Benzene Polyphosphates as Tools for Cell Signalling: Inhibition of Inositol 1,4,5‐Trisphosphate 5‐Phosphatase and Interaction with the PH Domain of Protein Kinase Bα

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Abstract
Novel benzene polyphosphates were synthesised as inositol polyphosphate mimics and evaluated against type‐I inositol 1,4,5‐trisphosphate 5‐phosphatase, which only binds soluble inositol polyphosphates, and against the PH domain of protein kinase Bα (PKBα), which can bind both soluble inositol polyphosphates and inositol phospholipids. The most potent trisphosphate 5‐phosphatase inhibitor is benzene 1,2,4‐trisphosphate (2, IC50 of 14 μM), a potential mimic of D‐myo‐inositol 1,4,5‐trisphosphate, whereas the most potent tetrakisphosphate Ins(1,4,5)P3 5‐phosphatase inhibitor is benzene 1,2,4,5‐tetrakisphosphate, with an IC50 of 4 μM. Biphenyl 2,3′,4,5′,6‐pentakisphosphate (4) was the most potent inhibitor evaluated against type I Ins(1,4,5)P3 5‐phosphatase (IC50 of 1 μM). All new benzene polyphosphates are resistant to dephosphorylation by type I Ins(1,4,5)P3 5‐phosphatase. Unexpectedly, all benzene polyphosphates studied bind to the PH domain of PKBα with apparent higher affinity than to type I Ins(1,4,5)P3 5‐phosphatase. The most potent ligand for the PKBα PH domain, measured by inhibition of biotinylated diC8‐PtdIns(3,4)P2 binding, is biphenyl 2,3′,4,5′,6‐pentakisphosphate (4, Ki=27 nm). The approximately 80‐fold enhancement of binding relative to parent benzene trisphosphate is explained by the involvement of a cation–π interaction. These new molecular tools will be of potential use in structural and cell signalling studies.