Improvements of ER-Ca2+ Based High-Throughput Screening Method for Searching Novel RyR2 Inhibitors

Abstract

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic reticulum (ER) and plays a central role in excitation-contraction coupling in the heart. When the RyR2 activity is abnormally enhanced by miss-sense mutations and/or excessive phosphorylation, spontaneous Ca2+ release occurs from the ER, which often results in fatal ventricular arrhythmias. Therefore, specific inhibitors of RyR2 are expected to have an antiarrhythmic effect on the RyR2-related arrhythmias, but currently such specific drugs have not been reported. We have recently established a screening method for isoform specific ryanodine receptor (RyR) inhibitors using ER Ca2+ indicator, R-CEPIA1er, in HEK293 cells (Murayama et al., Mol Pharmacol, 2018). Although this method is very useful, it requires 3 consecutive experimental days, i.e., culture of HEK293 cells expressing inducible RyR and R-CEPIA1er in 96-well plates on the first day, induction of protein expression by doxycycline on the next day, and assay using FlexStation3 on the third day. In order to screen a larger number of compounds more efficiently using a high-throughput screening device equipped with EM-CCD camera such as FDSS7000 (Hamamatsu), we made several improvements to this method. First, a novel bright green fluorescent indicator, G-CEPIA2er was used instead of R-CEPIA1er, which greatly improved signal-to-noise ratio. Second, measurements were done in 384 well plate with FDSS7000 just after seeding of cryopreserved cells which had been treated with doxycycline to induce RyR2 expression. These improvements resulted in a 48-fold increase in assay efficiency. This method, having allowed us to screen more than 20,000 compounds so far for both RyR1 and RyR2, proved to be a versatile and powerful tool for the development of specific RyR2-modifying reagents. This research was supported by JSPS Kakenhi grant (19K07105), AMED grant (19ek0109202s0203) and BINDS from AMED (0033).