Development of a homogenous high-throughput assay for inositol hexakisphosphate kinase 1 activity

Inositol pyrophosphates have been implicated in a wide range of cellular processes. Inositol hexakisphosphate kinase 1 catalyzes the pyrophosphorylation of inositol hexakisphosphate into inositol 5-diphospho-1,2,3,4,6-pentakisphosphate which is important in numerous areas of cell physiology such as DNA repair and glucose homeostasis. Furthermore, inositol 5-diphospho-1,2,3,4,6-pentakisphosphate is implicated in the pathology of diabetes and other human diseases. As such there is a demonstrated need in the field for a robust chemical probe to better understand the role of inositol hexakisphosphate kinase 1 and inositol pyrophosphate in physiology and disease. To aid in this effort we developed a homogenous coupled bioluminescence assay for measuring inositol hexakisphosphate kinase 1 activity in a 384-well format (Z’ = 0.62±0.05). Using this assay we were able to confirm the activity of a known inositol hexakisphosphate kinase 1 inhibitor N2-(m-trifluorobenzyl), N6-(p-nitroben-zyl)purine. We also screened the Sigma library of pharmacologically active compounds at 10μM concentration and found 24 hits. Two of the most potent compounds were found to have an IC₅₀ less than 5μM. The use of this high-throughput assay will accelerate the field towards the discovery of a potent inositol hexakisphosphate kinase 1 inhibitor.