High-Throughput Screening Yields Allosteric Inhibitors of Leaky RyRs for Therapeutic Development

Abstract

Using fluorescence lifetime detection of FRET, we have developed and validated high-throughput screening (HTS) assays to discover compounds that modulate the ryanodine receptor (RyR) calcium release channel, for therapeutic development. Intracellular calcium regulation is critical for striated muscle function, and RyR is a central player. Under resting calcium, increased propensity of channel opening due to RyR dysregulation is associated with severe cardiac and skeletal myopathies, diabetes and neurological disorders. This leaky state of the RyR is a high-value target for pharmacological agents to treat such pathologies. Our FRET-based HTS detects RyR binding of accessory proteins calmodulin or FKBP12.6. Under conditions that mimic a pathological state, we have carried out pilot screens of the 1280-compound library of pharmaceutically active compounds (LOPAC), and then progressed to larger screens of a 50k-compound library, to identify modulators of RyR in either skeletal or cardiac SR membrane preparations. These screens yielded compounds (hits) that are specific for either RyR1 (skeletal isoform) or RyR2 (cardiac isoform), or that act on both RyR1 and RyR2. Ongoing studies indicate good correlation between the FRET (structural) readout for the hits and their effects on RyR function in isolated sarcoplasmic reticulum, skeletal myofibers, and adult ventricular myocytes. This work is supported by NIH grants R01HL092097 and R01HL138539 (RLC/DMB) and R37AG26160 (DDT), and by American Heart Association Postdoctoral Fellowship 16POST31010019 (RTR).