Abstract

Racemic mixtures and enantiomerically pure d-isomers of both myo-inositol 1,3,6-trisphosphorothioate [Ins(1,3,6)PS₃] and myo-inositol 1,4,6-trisphosphorothioate [Ins(1,4,6)PS₃], prepared by total synthesis, were examined in Ca²⁺ flux and binding assays. Both d-Ins(1,3,6)PS₃ andd-Ins(1,4,6)PS₃ were shown to be low intrinsic activity partial agonists at the platelet myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] receptor, releasing less than 20% of the Ins(1,4,5)P₃-sensitive Ca²⁺ store. d-Ins(1,4,6)PS₃displaced specifically bound [³H]Ins(1,4,5)P₃from rat cerebellar membranes, although displacement was some 34-fold weaker than by d-Ins(1,4,5)P₃.d-Ins(1,4,6)PS₃ displaced [³H]Ins(1,4,5)P₃ from cerebellar membranes with roughly twice the affinity ofdl-Ins(1,4,6)PS₃ (IC₅₀ value = 1.4 ± 0.35 μM compared with 2.15 ± 0.13 μM), whereasd-Ins(1,3,6)PS₃ displaced [³H]Ins(1,4,5)P₃ with roughly twice the affinity of dl-Ins(1,3,6)PS₃ (IC₅₀value = 17.5 ± 5.8 μM compared with 34 ± 10 μM), confirming that the activity of both these phosphorothioates resides in their d-enantiomers. Increasing concentrations of either d-Ins(1,3,6)PS₃ ord-Ins(1,4,6)PS₃ were able to partially antagonize Ca²⁺ release induced by submaximal concentrations of Ins(1,4,5)P₃, an inhibition that could be overcome by increasing the concentration of Ins(1,4,5)P₃, suggesting competition for binding at the Ins(1,4,5)P₃-R. The only low-efficacy partial agonists at the Ins(1,4,5)P₃-R discovered to date have been phosphorothioates; the novel d-Ins(1,3,6)PS₃and d-Ins(1,4,6)PS₃ can now be added to this small group of analogs. However,d-Ins(1,4,6)PS₃ has a relatively high affinity for the Ins(1,4,5)P₃-R but maintains the lowest efficacy of all the partial agonists thus far identified. As such, it may be a useful tool for pharmacological intervention in the polyphosphoinositide pathway and an important lead compound for the development of further Ins(1,4,5)P₃-R antagonists.