Duchenne muscular dystrophy (DMD) 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 domain1 (ABD1). We engineered a novel fluorescence resonance energy transfer (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, high-throughput screening (HTS) fluorescence lifetime assay, identifying several compounds from a 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. 0.5 DTT. hDYS-ABD1 compound to the sensitivity of the cosedimentation assay. incubated 30 centrifuged 50K for 30 fraction of ABD1 actin quantified using SDS- and Coomassie blue staining Compound effect on ABD1 aggregation measured using a control sample lacked actin. simple linear single hyperbolic model, the maximum bound

Journal Pre-proof Enhancing interaction of actin and actin-binding domain 1 of dystrophin with modulators: toward improved gene therapy for Duchenne muscular dystrophy

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Journal Pre-proof Enhancing interaction of actin and actin-binding domain 1 of dystrophin with modulators: toward improved gene therapy for Duchenne muscular dystrophy