Identification of small molecule inhibitors of the mitotic kinase haspin by high-throughput screening using a homogeneous time-resolved fluorescence resonance energy transfer assay.
Identification of small molecule inhibitors of the mitotic kinase haspin by high-throughput screening using a homogeneous time-resolved fluorescence resonance energy transfer assay.
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DOI：
10.1177/1087057108326081
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发表时间：
2008-12
影响因子：
--
通讯作者：
Higgins JM
中科院分区：
文献类型：
--
作者：
Patnaik D;Jun Xian;Glicksman MA;Cuny GD;Stein RL;Higgins JM
Haspin/Gsg2是一种在有丝分裂过程中使组蛋白H3在Thr-3(H3T3ph)处磷酸化的激酶。它被RNA干扰耗尽，导致染色体配对失败和有丝分裂受阻。因此，Haspin是开发抗有丝分裂药物的新靶点。我们报道了一种高通量时间分辨荧光共振能量转移(TR-FRET)检测haspin的方法。以组蛋白H3多肽为底物，用Eu标记的H3T3ph磷酸化特异性单抗检测磷酸化。在三磷酸腺苷和多肽的Km浓度下筛选了一个包含137632个小分子的文库，以允许识别不同的抑制剂类型。用TRRET法和以重组组蛋白H3为底物的放射测定法对HITS和IC50进行再确认。特异性的初步评估是通过测试对两个不相关的激酶的抑制。用H3T3ph的细胞酶联免疫吸附试验测定细胞内的EC50值。基于效力和化学结构的考虑，选择了五个化合物作为先导化合物。这些先导化合物构成了开发Haspin特异性抑制剂的基础，这些抑制剂将在基础研究中具有明显的实用价值，并可能用作开发抗有丝分裂抗癌治疗药物的起点。
Haspin/Gsg2 is a kinase that phosphorylates Histone H3 at Thr-3 (H3T3ph) during mitosis. Its depletion by RNA interference results in failure of chromosome alignment and a block in mitosis. Haspin therefore is a novel target for development of anti-mitotic agents. We report the development of a high throughput time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assay for Haspin. Histone H3 peptide was used as a substrate, and a Europium-labeled H3T3ph phosphospecific monoclonal antibody was used to detect phosphorylation. A library of 137632 small molecules was screened at Km concentrations of ATP and peptide to allow identification of diverse inhibitor types. Reconfirmation of hits and IC50 determinations were carried out with the TR-FRET assay and by a radiometric assay using recombinant Histone H3 as the substrate. A preliminary assessment of specificity was made by testing inhibition of two unrelated kinases. EC50 values in cells were determined using a cell-based ELISA assay of H3T3ph. Five compounds were selected as leads based on potency and chemical structure considerations. These leads form the basis for the development of specific inhibitors of Haspin that will have clear utility in basic research and possible use as starting points for development of anti-mitotic anticancer therapeutics.