Modification at C2 of myo‐inositol 1,4,5‐trisphosphate produces inositol trisphosphates and tetrakisphosphates with potent biological activities

Novel 2‐position‐modified d‐myo‐inositol 1,4,5‐trisphosphate [Ins(1,4,5)P₃] analogues, dl‐2‐deoxy‐2‐fluoro‐myo‐inositol 1,4,5‐trisphosphate [dl‐2F‐Ins(1,4,5)P₃], dl‐myo‐inositol 1,2,4,5‐tetrakisphosphate [dl‐Ins(1,2,4,5)P₄], dl‐scyllo‐inositol 1,2,4‐trisphosphate [dl‐sc‐Ins(1,2,4)P₃], scyllo‐inositol 1,2,4,5‐tetrakisphosphate [sc‐Ins(1,2,4,5)P₄] and scyllo‐inositol 1,2,4,5‐tetrakisphos‐phorothioate [sc‐Ins(1,2,4,5)PS₄] were investigated for their ability to bind to the Ins(1,4,5)P₃ receptor, mobilise intracellular Ca²⁺ stores and interact with metabolic enzymes. With the exception of sc‐Ins(1,2,4,5)PS₄, all the Ins(1,4,5)P₃ analogues potently displaced [³H]Ins(1,4,5)P₃ from its receptor in bovine adrenal cortex and were apparently potent full agonists at the Ca²⁺ mobilising Ins(1,4,5)P₃ receptor of SH‐SY5Y cells, giving respective IC₅₀ and EC₅₀ values of: sc‐Ins(1,2,4,5)P₄ (IC₅₀ 14 nM, EC₅₀ 77 nM), dl‐2F‐Ins(1,4,5)P₃ (IC₅₀ 25 nM, EC₅₀ 105 nM), dl‐Ins(1,2,4,5)P₄ (IC₅₀ 26 nM, EC₅₀ 163 nM), dl‐sc‐Ins(1,2,4)P₃ (IC₅₀ 52 nM, EC₅₀ 171 nM), compared to Ins(1,4,5)P₃ (IC₅₀ 4 nM, EC₅₀ 52 nM). sc‐Ins(1,2,4,5)P₄ was equipotent to Ins(1,4,5)P₃ for Ca²⁺ release making it the most potent inositol tetrakisphosphate and indeed Ins(1,4,5)P₃ analogue yet characterised. In contrast, although sc‐Ins(1,2,4,5)PS₄ (IC₅₀ 425 nM, EC₅₀ 1603 nM) was a significantly weaker ligand and agonist than Ins(1,4,5)P₃, it was a partial agonist of high intrinsic activity with maximally effective concentrations releasing only about 80% of Ins(1,4,5)P₃‐sensitive Ca²⁺ stores of SH‐SY5Y cells. Ins(1,4,5)P₃ and sc‐Ins(1,2,4,5)P₄ were readily metabolised by Ins(1,4,5)P₃ 3‐kinase and 5‐phosphatase activities, dl‐2F‐Ins(1,4,5)P₃ and dl‐sc‐Ins(1,2,4)P₃ were resistant to 5‐phosphatase, while sc‐Ins(1,2,4,5)PS₄ and dl‐Ins(1,2,4,5)P₄ were resistant to both 3‐kinase and 5‐phosphatase activity and were potent inhibitors of the 5‐phosphatase enzyme (K_i= 300 nM and 2.9 μM, respectively). These results demonstrate that modification of the 2‐position of Ins(1,4,5)P₃, even with an anionic group, does not critically affect Ins(1,4,5)P₃ receptor binding interaction or Ca²⁺ release, suggesting that the 2‐OH of Ins(1,4,5)P₃ fails to interact significantly with the binding site of its receptor. However, modification remote from the crucial vicinal 4,5‐bisphosphate can affect analogue efficacy in Ca²⁺ release.