High-throughput discovery of actin-binding compounds for cardiac disorders

Interactions between actin and myosin generate contractile force in muscle, and their dysregulation contributes to severe myopathies. While small-molecule therapeutics targeting myosin have progressed, actin remains a largely unexplored target. We sought to identify small molecules that bind actin and differentially modulate cardiac and skeletal actomyosin interactions, with the goal of developing cardiac-specific therapeutics. We previously established a time-resolved fluorescence resonance energy transfer (TR-FRET) biosensor, consisting of fluorescently labeled actin (donor) and a 12-amino acid peptide (ANT) mimicking the myosin essential light chain N-terminus (acceptor). Following previous validation in a 384-well pilot screen, we have now miniaturized the FRET assay to a 1536-well format and screened a library of 2,684 FDA-approved compounds. Multiple compounds significantly altered the actin-myosin interaction, with several showing greater effects on cardiac than skeletal muscle. Identified compounds either enhanced or inhibited actomyosin activity-effects consistent with potential therapeutic mechanisms for dilated or hypertrophic cardiomyopathy, respectively. This study establishes a robust, high-throughput, actin-targeted screening platform and identifies actin-binding small molecules with isoform-specific effects. These findings support the feasibility of developing actin-directed therapeutics for cardiac disease.