RyR1-targeted drug discovery pipeline integrating FRET-based high-throughput screening and human myofiber dynamic Ca2+ assays

Abstract

Elevated cytoplasmic [Ca 2+ ] is characteristic in severe skeletal and cardiac myopathies, diabetes, and neurodegeneration, and partly results from increased Ca 2+ leak from sarcoplasmic reticulum stores via dysregulated ryanodine receptor (RyR) channels. Consequently, RyR is recognized as a high-value target for drug discovery to treat such pathologies. Using a FRET-based high-throughput screening assay that we previously reported, we identified small-molecule compounds that modulate the skeletal muscle channel isoform (RyR1) interaction with calmodulin and FK506 binding protein 12.6. Two such compounds, chloroxine and myricetin, increase FRET and inhibit [ 3 H]ryanodine binding to RyR1 at nanomolar Ca 2+. Both compounds also decrease RyR1 Ca 2+ leak in human skinned skeletal muscle fibers. Furthermore, we identified compound concentrations that reduced leak by > 50% but only slightly affected Ca 2+ release in excitation-contraction coupling, which is essential for normal muscle contraction. This report demonstrates a pipeline that effectively filters small-molecule RyR1 modulators towards clinical relevance. In striated muscle, contraction requires an intracellular Ca 2+-release event mediated by ryanodine receptors (RyR) that are embedded in the sarcoplasmic reticulum (SR) membrane. Dysregulation of skeletal (RyR1) and cardiac (RyR2) isoforms, via mutations or excess posttranslational modification, has been linked to severe muscle pathologies, including malignant hyperthermia (MH), central core disease, muscular dystrophy (MD), sarcopenia, catecholaminergic polymorphic ventricular tachycardia, heart failure, and more recently RyR2 has been recognized as a potentially significant contributor to diabetes and Alzheimer's disease 1-9. In most of these clinical indications, pathogenesis can be fueled by excess SR Ca 2+ "leak" via RyR under resting cellular conditions, which leads to toxic intracellular basal [Ca 2+ ] and insufficient SR Ca 2+ load. As a result, RyR is intensely studied as a therapeutic target. Indeed, the therapeutic potential of pharmaceutically targeting RyR1-mediated SR Ca 2+ leak in skeletal muscle has been shown in animal models of Duchenne MD, limb-girdle MD, and sarcopenia 5,10,11. The therapeutic potential of targeting RyR2-mediated SR Ca 2+ leak for treating heart failure and arrhythmia is also very well documented 12-16. Additionally, targeting RyR2 (which is abundant in the brain 17,18) may have therapeutic potential for treating neurodegenerative diseases. To introduce a systematic and efficient approach for identifying novel small-molecule chemical scaffolds with potential to mitigate RyR1 dysfunction, we developed and implemented a high-throughput screening (HTS) assay that uses fluorescence lifetime (FLT) detection of FRET 19. This assay was designed to identify compounds that bind to the RyR1 channel complex to allosterically correct its pathologically leaky state (without affecting normal channel function) 19. This FRET-based method is based on monitoring RyR binding of fluorescent...

Live-Cell Cardiac-Specific High-Throughput Screening Platform for Drug-Like Molecules That Enhance Ca2+ Transport

“…We solved the throughput problem by developing plate-readers that record FLT data, combining high precision (S/N > 100) and high throughput (>10 samples per s), as needed for HTS [8]. We applied this technology platform, combining biosensor engineering and FLT measurement technology, to disease-relevant protein targets, including SERCA (heart failure, sarcopenia) [7,9], SERCA2a-PLB (heart failure) [13], myosin (heart failure, sarcopenia) [14], myosin-actin (heart failure, sarcopenia) [14,15], ryanodine receptor (heart failure, muscular dystrophy) [16,17], tau (Alzheimer's disease) [18], TNF receptor (inflammation, arthritis) [19,20], and aurora kinase (cancer) [21,22]. In the present study, we describe two extensions of this technology.…”

Cardiac RyR N-terminal region biosensors for FRET-based high-throughput screening

“…Our results ( Figure 4b) indicate that these compounds may also directly interact with the RyRs, a hypothesis to be explored in future studies. Tacrolimus and Ro 90-7501 had already been identified as RyR regulators in our previous HTS campaigns (via a FRET HTS assay that uses fulllength RyRs), and their RyR activatory and inhibitory effect (respectively) have been reported (20,21 (30 µM), corresponding to the resting and contracting muscle conditions, respectively. This is to glean early insight in the potential effect of a hit on the resting RyR leak, as well as on RyR activation during excitation-contraction coupling.…”

“…In that work, fluorescence lifetime (FLT) detection of FRET between the two labeled regulators was used as an assay to identify small-molecule modulators of RyR1. Using this assay, compounds have been identified to decrease RyR1 Ca 2+ leak in skeletal muscle SR membrane vesicles and mechanically skinned muscle fibers (20,21), and were further shown to mitigate force loss in a muscular dystrophy mouse model (22). However, the binding sites of these molecules are difficult to identify within the full-length RyR, which limits progress on understanding their mechanisms of action and hinders structure-activity relationship studies.…”

Ryanodine receptor 1-related disorders: an historical perspective and proposal for a unified nomenclature

“…For RYR1-RM-affected individuals, a phase 1 clinical trial testing safety of the RyR stabilizing Rycal molecule S48168 (ARM210) is underway (NCT04141670). Moreover, several drugs already approved for other indications are in the clinical trial pipeline for potential repurposing, and novel compounds, identified by highthroughput screening, are being tested in pre-clinical studies [125][126][127]. The recent generation of murine model systems that more closely depict RYR1-RM clinical phenotypes [128,129] and greater understanding of RyR1 structure-function [130] will play a crucial role in identifying RYR1-RM-affected individuals who could benefit most from specific therapeutics.…”