A synthetic diphosphoinositol phosphate analogue of inositol trisphosphate

Diphosphoinositol phosphates (PP-InsPs) are inositol phosphates (InsPs) that contain PP (diphosphate) groups. Converting a phosphate group in an InsP into a diphosphate has been reported to enhance affinity for some binding proteins. We synthesised 1-PP-Ins(4,5)P(2), the first diphosphate analogue of the intracellular signalling molecule InsP(3), and examined its effects on InsP(3) receptors, which are intracellular Ca(2+) channels. 1-PP-Ins(4,5)P(2) was indistinguishable from InsP(3) in its ability to bind to and activate type 1 InsP(3) receptors, indicating that the diphosphate modification of InsP(3) affected neither affinity nor efficacy. Nevertheless, 1-PP-Ins(4,5)P(2) is the most potent 1-phosphate modified analogue of InsP(3) yet identified. PP-InsPs are generally hydrolysed by diphosphoinositol polyphosphate phosphohydrolases (DIPPs), but 1-PP-Ins(4,5)P(2) was not readily metabolised by human DIPPs. Differential scanning fluorimetry showed that 1-PP-Ins(4,5)P(2) stabilises DIPP proteins, but to a lesser extent than naturally occurring substrates 1-PP-InsP(5) and 5-PP-InsP(5). The non-hydrolysable InsP(7) analogues 1-PCP-InsP(5) and 5-PCP-InsP(5) showed comparable stabilising abilities to their natural counterparts and may therefore be promising substrate analogues for co-crystallisation with DIPPs.