A synthetic diphosphoinositol phosphate analogue of inositol trisphosphate

Abstract

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)P₂, the first diphosphate analogue of the intracellular signalling molecule InsP₃, and examined its effects on InsP₃ receptors, which are intracellular Ca²⁺ channels. 1-PP-Ins(4,5)P₂ was indistinguishable from InsP₃ in its ability to bind to and activate type 1 InsP₃ receptors, indicating that the diphosphate modification of InsP₃ affected neither affinity nor efficacy. Nevertheless, 1-PP-Ins(4,5)P₂ is the most potent 1-phosphate modified analogue of InsP₃ yet identified. PP-InsPs are generally hydrolysed by diphosphoinositol polyphosphate phosphohydrolases (DIPPs), but 1-PP-Ins(4,5)P₂ was not readily metabolised by human DIPPs. Differential scanning fluorimetry showed that 1-PP-Ins(4,5)P₂ stabilises DIPP proteins, but to a lesser extent than naturally occurring substrates 1-PP-InsP₅ and 5-PP-InsP₅. The non-hydrolysable InsP₇ analogues 1-PCP-InsP₅ and 5-PCP-InsP₅ showed comparable stabilising abilities to their natural counterparts and may therefore be promising substrate analogues for co-crystallisation with DIPPs.