Identification of a Small Compound Targeting PKM2-Regulated Signaling Using 2D Gel Electrophoresis-Based Proteome-wide CETSA
Identification of a Small Compound Targeting PKM2-Regulated Signaling Using 2D Gel Electrophoresis-Based Proteome-wide CETSA
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DOI：
10.1016/j.chembiol.2019.11.010
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发表时间：
2020-02-20
影响因子：
8.6
通讯作者：
Osada, Hiroyuki
中科院分区：
文献类型：
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作者：
Nagasawa, Ikuko;Muroi, Makoto;Osada, Hiroyuki
细胞热位移测定 (CETSA) 最近被设计为一种无标记方法，用于小化合物的靶标验证并监测由于与化合物结合而导致的蛋白质的热稳定或不稳定。在此，我们开发了一种结合 CETSA 和基于 2D 凝胶电泳的蛋白质组学分析的改进方法，即 2DE-CETSA，通过与新化合物结合来识别热稳定性转变的蛋白质。我们应用 2DE-CETSA 分析靶标未知化合物 NPD10084，该化合物在体外和体内对结直肠癌细胞发挥抗增殖活性，并确定丙酮酸激酶肌肉亚型 2 (PKM2) 作为候选靶蛋白。有趣的是，NPD10084 中断了 PKM2 和 β-catenin 或 STAT3 之间的蛋白质-蛋白质相互作用，随后抑制了下游信号传导。因此，我们证明我们的 2DE-CETSA 方法适用于鉴定通过表型筛选发现的目标化合物。
The cellular thermal shift assay (CETSA) has recently been devised as a label-free method for target validation of small compounds and monitoring the thermal stabilization or destabilization of proteins due to binding with the compound. Herein, we developed a modified method by combining the CETSA and proteomics analysis based on 2D gel electrophoresis, namely 2DE-CETSA, to identify the thermal stability-shifted proteins by binding with a new compound. We applied the 2DE-CETSA for analysis of a target-unknown compound, NPD10084, which exerts anti-proliferative activity against colorectal cancer cells in vitro and in vivo, and identified pyruvate kinase muscle isoform 2 (PKM2) as a candidate target protein. Interestingly, NPD10084 interrupted protein-protein interactions between PKM2 and beta-catenin or STAT3, with subsequent suppression of downstream signaling. We thus demonstrate that our 2DE-CETSA method is applicable for identification of target compounds discovered by phenotypic screening.