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

Skeletal Muscle

Liubov Parfenova¹  Judd R. Collado¹  Ekaterina Mokhonova²  Robert Damoiseaux³  Varghese John⁴  Jesus Campagna⁴  Cynthia Shu⁵  Rachelle H. Crosbie⁶ 

[1] Department of Integrative Biology and Physiology, University of California Los Angeles, 610 Charles E. Young Drive East, Terasaki Life Sciences Building, 90095, Los Angeles, CA, USA;Department of Integrative Biology and Physiology, University of California Los Angeles, 610 Charles E. Young Drive East, Terasaki Life Sciences Building, 90095, Los Angeles, CA, USA;Center for Duchenne Muscular Dystrophy, University of California Los Angeles, Los Angeles, CA, USA;Department of Molecular and Medicinal Pharmacology, University of California Los Angeles, Los Angeles, CA, USA;California NanoSystems Institute, University of California Los Angeles, Los Angeles, CA, USA;Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, 610 Charles E. Young Drive East, Terasaki Life Sciences Building, 90095, Los Angeles, CA, USA;Drug Discovery Lab, University of California Los Angeles, Los Angeles, CA, USA;Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, USA;Department of Integrative Biology and Physiology, University of California Los Angeles, 610 Charles E. Young Drive East, Terasaki Life Sciences Building, 90095, Los Angeles, CA, USA;Center for Duchenne Muscular Dystrophy, University of California Los Angeles, Los Angeles, CA, USA;Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, USA;Department of Integrative Biology and Physiology, University of California Los Angeles, 610 Charles E. Young Drive East, Terasaki Life Sciences Building, 90095, Los Angeles, CA, USA;Center for Duchenne Muscular Dystrophy, University of California Los Angeles, Los Angeles, CA, USA;Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, 610 Charles E. Young Drive East, Terasaki Life Sciences Building, 90095, Los Angeles, CA, USA;

关键词: Drug discovery;    Duchenne muscular dystrophy;    Dystrophin;    High-throughput screen;    Sarcolemma;    Sarcospan;    Small molecules;   

DOI  :  10.1186/s13395-020-00244-3

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY