A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance

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.

激酶（Kinase）是一类重要的药物靶点，在癌症研究领域尤为关键。为了在生理相关的环境中阐明潜在激酶抑制剂对其靶点的作用机制，亟需建立基于细胞的激酶检测方法。当前主流的基于细胞的激酶检测手段多依赖于内源性底物的抗体检测、精度不足的疾病模型，或是对药物作用的间接测定。本研究基于本实验室前期工作，开发了一种适配96孔板（96-well plate）的检测方法：通过外源性添加的多功能肽底物，可在疾病相关的人源慢性髓系白血病细胞系中测定细胞水平的激酶活性。本研究所用细胞模型可天然表达Bcr-Abl致癌基因，且分别对经过充分表征的Bcr-Abl酪氨酸激酶抑制剂（tyrosine kinase inhibitor）表现出敏感性或获得性耐药性。该方法测得的半数抑制浓度（IC50）与评估药物效力的成熟方法所得结果具有良好的一致性，其稳健性表明该方法可应用于药物研发场景。这种中通量检测手段可填补靶向单一靶点的高通量体外检测与低通量细胞水平后续验证实验之间的技术空白。