A synthetic diphosphoinositol phosphate analogue of inositol trisphosphate

Abstract

a 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-InsIJ4,5)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-InsIJ4,5)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-InsIJ4,5)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-PPInsIJ4,5)P 2 was not readily metabolised by human DIPPs. Differential scanning fluorimetry showed that 1-PP-InsIJ4,5)P 2 stabilises DIPP proteins, but to a lesser extent than naturally occurring substrates 1-PPInsP 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.

Metabolism and Functions of Inositol Pyrophosphates: Insights Gained from the Application of Synthetic Analogues

“…Biochemical assays have indicated that the 5-β-phosphate is preferentially cleaved from 1,5-InsP 7 [ 14 ]. Despite that, these enzymes show a greater preference for the 1-β-phosphate of 1-InsP 7 compared to the 5-β-phosphate of 5-InsP 7 [ 14 , 35 ]. Moreover, PCP analogs have been useful in validating the enzyme’s preference for 1-InsP 7 over 5-InsP 7 .…”

“…Moreover, PCP analogs have been useful in validating the enzyme’s preference for 1-InsP 7 over 5-InsP 7 . PCP analogs are not hydrolyzed by DIPPs, but by competing with the active site, they act as inhibitors of PP-InsP hydrolysis [ 25 , 26 , 35 ]. Fiedler and colleagues showed that 1-PCP-InsP 7 is an 80-fold more potent inhibitor of DIPP activity than is 5-PCP-InsP 7 [ 26 ].…”

“…It is noteworthy that these biochemical assays were performed in media containing the Mg 2+ that is necessary for activity. In contrast, Mg 2+ was absent when Potter and colleagues [ 35 ] assessed apparent ligand affinities for DIPP by using differential scanning fluorimetry. Under such conditions, the apparent K d values for 1-InsP 7 and 5-InsP 7 are very similar, while the apparent K d value for 5-PCP-InsP 7 is 3 to 4-fold lower than that for 1-PCP-InsP 7 [ 35 ].…”

“…In contrast, Mg 2+ was absent when Potter and colleagues [ 35 ] assessed apparent ligand affinities for DIPP by using differential scanning fluorimetry. Under such conditions, the apparent K d values for 1-InsP 7 and 5-InsP 7 are very similar, while the apparent K d value for 5-PCP-InsP 7 is 3 to 4-fold lower than that for 1-PCP-InsP 7 [ 35 ]. The fact that PCP versions of PP-InsPs recapitulate this Mg 2+ -dependence for DIPP’s preferential apparent affinity for 1-InsP 7 over 5-InsP 7 is testimony to their value as bioisosteres.…”

“…Potter and colleagues have used differential scanning calorimetry to show that the binding to DIPPs of either 1-PCP-InsP 7 or 5-PCP-InsP 7 elevates the protein’s thermal transitions temperature—which reflects increased protein stability—to almost the same degree as the corresponding PP-InsPs [ 35 ]. The authors posited that any protein stabilization by metabolically-stable PCP-InsPs might facilitate the formation of tractable crystal/ligand complexes for X-ray structural analysis.…”