RyR1-targeted drug discovery pipeline integrating FRET-based high-throughput screening and human myofiber dynamic Ca assays.

Elevated cytoplasmic [Ca] is characteristic in severe skeletal and cardiac myopathies, diabetes, and neurodegeneration, and partly results from increased Ca leak from sarcoplasmic reticulum stores via dysregulated ryanodine receptor (RyR) channels. Consequently, RyR is recognized as a high-value target for drug discovery to treat such pathologies. Using a FRET-based high-throughput screening assay that we previously reported, we identified small-molecule compounds that modulate the skeletal muscle channel isoform (RyR1) interaction with calmodulin and FK506 binding protein 12.6. Two such compounds, chloroxine and myricetin, increase FRET and inhibit [H]ryanodine binding to RyR1 at nanomolar Ca. Both compounds also decrease RyR1 Ca leak in human skinned skeletal muscle fibers. Furthermore, we identified compound concentrations that reduced leak by > 50% but only slightly affected Ca release in excitation-contraction coupling, which is essential for normal muscle contraction. This report demonstrates a pipeline that effectively filters small-molecule RyR1 modulators towards clinical relevance.