A Structure‐and‐Function‐Based Approach to Target Ryanodine Receptor 2 for Heart Failure

• Published in: The FASEB journal Vol. 34; no. S1; p. 1
• Main Authors: Sun, Qing, Roosild, Tarmo, Bobkov, Andrey, Ma, Chen-Ting, Holleran, John, Sergienko, Eduard
• Format: Journal Article
• Language: English
• Published: 01-04-2020
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.2020.34.s1.08949

Abstract only
Ryanodine receptor 2 (RYR2) controls transient calcium release (calcium spark) from the sarcoplasmic reticulum (SR) into the cytoplasm in cardiomyocytes. It is an essential part of the calcium handling machinery which drives cardiac contraction. In pathological conditions caused by hyperphosphorylation or mutation, RYR2 can become leaky, resulting in cytosolic calcium overload and abnormal calcium sparks, manifesting as loss of cardiac contractility, arrhythmia and cardiac hypertrophy, leading to cardiomyocyte injury and death. This is a key dysfunction underlying heart failure, a disease that has a five‐year mortality rate of over 50% and affects 26 million people worldwide. Current standard‐of‐care drugs alleviate symptoms, but do not directly address pathophysiology of the disease. Efforts in targeting RYR2 in the past have met considerable challenges, given the extremely large size of this tetrameric calcium channel, its complex structure and regulatory mechanisms involving a multitude of binding partners and modifiers. There is a clear unmet need for direct modulators of RYR2 able to suppress excessive calcium transport caused by hyperphosphorylation or mutations.
Here we search for modulators of RYR2, which can reduce calcium leak from the SR, restore calcium homeostasis and normalize afterdepolarization in the cardiomyocyte. We utilized a twotier approach, taking advantage of protein thermal shift (PTS) assays to identify initial hits that bind to the protein and relying on cell‐based assays to establish the functional outcome of the binding. To overcome the large size of RYR2, we designed protein constructs corresponding to various domains with hotspots of pathogenic mutations and hyperphosphorylation, as well as binding sites of key regulatory proteins. We successfully produced six soluble domains or domain complexes and developed PTS assays for five of them. In a proof of concept study, four of these domains were utilized for pilot screening with a twenty‐thousand compound collection. The hits for each domain were profiled in PTS assays on all four domains, and select representatives were reconfirmed in isothermal titration calorimetry assays.
The second tier of the approach utilized RYR2‐expressing cells. We built cell‐based assays to monitor cytosolic and endoplasmic reticulum (ER) calcium basal levels and transients using the HL‐1 mouse cardiomyocyte cell line and an RYR2‐expressing HEK‐293 cell line. We then validated the assays using known RYR2 modulators. Pathophysiological cell models are being established that carry hyperphosphorylation on S2814 and S2808 on the RYR2 protein, which are the key abnormalities causing SR calcium leakage in diseased cardiomyocytes. Our goal is to identify RYR2‐binding compounds that restores intracellular calcium in these hyperphosphorylated cells to wildtype levels.