kelley et al, 2004;Sleigh et al, 2011;Konieczny and Artero, 2020). Afterward, once screened in search of specific cellular/ molecular readouts, the substances can be firstly tested on "simplified" SMA models (cell cultures and/or invertebrates models), and then (or directly, in some cases) on SMA mice and/or patient-derived iPSCs (Figure 1).…”

“…However, the most recent fly SMA model has been developed by inserting the human SMN2 minigene reporter, fused to luciferase, into the Drosophila genome, in order to easily obtain the exon 7inclusion during SMN2 splicing process. This cheap and feasible model allows to rapidly screen thousands of chemicals, possibly increasing FL-SMN protein levels (Konieczny and Artero, 2020).…”

“…Indeed, Aclarubicin seems to act as a transcriptional activator, inducing by indirect pathways SMN2 exon seven inclusion (Morceau et al, 1996;Andreassi et al, 2001). Likewise, Moxifloxacin, a synthetic fluoroquinolone antibiotic used to treat several infections, was identified by performing Highthroughput screening study in a Drosophila SMN2 minigene reporter model, as described above (Konieczny and Artero, 2020). Moxifloxacin modulates SMN2 splicing by promoting exon seven inclusion and crosses the Drosophila BBB; moreover, it increases FL-SMN expression in HeLa cell lines.…”

“…Moxifloxacin modulates SMN2 splicing by promoting exon seven inclusion and crosses the Drosophila BBB; moreover, it increases FL-SMN expression in HeLa cell lines. The authors showed that Moxifloxacin exerts a dose-dependent increase of Serine/arginine Rich Splicing Factor 1 (SRSF1) levels promoting the SMN2 exon seven inclusion (Konieczny and Artero, 2020).…”