Poster - #UCLA-74 - Keywords: Duchenne Muscular Dystrophy Sarcospan Small Molecules Therapy

Small molecules stabilizing cell membrane for treatment of Duchenne muscular dystrophy

Ekaterina Mokhonova, Ravinder Malik, Cynthia Shu, Jesus Campagna, Whitaker Cohn, Dongwook Wi, Barbara Jagodzinska, Varghese John, Robert Damoiseaux, Rachelle H. Crosbie
Department of Integrative Biology and Physiology, Molecular Biology Institute, Department of Molecular and Medicinal Pharmacology, California NanoSystems Institute, Drug Discovery Lab, Department of Neurology, David Geffen School of Medicine, University of California Los Angeles

ABSTRACT:
Duchenne muscular dystrophy (DMD) is the most common lethal genetic disorder in children, characterized by progressive muscle wasting leading to severe muscle weakness, cardiac, and pulmonary failure. DMD is caused by loss of dystrophin, which normally forms a protein complex that connects the actin cytoskeleton, muscle membrane, and extracellular matrix. Sarcospan (SSPN) is a transmembrane protein that interacts with several major adhesion complexes. Overexpression of SSPN in the mouse model of DMD prevents the disease by restoring membrane stability. To identify pharmacological methods of increasing SSPN expression, we created a hSSPN promoter reporter assay and conducted an screened over 200,000 small molecules and rescreened the top 1,000 hits in two reporter cell lines. 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) sarcospan gene expression and 2) sarcospan protein expression. A membrane stability assay using osmotic shock was used to validate the functional effects of treatment. Dystrophin-deficient mdx mice were treated with compound and muscle was subjected to quantitative PCR to assess SSPN gene expression. The lead compound OT-9 increased cell membrane localization of compensatory laminin-binding adhesion complexes and improved membrane stability in DMD myotubes. Intramuscular injection of OT-9 in the mouse model of DMD increased sarcospan gene expression. More soluble analogue OT-9m increased sarcospan gene expression in mdx mice after local and systemic administration by subcutaneous injections and by oral administration. Systemic oral treatment by OT-9m increases level of sarcospan gene 1.7 to 2-fold in skeletal muscles and rescues sarcolemma localization of sarcospan, utrophin, b-Integrin and dystroglycans. This study identifies a pharmacological approach to treat DMD and sets the path for the development of SSPN-based therapies