CCDI: A new ligand that modulates mammalian type 1 ryanodine receptor (RyR1)

Background and Purpose Ryanodine receptors (RyRs) are Ca ²⁺-release channels on the sarco(endo)plasmic reticulum that modulate a wide array of physiological functions. Three RyR isoforms are present in cells: RyR1, RyR2 and RyR3. To date, there are no reports on ligands that modulate RyR in an isoform-selective manner. Such ligands are not only valuable research tools, but could serve as intermediates for development of therapeutics. Experimental approach Pyrrole-2-carboxylic acid and 1,3-dicyclohexylcarbodiimide were allowed to react in carbon tetrachloride for 24-h at low temperatures and pressures. The chemical structures of the two products isolated were elucidated using NMR spectrometry, mass spectrometry and elemental analyses. [³H]-ryanodine binding, lipid bilayer and time-lapsed confocal imaging were used to determine their effects on RyR isoforms. Key results The major product, 2-cyclohexyl-3-cyclohexylimino-2, 3, dihydro-pyrrolo[1,2-c]imidazol-1-one (CCDI) dose-dependently potentiated Ca ²⁺-dependent binding of [³H]-ryanodine to RyR1, with no significant effects on [³H]-ryanodine binding to RyR2 or RyR3. CCDI also reversibly increased the open probability (P_o) of RyR1 with minimal effects on RyR2 and RyR3. CCDI induced Ca²⁺ transients in C2C12 skeletal myotubes, but not in rat ventricular myocytes. This effect was blocked by pretreating cells with ryanodine. The minor product 2-cyclohexyl-pyrrolo[1,2-c]imidazole-1,3-dione had no effect on either [ ³H]-ryanodine binding or P_o of RyR1, RyR2 and RyR3. Conclusions and implications A new ligand that preferentially modulates RyR1 was identified. In addition to being an important research tool, the pharmacophore of this small molecule could serve as a template for the synthesis of other isoform-selective modulators of RyRs.