A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance.
A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance.
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DOI：
10.1371/journal.pone.0161748
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Parker LL
中科院分区：
文献类型：
--
作者：
Ouellette SB;Noel BM;Parker LL
激酶是一类重要的药物靶标，特别是在癌症中。需要基于细胞的激酶测定来了解潜在的激酶抑制剂如何在生理学相关的背景下作用于其靶点。目前基于细胞的激酶测定依赖于内源性底物的基于抗体的检测、不准确的疾病模型或药物作用的间接测量。在这里，我们扩展了我们实验室以前的工作，介绍了一种96孔板兼容的方法，用于使用外源性添加的多功能肽底物测量疾病相关的人类慢性髓性白血病细胞系中基于细胞的激酶活性。我们的细胞模型天然表达BcrAbl癌基因，并且对充分表征的BcrAbl酪氨酸激酶抑制剂敏感或具有获得性抗性。这种方法测量的IC50值与已建立的评估药物效力的方法相当，其稳健性表明其可用于药物发现应用。这种中等通量测定可以弥合单靶点聚焦、高通量体外测定和低通量基于细胞的后续实验之间的差距。
Kinase enzymes are an important class of drug targets, particularly in cancer. Cell-based kinase assays are needed to understand how potential kinase inhibitors act on their targets in a physiologically relevant context. Current cell-based kinase assays rely on antibody-based detection of endogenous substrates, inaccurate disease models, or indirect measurements of drug action. Here we expand on previous work from our lab to introduce a 96-well plate compatible approach for measuring cell-based kinase activity in disease-relevant human chronic myeloid leukemia cell lines using an exogenously added, multi-functional peptide substrate. Our cellular models natively express the BcrAbl oncogene and are either sensitive or have acquired resistance to well-characterized BcrAbl tyrosine kinase inhibitors. This approach measures IC50 values comparable to established methods of assessing drug potency, and its robustness indicates that it can be employed in drug discovery applications. This medium-throughput assay could bridge the gap between single target focused, high-throughput in vitro assays and lower-throughput cell-based follow-up experiments.
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影响因子：
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