Abstract
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-nitrobenzyl)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.
Highlights
Inositol phosphates have been recognized as important signaling molecules that are involved in a whole host of cellular processes ranging from growth to apoptosis [1,2,3]
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
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)
Summary
Inositol phosphates have been recognized as important signaling molecules that are involved in a whole host of cellular processes ranging from growth to apoptosis [1,2,3]. Inositol hexakisphosphate (IP6) can be pyrophosphorylated into a molecule called inositol 5-diphospho-1,2,3,4,6-pentakisphosphate (5PP-IP5) [4]. This compound contains a high-energy phosphoanhydride bond that allows it to participate in a different, but diverse, set of physiological processes than the inositol phosphates. Three inositol hexakisphosphate kinases (IP6K1, IP6K2 and IP6K3) have been found to catalyze the phosphorylation of IP6 to 5PP-IP5 and produce the vast majority of these molecules in the cell [4].
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