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Synergistic FRET assays for drug discovery targeting RyR2 channels
RobynT Rebbeck1, Kenneth S Ginsburg2, Christopher Y Ko2
1Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, MN, USA.
Abstract:
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.
Insights
Researchers developed a high-throughput screening method to find drugs targeting leaky ryanodine receptor 2 (RyR2) calcium channels, crucial for heart failure. The study identified Ro 90-7501 as a promising compound that inhibits RyR2 leak without harming heart cells.
Area of Science:
- Cardiovascular Biology
- Pharmacology
- Biophysics
Background:
- The sarcoplasmic reticulum (SR) ryanodine receptor 2 (RyR2) calcium release channel is a key target for heart failure and arrhythmia therapies.
- Pathological leaks in RyR2 contribute to cardiac dysfunction.
- Previous methods monitored RyR2 binding to calmodulin (CaM) or DPc10 to detect leaky states.
Purpose of the Study:
- To evaluate complementary binding measurements as high-throughput screening (HTS) assays for discovering small molecules targeting leaky RyR2.
- To identify modulators of RyR2 in cardiac SR membrane preparations using HTS.
- To develop a drug discovery platform for RyR2 inhibitors.
Main Methods:
- Developed and validated FRET-based HTS procedures mimicking pathological RyR2 leak.
- Screened a library of 1280 pharmaceutically active compounds (LOPAC).
- Utilized complementary FRET assays with CaM and DPc10 for Hit prioritization.
Main Results:
- Identified Ro 90-7501 as a Hit compound, showing increased RyR2-CaM binding and decreased DPc10 binding.
- Ro 90-7501 did not adversely affect myocyte calcium transients.
- Ro 90-7501 partially inhibited Ca2+ leak, as evidenced by reduced Ca2+ sparks in cardiomyocytes.
Conclusions:
- The developed HTS approach, using synergistic complementary assays, is effective for Hit ranking.
- Ro 90-7501 is a promising therapeutic candidate for inhibiting RyR2 leak.
- The study presents a drug discovery platform combining in vitro and in situ assays for large-scale HTS campaigns targeting RyR2.
