PT - JOURNAL ARTICLE AU - Takashi Murayama AU - Nagomi Kurebayashi AU - Mari Ishigami-Yuasa AU - Shuichi Mori AU - Yukina Suzuki AU - Ryunosuke Akima AU - Haruo Ogawa AU - Junji Suzuki AU - Kazunori Kanemaru AU - Hideto Oyamada AU - Yuji Kiuchi AU - Masamitsu Iino AU - Hiroyuki Kagechika AU - Takashi Sakurai TI - Efficient High-Throughput Screening by Endoplasmic Reticulum Ca<sup>2+</sup> Measurement to Identify Inhibitors of Ryanodine Receptor Ca<sup>2+</sup>-Release Channels AID - 10.1124/mol.117.111468 DP - 2018 Jul 01 TA - Molecular Pharmacology PG - 722--730 VI - 94 IP - 1 4099 - http://molpharm.aspetjournals.org/content/94/1/722.short 4100 - http://molpharm.aspetjournals.org/content/94/1/722.full SO - Mol Pharmacol2018 Jul 01; 94 AB - Genetic mutations in ryanodine receptors (RyRs), Ca2+-release channels in the sarcoplasmic reticulum essential for muscle contractions, cause various skeletal muscle and cardiac diseases. Because the main underlying mechanism of the pathogenesis is overactive Ca2+ release by gain-of-function of the RyR channel, inhibition of RyRs is expected to be a promising treatment of these diseases. Here, to identify inhibitors specific to skeletal muscle type 1 RyR (RyR1), we developed a novel high-throughput screening (HTS) platform using time-lapse fluorescence measurement of Ca2+ concentrations in the endoplasmic reticulum (ER) ([Ca2+]ER). Because expression of RyR1 carrying disease-associated mutation reduces [Ca2+]ER in HEK293 cells through Ca2+ leakage from RyR1 channels, specific drugs that inhibit RyR1 will increase [Ca2+]ER by preventing such Ca2+ leakage. RyR1 carrying the R2163C mutation and R-CEPIA1er, a genetically encoded ER Ca2+ indicator, were stably expressed in HEK293 cells, and time-lapse fluorescence was measured using a fluorometer. False positives were effectively excluded by using cells expressing wild-type (WT) RyR1. By screening 1535 compounds in a library of well characterized drugs, we successfully identified four compounds that significantly increased [Ca2+]ER. They include dantrolene, a known RyR1 inhibitor, and three structurally different compounds: oxolinic acid, 9-aminoacridine, and alexidine. All the hit compounds, except for oxolinic acid, inhibited [3H]ryanodine binding of WT and mutant RyR1. Interestingly, they showed different dose dependencies and isoform specificities. The highly quantitative nature and good correlation with the channel activity validated this HTS platform by [Ca2+]ER measurement to explore drugs for RyR-related diseases.