BPS2026 – Calcium channel and transporter targeted modulators identified by high-throughput screening

Using high-throughput screening (HTS) assays, we have identified compounds that modulate sarcoplasmic reticulum’s (SR) Ca 2+ cycling, either through the ryanodine receptor (RyR) channel or S/ER Ca 2+ ATPase transporter. Intracellular Ca 2+ regulation is critical for striated muscle contraction, with SR Ca 2+ release via RyR opening and Ca 2+ uptake via SERCA. Under cellular rest, increased propensity of channel opening due to RyR dysregulation and/or decreased SERCA activity/expression is associated with severe cardiac and skeletal myopathies, and neurodegenerative diseases. Thus, the leaky states of the RyR or reduced SERCA activity are high-value pharmacological targets for treating such pathologies. Our RyR-targeted FRET-based HTS platform detects the RyR leaky state in skeletal or cardiac SR membrane preparations by monitoring binding of the accessory proteins calmodulin, FKBP12.0, FKBP12.6, and DPc10. Our SERCA-targeted FRET-based HTS platform involves monitoring structural states of skeletal SERCA (SERCA1a) and cardiac SERCA (SERCA2a) and binding of small peptide regulators phospholamban, DWORF and sarcolipin. For multi-dimensional data analysis, we have a recently developed application that includes analysis, Hit selection, plate visualization, chemical clustering, and data comparison across multiple screening data sets. We have screened two distinct 50,000-compound libraries for modulators of RyR and SERCA. With excellent screening performance, these screens yielded 100s of compounds (Hits) that reproducibly altered FRET in a dose-responsive manner. The integration of chemical clustering and multidimensional data analysis has greatly facilitated Hit prioritization for functional testing. Of Hits validated in more physiological conditions, we are pursuing analogues to identify more potent RyR stabilizers and/or SERCA activators. Good candidate compounds for treatment of striated muscle myopathies are progressing to testing on resting Ca 2+ and Ca 2+ cycling in skeletal muscle fibers and adult ventricular myocytes. This work is supported by NIH grants R01HL139065, R01HL092097, and R01AR082533.