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High-throughput screening identifies modulators of sarcospan that stabilize muscle cells and exhibit activity in the mouse model of Duchenne muscular dystrophy
BACKGROUND: 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. Overexpression of sarcospan (SSPN) in the mouse...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499884/ https://www.ncbi.nlm.nih.gov/pubmed/32948250 http://dx.doi.org/10.1186/s13395-020-00244-3 |
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author | Shu, Cynthia Parfenova, Liubov Mokhonova, Ekaterina Collado, Judd R. Damoiseaux, Robert Campagna, Jesus John, Varghese Crosbie, Rachelle H. |
author_facet | Shu, Cynthia Parfenova, Liubov Mokhonova, Ekaterina Collado, Judd R. Damoiseaux, Robert Campagna, Jesus John, Varghese Crosbie, Rachelle H. |
author_sort | Shu, Cynthia |
collection | PubMed |
description | BACKGROUND: 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. 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. METHODS: Using 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. RESULTS: We 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. CONCLUSIONS: This study identifies a pharmacological approach to treat DMD and sets the path for the development of SSPN-based therapies. |
format | Online Article Text |
id | pubmed-7499884 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-74998842020-09-21 High-throughput screening identifies modulators of sarcospan that stabilize muscle cells and exhibit activity in the mouse model of Duchenne muscular dystrophy Shu, Cynthia Parfenova, Liubov Mokhonova, Ekaterina Collado, Judd R. Damoiseaux, Robert Campagna, Jesus John, Varghese Crosbie, Rachelle H. Skelet Muscle Research BACKGROUND: 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. 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. METHODS: Using 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. RESULTS: We 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. CONCLUSIONS: This study identifies a pharmacological approach to treat DMD and sets the path for the development of SSPN-based therapies. BioMed Central 2020-09-18 /pmc/articles/PMC7499884/ /pubmed/32948250 http://dx.doi.org/10.1186/s13395-020-00244-3 Text en © The Author(s) 2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Shu, Cynthia Parfenova, Liubov Mokhonova, Ekaterina Collado, Judd R. Damoiseaux, Robert Campagna, Jesus John, Varghese Crosbie, Rachelle H. High-throughput screening identifies modulators of sarcospan that stabilize muscle cells and exhibit activity in the mouse model of Duchenne muscular dystrophy |
title | High-throughput screening identifies modulators of sarcospan that stabilize muscle cells and exhibit activity in the mouse model of Duchenne muscular dystrophy |
title_full | High-throughput screening identifies modulators of sarcospan that stabilize muscle cells and exhibit activity in the mouse model of Duchenne muscular dystrophy |
title_fullStr | High-throughput screening identifies modulators of sarcospan that stabilize muscle cells and exhibit activity in the mouse model of Duchenne muscular dystrophy |
title_full_unstemmed | High-throughput screening identifies modulators of sarcospan that stabilize muscle cells and exhibit activity in the mouse model of Duchenne muscular dystrophy |
title_short | High-throughput screening identifies modulators of sarcospan that stabilize muscle cells and exhibit activity in the mouse model of Duchenne muscular dystrophy |
title_sort | high-throughput screening identifies modulators of sarcospan that stabilize muscle cells and exhibit activity in the mouse model of duchenne muscular dystrophy |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499884/ https://www.ncbi.nlm.nih.gov/pubmed/32948250 http://dx.doi.org/10.1186/s13395-020-00244-3 |
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