CNS-focused high throughput screening for RyR-targeted therapeutics.

Using high-throughput screening (HTS) assays based on fluorescence lifetime (FLT)-detected FRET, we have identified compounds that allosterically modulate ryanodine receptor (RyR) calcium release channels, to prevent or reduce the elevated Ca2+ leak that fuels age-related neurodegeneration. RyRs are responsible for Ca release from intracellular storage organelles (endoplasmic reticulum – ER; sarcoplasmic reticulum – SR), and the resulting Ca signaling is essential for many cellular processes. RyRs have been intensively studied for decades in SR vesicles purified from skeletal muscle (RyR1) or heart (RyR2), where dysregulated RyR function – particularly a leaky state of the resting RyR – has been identified as a targetable culprit to interrupt the vicious cycle that compromises cell physiology in age-related pathologies. Recently RyR2 dysfunction in neurons has been implicated as a major contributor to Alzheimer's disease. Our FRET-based HTS detects the RyR leaky state by monitoring binding of the accessory proteins calmodulin, FKBP12.6, and DPc10. Under conditions that mimic a pathological state, we have carried out HTS of the 50k-compound CNS library (ChemBridge), to identify modulators of RyR2 in native SR membranes. This screen yielded 603 compounds (Hits) that reproducibly altered FRET between screens. With therapeutic feasibility a priority, freshly prepared stock solutions of 85 of the most promising compounds, validated for purity and chemical structure, were then evaluated for their dose-response in FRET and functional assays measuring 3[H]ryanodine binding. The resulting Hit compounds comprised seven unique chemical clusters that were either RyR isoform-specific or altered both RyR1 and RyR2 activity. Structure-activity relationship studies are ongoing. Good candidate compounds for treatment of neurodegeneration will progress to testing on RyR function in neurons from AD mouse models. This work is supported by NIH grant R43AG069582 (JJT/RLC).