Duchenne muscular dystrophy is a lethal muscle disease, caused by mutations in the gene encoding dystrophin, an actin-binding cytoskeletal protein. Absence of functional dystrophin results in muscle weakness and degeneration, eventually leading to cardiac and respiratory failure. Strategies to replace the missing dystrophin via gene therapy have been intensively pursued. However, the dystrophin gene is too large for current gene therapy approaches. Currently available micro-dystrophin constructs lack the actin-binding domain 2 and show decreased actin-binding affinity in vitro compared to full-length dystrophin. Thus, increasing the actin-binding affinity of micro-dystrophin, using small molecules, could be a beneficial therapeutic approach. Here, we have developed and validated a novel high-throughput screening (HTS) assay to discover small molecules that increase the binding affinity of dystrophin's actin-binding domain 1 (ABD1). We engineered a novel FRET biosensor, consisting of the mClover3, fluorescent protein (donor) attached to the C-terminus of dystrophin ABD1, and Alexa Fluor 568 (acceptor) attached to the C-terminal cysteine of actin. We used this biosensor in small-molecule screening, using a unique high-precision, HTS fluorescence lifetime assay, identifying several compounds from an FDA-approved library that significantly increase the binding between actin and ABD1. This HTS assay establishes feasibility for the discovery of small-molecule modulators of the actin–dystrophin interaction, with the ultimate goal of developing therapies for muscular dystrophy.
|Original language||English (US)|
|Journal||Journal of Biological Chemistry|
|State||Published - Dec 2022|
Bibliographical noteFunding Information:
We thank Samantha Yuen for technical assistance with the fluorescence lifetime plate reader and Dr Robyn Rebbeck for helpful discussion. This work was funded by Sarepta Therapeutics (215 First Street, Cambridge, MA 02142).
© 2022 The Authors
- high-throughput screening
- time-resolved FRET
PubMed: MeSH publication types
- Journal Article
- Research Support, Non-U.S. Gov't