Large-scale high-throughput screen for cardiac ryanodine receptor targeted therapeutics.

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 Ca2+ that fuels arrhythmia, heart failure, and age-related neurodegeneration. RyRs are responsible for intracellular Ca2+ release from endoplasmic/sarcoplasmic reticulum (ER/SR). The resulting [Ca2+] pulse is a signal for many cellular processes, whereas sustained elevated [Ca2+] 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 interdomain 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 [3H]ryanodine binding. Ca2+ 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.