Cardiac ryanodine receptor N-terminal region biosensors identify novel inhibitors via FRET-based high-throughput screening. Journal Article


Authors: Zhang, J; Singh, DP; Ko, CY; Nikolaienko, R; Wong King Yuen, SM; Schwarz, JA; Treinen, LM; Tung, CC; Rožman, K; Svensson, B; Aldrich, CC; Zima, AV; Thomas, DD; Bers, DM; Launikonis, BS; Van Petegem, F; Cornea, RL
Article Title: Cardiac ryanodine receptor N-terminal region biosensors identify novel inhibitors via FRET-based high-throughput screening.
Abstract: The N-terminal region (NTR) of ryanodine receptor (RyR) channels is critical for the regulation of Ca release during excitation-contraction (EC) coupling in muscle. The NTR hosts numerous mutations linked to skeletal (RyR1) and cardiac (RyR2) myopathies, highlighting its potential as a therapeutic target. Here, we constructed two biosensors by labeling the mouse RyR2 NTR at domains A, B, and C with FRET pairs. Using fluorescence lifetime (FLT) detection of intramolecular FRET signal, we developed high-throughput screening (HTS) assays with these biosensors to identify small-molecule RyR modulators. We then screened a small validation library and identified several hits. Hits with saturable FRET dose-response profiles and previously unreported effects on RyR were further tested using [H]ryanodine binding to isolated sarcoplasmic reticulum vesicles to determine effects on intact RyR opening in its natural membrane. We identified three novel inhibitors of both RyR1 and RyR2, and two RyR1-selective inhibitors effective at nanomolar Ca. Two of these hits activated RyR1 only at micromolar Ca, highlighting them as potential enhancers of EC coupling. To determine whether such hits can inhibit RyR leak in muscle, we further focused on one, an FDA-approved natural antibiotic, fusidic acid (FA). In skinned skeletal myofibers and permeabilized cardiomyocytes, FA was demonstrated to inhibit RyR leak with no detrimental effect on skeletal myofiber EC coupling. However, in intact cardiomyocytes FA induced arrhythmogenic Ca transients, a cautionary observation for a compound with an otherwise solid safety record. These results indicate that HTS campaigns using the NTR biosensor can identify compounds with therapeutic potential.
Journal Title: The Journal of biological chemistry
ISSN: 1083-351X; 0021-9258
Publisher: Unknown  
Date Published: 2021