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

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Abstract
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 (Ki= 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.

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