Gagestein Berend, Rüegger Joel, Janssen Antonius P A, Kirchner Stephan, Grether Uwe, Rufer Arne C, van der Stelt Mario
Department of Molecular Physiology, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
Oncode Institute, Amsterdam, The Netherlands.
Methods Mol Biol. 2025;2921:231-253. doi: 10.1007/978-1-0716-4502-4_12.
The human genome encodes for over 600 kinases, which are important targets for drug discovery in many diseases, such as cancer and autoimmune disorders. The majority of kinase inhibitors target the conserved ATP-binding pocket, which contributes to the difficulty of developing selective inhibitors. A lack of selectivity may contribute to high drug candidate attrition rates due to an unfavorable off-target profile. To prevent unforeseen side effects, as well as to accurately link inhibitor function to pharmacological effects and phenotypic readouts, efficient methods for determining target engagement across the cellular kinome are necessary. One way to address this problem is to profile the interaction landscape of kinase inhibitors in living cells using the broad-spectrum kinase probe XO44. Here, we describe a detailed protocol of the CellEKT workflow using XO44 to profile kinase inhibitors in a full dose-response manner across 200+ kinases. The protocol allows to determine the EC of up to three kinase inhibitors in a cell line of interest in 4 days.
人类基因组编码600多种激酶，它们是许多疾病（如癌症和自身免疫性疾病）药物研发的重要靶点。大多数激酶抑制剂靶向保守的ATP结合口袋，这导致开发选择性抑制剂存在困难。缺乏选择性可能会因不良的脱靶效应导致药物候选物的高淘汰率。为了防止不可预见的副作用，并准确地将抑制剂功能与药理作用和表型读数联系起来，需要有效的方法来确定整个细胞激酶组中的靶点结合情况。解决这个问题的一种方法是使用广谱激酶探针XO44在活细胞中描绘激酶抑制剂的相互作用图谱。在这里，我们描述了一个详细的CellEKT工作流程方案，该方案使用XO44以全剂量反应方式对200多种激酶的激酶抑制剂进行分析。该方案能够在4天内确定感兴趣细胞系中多达三种激酶抑制剂的半数有效浓度（EC）。
