Wormald Michael, Liao Gangling, Kimos Martha, Barrow James, Wei Huijun
Department of Pharmacology and Molecular Sciences, Johns Hopkins University, Baltimore, Maryland, United States of America.
Drug Discovery Division, Lieber Institute for Brain Development, Baltimore, Maryland, United States of America.
PLoS One. 2017 Nov 29;12(11):e0188852. doi: 10.1371/journal.pone.0188852. eCollection 2017.
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.
肌醇焦磷酸已被证明参与多种细胞过程。肌醇六磷酸激酶1催化肌醇六磷酸焦磷酸化生成5-二磷酸-1,2,3,4,6-五磷酸肌醇，这在细胞生理学的许多领域如DNA修复和葡萄糖稳态中都很重要。此外，5-二磷酸-1,2,3,4,6-五磷酸肌醇与糖尿病及其他人类疾病的病理过程有关。因此，该领域迫切需要一种强大的化学探针，以更好地了解肌醇六磷酸激酶1和肌醇焦磷酸在生理和疾病中的作用。为了助力这一工作，我们开发了一种均相偶联生物发光测定法，用于在384孔板中测量肌醇六磷酸激酶1的活性（Z' = 0.62±0.05）。使用该测定法，我们能够证实已知的肌醇六磷酸激酶1抑制剂N2-（间三氟苄基），N6-（对硝基苄基）嘌呤的活性。我们还在10μM浓度下筛选了Sigma药理活性化合物库，发现了24个命中物。发现其中两种最有效的化合物的IC50小于5μM。这种高通量测定法的使用将加速该领域发现一种有效的肌醇六磷酸激酶1抑制剂。
