High-throughput screening identified the isoxazole group of compounds as potential therapeutics targeting the pathological RyR2 calcium leak associated with arrhythmia and heart failure.
In high-throughput screening (HTS) assays using fluorescence lifetime (FLT)-detected FRET, we have identified compounds that allosterically modulate the pathologically leaky ryanodine receptor (RyR) calcium release channels. These compounds may prevent or reduce the elevated Ca 2+ that fuels arrhythmia, heart failure, and age-related neurodegeneration. RyRs are responsible for intracellular Ca 2+ release from endoplasmic/sarcoplasmic reticulum (ER/SR). The resulting Ca 2+ pulse is a signal for many cellular processes, whereas sustained elevated Ca 2+ is pathologic. Our FRET-based HTS detects the pathology-linked RyR leaky state by monitoring binding of the accessory protein calmodulin and the DPc10 peptide (corresponding to RyR2 residues 2460–2495) known to perturb inter-domain interactions within RyR2. Under conditions mimicking a pathological state, we have screened a 50,000-compound chemical library to identify small-molecule modulators of RyR2 in cardiac SR membranes. This screen yielded 603 compounds that reproducibly altered FRET. Based on FRET response profiles that align with therapeutic potential, 83 of those most promising compounds were purchased and validated by FRET dose response evaluation. Focusing on ten chemical scaffolds that desirably increase A-CaM binding, six representative compounds reduced RyR2 activity as measured by 3 Hryanodine binding. Ca 2+ dynamics in HEK293 cells expressing human RyR2 or in cardiomyocytes highlighted the isoxazole group of hits as potentially therapeutic by targeting the pathological RyR2 leak state.
Nikolaienko et al. (Thu,) studied this question.