Using Functional Assays to Investigate Therapeutic Potential of Hit Compounds for Leaky Skeletal RyR1 Inhibition
Many myopathies of skeletal muscle have been traced back to a leaky Skeletal Isoform Ryanodine Receptor (RyR1) ion channel that allows Ca2+ to flow out of the Sarcoplasmic Reticulum (SR) when the cell is in its relaxed state. In order to identify therapeutic compounds that inhibit RyR1 activity under relaxed conditions, the functional effects of a DIVERSET Hit compound and 15 of its analogues were tested on heavy SR membranes containing the RyR1 isoform at low [Ca2+]. This was done using isolated SR membranes from pig latissimus dorsi skeletal muscle, which were treated with compound, and oxidative GSSG to express the receptor’s pathological state. Cells were then incubated in [3H]ryanodine, after which they were harvested and run through a scintillation detector. Of the 16 compounds tested, 5 were found to have the desired inhibitory effect on RyR1 relaxed conformation, while 8 displayed activating effects and 3 displayed no significant effect. The compounds that showed inhibitory effects could present a solution to current RyR related myopathies, and will have their functional effects further tested with the RyR2 cardiac isoform and with high [Ca2+] conditions to establish their effects on Ca2+ modulation in the heart, and at contracted muscle state respectively.