High-Throughput Screening And Drug Development To Fix Ryr Pathology

Using fluorescence lifetime FRET-based high-throughput screening (HTS) assays, we identified compounds that modulate the sarcoplasmic reticulum's (SR) Ca2+ gatekeeper, the ryanodine receptor (RyR) channel. Intracellular Ca2+ regulation is critical for striated muscle function, and SR Ca2+ release via opening of RyR is essential for triggering muscle contraction. Under cellular rest, increased propensity of channel opening due to RyR dysregulation is associated with severe cardiac and skeletal myopathies, diabetes and neurological disorders. Thus, this leaky state of the RyR is a high-value pharmacological target for treating such pathologies. Our FRET-based HTS detects the RyR leaky state by monitoring binding of the accessory proteins calmodulin, FKBP12.0, FKBP12.6, and DPc10. Under conditions that mimic a pathological state, we carried out screens of a 50,000-compound library, to identify modulators of RyR in either skeletal or cardiac SR membrane preparations. These screens yielded >60 compounds (Hits) that reproducibly altered FRET. Following functional validation, the resulting 37 Hits fall within 10 chemical scaffold groups that are specific for either RyR1 (skeletal isoform) or RyR2 (cardiac isoform), or that act on both RyR1 and RyR2. With the first round of Hit analogue testing, we are pursuing >80 compounds to further inform medicinal chemistry synthesis of more potent RyR stabilizers. Good candidate compounds for treatment of striated muscle myopathies will progress to testing on RyR function in skeletal muscle fibers and adult ventricular myocytes. This work is supported by NIH grants R01HL092097 and R01HL138539 (RLC/DMB) and R37AG026160 (DDT).