Discovery Sciences, BioPharmaceutical R&D , AstraZeneca , Boston , United States.
Discovery Sciences, BioPharmaceutical R&D , AstraZeneca , Pepparedsleden 1 , Gothenburg , Sweden.
ACS Chem Biol. 2019 Sep 20;14(9):1913-1920. doi: 10.1021/acschembio.9b00399. Epub 2019 Aug 16.
Demonstration of target binding is a key requirement for understanding the mode of action of new therapeutics. The cellular thermal shift assay (CETSA) has been introduced as a powerful label-free method to assess target engagement in physiological environments. Here, we present the application of live-cell CETSA to different classes of integral multipass transmembrane proteins using three case studies, the first showing a large and robust stabilization of the outer mitochondrial five-pass transmembrane protein TSPO, the second being a modest stabilization of SERCA2, and the last describing an atypical compound-driven stabilization of the GPCR PAR2. Our data demonstrated that using modified protocols with detergent extraction after the heating step, CETSA can reliably be applied to several membrane proteins of different complexity. By showing examples with distinct CETSA behaviors, we aim to provide the scientific community with an overview of different scenarios to expect during CETSA experiments, especially for challenging, membrane bound targets.
证明靶标结合是理解新型治疗药物作用模式的关键要求。细胞热转移分析 (CETSA) 已被引入作为一种强大的无标记方法来评估生理环境中的靶标结合。在这里，我们通过三个案例研究展示了活细胞 CETSA 在不同类别整体多次跨膜蛋白中的应用，第一个案例显示了外线粒体五跨膜蛋白 TSPO 的大幅和稳健稳定，第二个案例显示了 SERCA2 的适度稳定，最后一个案例描述了 GPCR PAR2 的非典型化合物驱动稳定。我们的数据表明，使用经过加热步骤后用去污剂提取的改良方案，CETSA 可以可靠地应用于不同复杂性的几种膜蛋白。通过展示具有不同 CETSA 行为的例子，我们旨在为科学界提供 CETSA 实验中可能出现的不同情况的概述，特别是对于具有挑战性的膜结合靶标。
