Optimization and validation of a DYRK1A TR-FRET assay for high-throughput screening.
Optimization and validation of a DYRK1A TR-FRET assay for high-throughput screening.
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DOI：
10.1016/j.mex.2021.101383
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发表时间：
2021
期刊：
影响因子：
1.9
通讯作者：
Williams KP
中科院分区：
文献类型：
--
作者：
Tarpley M;Caligan TB;Onyenwoke RU;Williams KP
结果是DYRK 1A的优化的低体积、均质和经验证的测定。由于DYRK 1A激酶（双特异性酪氨酸磷酸化调节激酶1a）在大脑发育中的作用，DYRK 1A激酶已被提议作为唐氏综合征以及与神经变性相关的疾病（包括阿尔茨海默氏症和帕金森氏症）的药物靶标。其他与DYRK 1A有关的疾病包括癌症和糖尿病。因此，需要有效和选择性的DYRK 1A抑制剂。与DYRK 1A相比，筛选大多样性化合物库需要开发一种具有成本效益的高通量筛选。在这项研究中，我们已经采取了商业的时间分辨荧光能量转移（TR-FRET）为基础的DYRK 1A的测定和优化，在室温下的小体积和均匀的格式。测定示踪剂和酶浓度。预组合DYRK 1A-GST、抗GST Ab和示踪剂，总测定体积减少2倍。使用Z'为0.7-0.8的全板最小和最大信号威尔斯孔验证测定。总体而言，这种方法：图形摘要
Results in an optimized low volume, homogenous and validated assay for DYRK1A. Delivers a cost effective high-throughput assay format for DYRK1A inhibitor screening Due to its role in brain development, the DYRK1A kinase (dual-specificity tyrosine phosphorylation-regulated kinase 1a) has been proposed as a drug target for Down syndrome, and diseases associated with neurodegeneration including Alzheimer's and Parkinson's. Other diseases in which DYRK1A is implicated include cancer and diabetes. Hence, there is need for potent and selective DYRK1A inhibitors. To screen large diversity compound libraries versus DYRK1A requires the development of a cost-effective high-throughput screen. In this study, we have taken a commercial time-resolved fluorescence energy transfer (TR-FRET)-based assay for DYRK1A and optimized for smaller volumes and homogenous format at room temperature. Tracer and enzyme concentrations were determined. DYRK1A-GST, anti-GST Ab and tracer were pre-combined and total assay volume reduced 2-fold. The assay was validated using whole plate minimum and maximum signal wells with a Z’ of 0.7-0.8 determined. Overall, this method: Graphical abstract
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