Elucidating Diphosphoinositol Polyphosphate Function with Nonhydrolyzable Analogues

The diphosphoinositol polyphosphates (PP-IPs) represent a novel class of high-energy phosphate-containing messengers which control a wide variety of cellular processes. It is thought that PP-IPs exert their pleiotropic effects as allosteric regulators and through pyrophosphorylation of protein substrates. However, most details of PP-IP signaling have remained elusive because of a paucity of suitable tools. We describe the synthesis of PP-IP bisphosphonate analogues (PCP-IPs), which are resistant to chemical and biochemical degradation. While the two regioisomers 1PCP-IP5 and 5PCP-IP5 inhibited Akt phosphorylation with similar potencies, 1PCP-IP5 was much more effective at inhibiting its cognate phosphatase hDIPP1. Furthermore, the PCP analogues inhibit protein pyrophosphorylation because of their inability to transfer the beta-phosphoryl group, and thus enable the distinction between PP-IP signaling mechanisms. As such, the PCP analogues will find widespread applications for the structural and biochemical characterization of PP-IP signaling properties.