High-throughput screening identifies modulators of sarcospan that stabilize muscle cells and exhibit activity in the mouse model of Duchenne muscular dystrophy

Cynthia Shu,Jesus Campagna,Robert Damoiseaux,Rachelle H Crosbie,Ekaterina Mokhonova,Liubov Parfenova,Varghese John,Judd R Collado

doi:10.1186/s13395-020-00244-3

Abstract

BackgroundDuchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by mutations in the dystrophin gene. Loss of dystrophin prevents the formation of a critical connection between the muscle cell membrane and the extracellular matrix. Overexpression of sarcospan (SSPN) in the mouse model of DMD restores the membrane connection and reduces disease severity, making SSPN a promising therapeutic target for pharmacological upregulation.MethodsUsing a previously described cell-based promoter reporter assay of SSPN gene expression (hSSPN-EGFP), we conducted high-throughput screening on libraries of over 200,000 curated small molecules to identify SSPN modulators. The hits were validated in both hSSPN-EGFP and hSSPN-luciferase reporter cells. Hit selection was conducted on dystrophin-deficient mouse and human myotubes with assessments of (1) SSPN gene expression using quantitative PCR and (2) SSPN protein expression using immunoblotting and an ELISA. A membrane stability assay using osmotic shock was used to validate the functional effects of treatment followed by cell surface biotinylation to label cell surface proteins. Dystrophin-deficient mdx mice were treated with compound, and muscle was subjected to quantitative PCR to assess SSPN gene expression.ResultsWe identified and validated lead compounds that increased SSPN gene and protein expression in dystrophin-deficient mouse and human muscle cells. The lead compound OT-9 increased cell membrane localization of compensatory laminin-binding adhesion complexes and improved membrane stability in DMD myotubes. We demonstrated that the membrane stabilizing benefit is dependent on SSPN. Intramuscular injection of OT-9 in the mouse model of DMD increased SSPN gene expression.ConclusionsThis study identifies a pharmacological approach to treat DMD and sets the path for the development of SSPN-based therapies.

Highlights

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by mutations in the dystrophin gene
We demonstrated that the membrane stabilizing benefit is dependent on SSPN
The reporter cells used in the assay were C2C12 murine myoblasts stably transfected with a construct containing the human SSPN promoter region followed by the coding sequence for enhanced green fluorescent protein

Summary

Introduction

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by mutations in the dystrophin gene. Loss of dystrophin prevents the formation of a critical connection between the muscle cell membrane and the extracellular matrix. Duchenne muscular dystrophy (DMD) is a degenerative muscle disease that affects 1 in every 5700 males [1]. DMD is caused by mutations in the gene that encodes the dystrophin protein. The only Food and Drug Administration approved treatments for DMD are corticosteroids and exon skipping therapies. Corticosteroids extended the age of loss of ambulation by several years, and exon skipping therapies slowed disease progression, but neither is considered a cure [2, 3].

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