Liu Yong, Adayev Tatyana, Hwang Yu-Wen
Molecular Biology Department, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA.
F1000Res. 2017 Jan 13;6:42. doi: 10.12688/f1000research.10582.2. eCollection 2017.
The DYRK1A (dual specificity tyrosine phosphorylation-regulated kinase 1A) gene encodes a proline-directed Ser/Thr kinase. Elevated expression and/or altered distribution of the kinase have been implicated in the neurological impairments associated with Down syndrome (DS) and Alzheimer's disease (AD). Consequently, DYRK1A inhibition has been of significant interest as a potential strategy for therapeutic intervention of DS and AD. Many classes of novel inhibitors have been described in the past decade. Although non-radioactive methods for analyzing DYRK1A inhibition have been developed, methods employing radioactive tracers are still commonly used for quantitative characterization of DYRK1A inhibitors. Here, we present a non-radioactive ELISA assay based on the detection of DYRK1A-phosphorylated dynamin 1a fragment using a phosphorylation site-specific antibody. The assay was verified by the use of two well-characterized DYRK1A inhibitors, epigallocatechin gallate (EGCG) and harmine. The IC s for EGCG and harmine determined by the ELISA method were found to be comparable to those previously measured by radioactive tracing methods. Furthermore, we determined the mode of inhibition for EGCG and harmine by a modification of the ELISA assay. This assay confirms the mode of inhibition of EGCG (non-ATP-competitive) and harmine (ATP-competitive), as previously determined. We conclude that the ELISA platform demonstrated here is a viable alternative to the traditional radioactive tracer assays for analyzing DYRK1A inhibitors.
双特异性酪氨酸磷酸化调节激酶1A（DYRK1A）基因编码一种脯氨酸定向的丝氨酸/苏氨酸激酶。该激酶表达升高和/或分布改变与唐氏综合征（DS）和阿尔茨海默病（AD）相关的神经功能障碍有关。因此，抑制DYRK1A作为DS和AD治疗干预的潜在策略备受关注。在过去十年中，已描述了许多类新型抑制剂。尽管已开发出用于分析DYRK1A抑制作用的非放射性方法，但使用放射性示踪剂的方法仍常用于定量表征DYRK1A抑制剂。在此，我们基于使用磷酸化位点特异性抗体检测DYRK1A磷酸化的发动蛋白1a片段，提出了一种非放射性ELISA测定法。通过使用两种已充分表征的DYRK1A抑制剂表没食子儿茶素没食子酸酯（EGCG）和骆驼蓬碱验证了该测定法。发现通过ELISA方法测定的EGCG和骆驼蓬碱的半数抑制浓度与先前通过放射性示踪法测量的浓度相当。此外，我们通过改进ELISA测定法确定了EGCG和骆驼蓬碱的抑制模式。该测定法证实了EGCG（非ATP竞争性）和骆驼蓬碱（ATP竞争性）的抑制模式，如先前所确定。我们得出结论，此处展示的ELISA平台是分析DYRK1A抑制剂的传统放射性示踪剂测定法的可行替代方法。
