Promega Corporation, Madison, WI, USA.
Methods Mol Biol. 2023;2706:97-124. doi: 10.1007/978-1-0716-3397-7_8.
Kinases represent one of the most therapeutically tractable targets for drug discovery in the twenty-first century. However, confirming engagement and achieving intracellular kinase selectivity for small-molecule kinase inhibitors can represent noteworthy challenges. The NanoBRET platform enables broad-spectrum live-cell kinase selectivity profiling in most laboratory settings, without advanced instrumentation or expertise. However, the prototype workflow for this selectivity profiling is currently limited to manual liquid handling and 96-well plates. Herein, we describe a scalable workflow with automation and acoustic dispensing, thus dramatically improving the throughput. Such adaptations enable profiling of larger compound sets against 192 full-length protein kinases in live cells, with statistical robustness supporting quantitative analysis.
激酶是 21 世纪药物发现中最具治疗潜力的靶标之一。然而，对于小分子激酶抑制剂来说，确认其与靶点的结合并实现细胞内激酶选择性仍然是一个挑战。NanoBRET 平台可在大多数实验室环境中进行广谱的活细胞激酶选择性分析，而无需使用先进的仪器或专业知识。然而，目前该选择性分析的原型工作流程仅限于手动移液和 96 孔板。在此，我们描述了一种具有自动化和声波分配功能的可扩展工作流程，从而显著提高了通量。这些改进使得可以对更大的化合物集进行分析，以研究 192 种全长蛋白激酶在活细胞中的情况，并且具有统计学稳健性的定量分析支持。
