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 β-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.

中文翻译：

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使用基于2D凝胶电泳的全蛋白质组CETSA鉴定靶向PKM2调控信号的小化合物。

细胞热位移测定法（CETSA）最近已被设计为一种无标记方法，用于小化合物的目标验证以及监测由于与该化合物结合而引起的蛋白质的热稳定或不稳定。在本文中，我们结合了CETSA和基于2D凝胶电泳的蛋白质组学分析（即2DE-CETSA），开发了一种改进的方法，以通过与新化合物结合来鉴定热稳定性改变的蛋白质。我们将2DE-CETSA应用于目标未知化合物NPD10084的分析，该化合物在体外和体内对结肠直肠癌细胞均具有抗增殖活性，并确定了丙酮酸激酶肌肉同工型2（PKM2）作为候选目标蛋白。有趣的是，NPD10084中断了PKM2和β-catenin或STAT3之间的蛋白相互作用，并随后抑制下游信令。因此，我们证明了我们的2DE-CETSA方法适用于鉴定通过表型筛选发现的目标化合物。