Key result
A FRET-based high-throughput screen of 727 compounds identified six hits that reproducibly changed FRET by >3 SD, validating the method to discover ryanodine receptor modulators.
A novel FRET-based high-throughput screening method successfully identified small-molecule modulators of the ryanodine receptor, providing a tool for discovering therapies for cardiac and skeletal myopathies associated with RyR dysregulation.
Opens RyR drug discovery; leaves open clinical translation for cardiac Ca2+ disorders.
Using time-resolved fluorescence resonance energy transfer (FRET), we have developed and validated the first high-throughput screening (HTS) method to discover compounds that modulate an intracellular Ca 2+ channel, the ryanodine receptor (RyR), for therapeutic applications. Intracellular Ca 2+ regulation is critical for striated muscle function, and RyR is a central player. At resting [Ca 2+ ], an 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 an attractive target for pharmacological agents to treat such pathologies. Our FRET-based HTS detects RyR binding of accessory proteins calmodulin (CaM) or FKBP12.6. Under conditions that mimic a pathological state, we carried out a screen of the 727-compound NIH Clinical Collection, which yielded six compounds that reproducibly changed FRET by >3 SD. Dose–response of FRET and [ 3 H]ryanodine binding readouts reveal that five hits reproducibly alter RyR1 structure and activity. One compound increased FRET and inhibited RyR1, which was only significant at nM [Ca 2+ ], and accentuated without CaM present. These properties characterize a compound that could mitigate RyR1 leak. An excellent Z′ factor and the tight correlation between structural and functional readouts validate this first HTS method to identify RyR modulators.
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Rebbeck et al. (2016) studied Ryanodine receptor dysregulation. FRET-based high-throughput screening was evaluated on Identification of compounds that reproducibly changed FRET by >3 SD. A FRET-based high-throughput screen of 727 compounds identified six hits that reproducibly changed FRET by >3 SD, validating the method to discover ryanodine receptor modulators.
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