School of Biological Sciences , University of East Anglia , Norwich Research Park , Norwich NR4 7TJ , U.K.
Medicinal Chemistry & Drug Discovery, Department of Pharmacology , University of Oxford , Mansfield Road , Oxford OX1 3QT , U.K.
J Med Chem. 2018 Oct 11;61(19):8838-8846. doi: 10.1021/acs.jmedchem.8b01022. Epub 2018 Sep 19.
Inositol pentakisphosphate 2-kinase catalyzes the phosphorylation of the axial 2-OH of myo-inositol 1,3,4,5,6-pentakisphosphate for de novo synthesis of myo-inositol hexakisphosphate. Disruption of inositol pentakisphosphate 2-kinase profoundly influences cellular processes, from nuclear mRNA export and phosphate homeostasis in yeast and plants to establishment of left-right asymmetry in zebrafish. We elaborate an active site fluorescent probe that allows high throughput screening of Arabidopsis inositol pentakisphosphate 2-kinase. We show that the probe has a binding constant comparable to the K values of inositol phosphate substrates of this enzyme and can be used to prospect for novel substrates and inhibitors of inositol phosphate kinases. We identify several micromolar K inhibitors and validate this approach by solving the crystal structure of protein in complex with purpurogallin. We additionally solve structures of protein in complexes with epimeric higher inositol phosphates. This probe may find utility in characterization of a wide family of inositol phosphate kinases.
肌醇五磷酸 2-激酶催化肌醇 1,3,4,5,6-五磷酸轴向 2-OH 的磷酸化，用于肌醇六磷酸的从头合成。肌醇五磷酸 2-激酶的破坏深刻影响着细胞过程，从酵母和植物中的核 mRNA 输出和磷酸盐稳态到斑马鱼左右不对称的建立。我们详细阐述了一种活性位点荧光探针，可用于高通量筛选拟南芥肌醇五磷酸 2-激酶。我们表明，该探针具有与该酶的肌醇磷酸盐底物的 K 值相当的结合常数，并且可用于寻找新型肌醇磷酸盐激酶的底物和抑制剂。我们鉴定了几种微摩尔 K 抑制剂，并通过与 purpurogallin 结合的蛋白质的晶体结构解决来验证该方法。我们还解决了蛋白质与差向异构的更高肌醇磷酸盐复合物的结构。该探针在广泛的肌醇磷酸盐激酶家族的表征中可能具有实用性。
