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 Balpha.

Stephen J Mills, Fabrice Vandeput, Melanie N Trusselle, Stephen T Safrany, Christophe Erneux, Barry V L Potter

Index: ChemBioChem. 9(11) , 1757-66, (2008)

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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 Balpha (PKBalpha), which can bind both soluble inositol polyphosphates and inositol phospholipids. The most potent trisphosphate 5-phosphatase inhibitor is benzene 1,2,4-trisphosphate (2, IC(50) of 14 microM), a potential mimic of D-myo-inositol 1,4,5-trisphosphate, whereas the most potent tetrakisphosphate Ins(1,4,5)P(3) 5-phosphatase inhibitor is benzene 1,2,4,5-tetrakisphosphate, with an IC(50) of 4 microM. Biphenyl 2,3',4,5',6-pentakisphosphate (4) was the most potent inhibitor evaluated against type I Ins(1,4,5)P(3) 5-phosphatase (IC(50) of 1 microM). All new benzene polyphosphates are resistant to dephosphorylation by type I Ins(1,4,5)P(3) 5-phosphatase. Unexpectedly, all benzene polyphosphates studied bind to the PH domain of PKBalpha with apparent higher affinity than to type I Ins(1,4,5)P(3) 5-phosphatase. The most potent ligand for the PKBalpha PH domain, measured by inhibition of biotinylated diC(8)-PtdIns(3,4)P(2) binding, is biphenyl 2,3',4,5',6-pentakisphosphate (4, K(i)=27 nm). The approximately 80-fold enhancement of binding relative to parent benzene trisphosphate is explained by the involvement of a cation-pi interaction. These new molecular tools will be of potential use in structural and cell signalling studies.