Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO, 347 UCB, USA.
Commun Biol. 2020 Feb 14;3(1):75. doi: 10.1038/s42003-020-0795-6.
Most small molecule drugs act on living systems by physically interacting with specific proteins and modulating target function. Identification of drug binding targets, within the complex milieu of the human proteome, remains a challenging task of paramount importance in drug discovery. Existing approaches for target identification employ complex workflows with limited throughput. Here, we present the isothermal shift assay (iTSA), a mass spectrometry method for proteome-wide identification of drug targets within lysates or living cells. Compared with prevailing methods, iTSA uses a simplified experimental design with increased statistical power to detect thermal stability shifts that are induced by small molecule binding. Using a pan-kinase inhibitor, staurosporine, we demonstrate improved performance over commonly used thermal proteome profiling methods, identifying known targets in cell lysates and living cells. We also demonstrate the identification of both known targets and additional candidate targets for the kinase inhibitor harmine in cell and tissue lysates.
大多数小分子药物通过与特定蛋白质物理相互作用并调节靶标功能来作用于生命系统。在人类蛋白质组的复杂环境中，确定药物结合靶标仍然是药物发现中至关重要的挑战性任务。现有的靶标识别方法采用具有有限通量的复杂工作流程。在这里，我们提出了等温位移分析（iTSA），这是一种用于在裂解物或活细胞中鉴定药物靶标的质譜法。与现有方法相比，iTSA 使用简化的实验设计，具有更高的统计能力来检测小分子结合诱导的热稳定性变化。使用泛激酶抑制剂 staurosporine，我们证明了在识别细胞裂解物和活细胞中的已知靶标方面优于常用的热蛋白质组分析方法的性能。我们还证明了在细胞和组织裂解物中鉴定激酶抑制剂 harmine 的已知靶标和其他候选靶标的能力。
