Efficient High-Throughput Screening for Type 1 Ryanodine Receptor Inhibitors using ER Ca2+Measurements

Abstract

Type 1 ryanodine receptor (RyR1) is a Ca2+release channel in the sarcoplasmic reticulum and plays an important role in excitation-contraction coupling. Genetic mutations in RyR1 cause various skeletal muscle diseases including malignant hyperthermia (MH). Because the main underlying mechanism of the pathogenesis is hyperactive Ca2+release by gain-of-function of the RyR1 channel, inhibition of RyR1 is expected to be a promising treatment for these diseases. We here developed a novel high-throughput screening (HTS) platform using time-lapse fluorescence measurement of Ca2+in the endoplasmic reticulum (ER) to identify RyR1 inhibitors. RyR1 carrying an MH 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. Since MH mutant RyR1 reduces ER Ca2+in HEK293 cells through Ca2+leakage, specific drugs that inhibit RyR1 increase ER Ca2+by preventing Ca2+leakage. By screening 1,535 compounds in a library of well-characterized drugs, we successfully identified four compounds including dantrolene, a known RyR1 inhibitor, and three structurally-different compounds; oxolinic acid, 9-aminoacridine and alexidine. Interestingly, they showed different dose-dependencies and isoform specificities. The highly quantitative characteristic and good correlation with the channel activity validate this HTS platform by ER Ca2+measurement to explore novel drugs for RyR1-linked diseases.