Synergistic FRET assays for drug discovery targeting RyR2 channels

Robyn T. Rebbeck, Kenneth S. Ginsburg, Christopher Y. Ko, Anna Fasoli, Katherine Rusch, George F. Cai, Xiaoqiong Dong, David D. Thomas, Donald M. Bers, Razvan L. Cornea

Research output: Contribution to journalArticlepeer-review


A key therapeutic target for heart failure and arrhythmia is the deleterious leak through sarcoplasmic reticulum (SR) ryanodine receptor 2 (RyR2) calcium release channels. We have previously developed methods to detect the pathologically leaky state of RyR2 in adult cardiomyocytes by monitoring RyR2 binding to either calmodulin (CaM) or a biosensor peptide (DPc10). Here, we test whether these complementary binding measurements are effective as high-throughput screening (HTS) assays to discover small molecules that target leaky RyR2. Using FRET, we developed and validated HTS procedures under conditions that mimic a pathological state, to screen the library of 1280 pharmaceutically active compounds (LOPAC) for modulators of RyR2 in cardiac SR membrane preparations. Complementary FRET assays with acceptor-labeled CaM and DPc10 were used for Hit prioritization based on the opposing binding properties of CaM vs. DPc10. This approach narrowed the Hit list to one compound, Ro 90–7501, which altered FRET to suggest increased RyR2-CaM binding and decreased DPc10 binding. Follow-up studies revealed that Ro 90–7501 does not detrimentally affect myocyte Ca2+ transients. Moreover, Ro 90–7501 partially inhibits overall Ca2+ leak, as assessed by Ca2+ sparks in permeabilized rat cardiomyocytes. Together, these results demonstrate (1) the effectiveness of our HTS approach where two complementary assays synergize for Hit ranking and (2) a drug discovery process that combines high-throughput, high-precision in vitro structural assays with in situ myocyte assays of the pathologic RyR2 leak. These provide a drug discovery platform compatible with large-scale HTS campaigns, to identify agents that inhibit RyR2 for therapeutic development.

Original languageEnglish (US)
Pages (from-to)13-23
Number of pages11
JournalJournal of Molecular and Cellular Cardiology
StatePublished - Jul 2022

Bibliographical note

Funding Information:
This work was supported by NIH grants R01-HL138539 and R01-HL092097 (RLC and DMB), P01-HL141084 and R01-HL142282 (DMB), F32-HL144017 (CYK), R37-AG026160 (DDT), and R43-AG069582 (RLC and DDT).

Publisher Copyright:
© 2022 The Authors


  • Calcium channel
  • Calcium leak
  • Fluorescence lifetime
  • RyR
  • Sarco/endoplasmic reticulum

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural


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