High affinity C₁₀-O_eq ester derivatives of ryanodine: Activator-selective agonists of the sarcoplasmic reticulum calcium release channel

The plant alkaloids ryanodine and dehydroryanodine are specific and potent modulators of the sarcoplasmic reticulum calcium release channel. In the present study, acidic, basic, and neutral side chains esters of these diterpene compounds were prepared and their pharmacologic activities were assessed. Binding affinities of the novel C₁₀-O_eq ester derivatives for the sarcoplasmic reticulum Ca²⁺ release channel were evaluated with sarcoplasmic reticular vesicles prepared from rabbit skeletal muscle. K_d values of the derivatives varied 500-fold, ranging from 0.5 to 244 nM. In comparison, K_d values for ryanodine and dehydroryanodine were 4.4 nM and 5.4 nM, respectively. Basic substituents at the C₁₀-O_eq side chain terminus produced the highest affinity derivatives (K_d values from 0.5 to 1.3 nM). Neutral and/or hydrophobic side chain derivatives exhibited intermediate affinities for the high affinity ryanodine receptor site (K_d values from 2.5 to 39 nM), whereas a derivative with a terminal acidic group had the lowest affinity (K_d value >100 nM). Certain of the higher affinity C₁₀-O_eq derivatives were evaluated more extensively for their pharmacologie activity on the sarcoplasmic reticular Ca²⁺ release channel. Both channel activating (opening) and deactivating (closing) actions were assessed from the ability of the ryanoids to alter Ca²⁺ efflux rates from skeletal junctional sarcoplasmic reticular vesicles that had been passively loaded with Ca²⁺. The natural Ryania secondary metabolites ryanodine, dehydroryanodine and esters E and F, all exhibit antithetical concentration-effect curves, indicating both activator and deactivator actions. In contrast, the semi-synthetic C₁₀-O_eq esters selectively activate the Ca²⁺ release channel. Half-maximal concentrations for such activation (EC_{50 act}) ranged from 0.87 μM to 4.2 μM, compared with an EC_{50 act} of 1.3 μM for ryanodine. These derivatives were also evaluated for their ability to augment ATP-dependent Ca²⁺ accumulation by cardiac junctional sarcoplasmic reticular vesicles, an effect that results from deactivation of the Ca²⁺ release channels. None of the derivatives tested was able to significantly augment Ca²⁺ accumulation, further substantiating their inability to deactivate the sarcoplasmic reticular Ca²⁺ release channel. Additionally, these derivatives functionally antagonized the action of ryanodine to close the Ca²⁺ release channel. The results presented demonstrate that these C₁₀-O_eq ester derivatives of ryanodine and dehydroryanodine bind specifically to the SR Ca²⁺ release channel, selectively activate the channel, and, although they fail to effect channel closure, they nevertheless functionally compete with ryanodine at its low affinity (deactivator) site(s).