Development of Ryanodine Receptor (RyR) Inhibitors for Skeletal Muscle and Heart Diseases

Figure 1. Structure of existing RyR inhibitors

Figure 2. High-throughput assays for identifying RyR1 inhibitors by endoplasmic reticulum Ca2+ measurement (A) Schematic drawing of an endoplasmic reticulum (ER) Ca2+-based assay. Stable HEK293 cells were generated that express the gain-of-function mutant, RyR1, and R-CEPIA1er, a genetically encoded ER Ca2+ indicator. Ca2+ leakage via mutant RyR1 channels causes a reduction in ER Ca2+ content, therefore R-CEPIA1er fluorescence is low. RyR1 inhibitors prevent Ca2+ leakage, which causes an increase in ER Ca2+ content (via active transport through a Ca2+ pump) and increases R-CEPIA1er fluorescence. (B) Typical results for a highthroughput assay for RyR1 inhibitors. Oxolinic acid was identified and shown to increase R-CEPIA1er fluorescence. (C) Development of Cpd1. Among many oxolinic acid derivatives, Cpd1 exhibited 70-fold greater potency than oxolinic acid. (This figure and its legend were modified from Murayama et al., Mol Pharmacol, 2018; 94: 722-73053); Murayama and Kurebayashi, Curr Protoc Pharmacol, 2019; 87: e7154); Mori et al., Eur J Med Chem, 2019; 179: 837-84859). The authors have permission to reproduce images from the copyright owner.)

Figure 3. Therapeutic effects of Cpd1 on malignant hyperthermia model mice (A) Cpd1 was administered to malignant hyperthermia (MH) model mice during isofluraneinduced episodes of MH. Cpd1 reduced the body temperature of the mice. (B) Cpd1 improved survival rate in isoflurane-induced MH episodes. (C) Change in plasma Cpd1. Cpd1 rapidly declined from plasma with a half-life of ~10 min. (D) Cpd1 and muscle weakness in mice. Muscle weakness was observed at 10 min after administration of Cpd1, but disappeared by 60 min. (This figure and its legend were modified from Yamazawa T et al., Nat Commun, 2021; 12: 429361) under a CC BY license［ Creative Commons Attribution 4.0 International license］).