Synergistic FRET assays for drug discovery targeting RyR2 channels.

Autor: Rebbeck R; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, MN, USA., Ginsburg KS; Department of Pharmacology, University of California, Davis, CA, USA., Ko CY; Department of Pharmacology, University of California, Davis, CA, USA., Fasoli A; Department of Pharmacology, University of California, Davis, CA, USA., Rusch K; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, MN, USA., Cai GF; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, MN, USA., Dong X; Department of Pharmacology, University of California, Davis, CA, USA., Thomas DD; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, MN, USA; Photonic Pharma LLC, Minneapolis, MN, USA., Bers DM; Department of Pharmacology, University of California, Davis, CA, USA., Cornea RL; Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, MN, USA; Photonic Pharma LLC, Minneapolis, MN, USA. Electronic address: corne002@umn.edu.
Jazyk: angličtina
Zdroj: Journal of molecular and cellular cardiology [J Mol Cell Cardiol] 2022 Jul; Vol. 168, pp. 13-23. Date of Electronic Publication: 2022 Apr 09.
DOI: 10.1016/j.yjmcc.2022.04.002
Abstrakt: 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 Ca 2+ transients. Moreover, Ro 90-7501 partially inhibits overall Ca 2+ leak, as assessed by Ca 2+ 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.
(Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)
Databáze: MEDLINE
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