Elevated cytoplasmic [Ca2+] is characteristic in severe skeletal and cardiac myopathies, diabetes, and neurodegeneration, and partly results from increased Ca2+ 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 [3H]ryanodine binding to RyR1 at nanomolar Ca2+. Both compounds also decrease RyR1 Ca2+ leak in human skinned skeletal muscle fibers. Furthermore, we identified compound concentrations that reduced leak by > 50% but only slightly affected Ca2+ 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.
Bibliographical noteFunding Information:
This work was supported by American Heart Association Grant-in-Aid 15GRNT25610022 (R.L.C.) and Postdoctoral Fellowship 16POST31010019 (R.T.R.), NIH grants R01HL092097 and R01HL138539 (R.L.C., D.M.B.), R01GM027906, R37AG026160, and R01HL129814 (D.D.T). Samantha Yuen and Ji Li assisted with library formatting. Kurt Peterson, Benjamin Grant, and Ji Li provided helpful discussions for undertaking the HTS. HTS was performed using the facilities provided by Fluorescence Innovations, Inc. (Minneapolis, MN, USA), with assistance from Benjamin Grant and Kurt Peterson. Gabrielle Evans assisted with FRET dose response. Gabrielle Evans and Opoku Akyeampong assisted with FKBP binding assays.
© 2020, The Author(s).
- Anti-Bacterial Agents/pharmacology
- Calcium Signaling/drug effects
- Drug Discovery
- Fluorescence Resonance Energy Transfer
- Muscle, Skeletal/drug effects
- Ryanodine Receptor Calcium Release Channel/metabolism
PubMed: MeSH publication types
- Research Support, Non-U.S. Gov't
- Journal Article
- Research Support, N.I.H., Extramural