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.