Development of a Cell-Based Ligand-Screening System for Identifying Hsp90 Inhibitors
Development of a Cell-Based Ligand-Screening System for Identifying Hsp90 Inhibitors
复制标题
DOI：
10.1021/acs.biochem.9b00781
复制
发表时间：
2020-01-21
期刊：
影响因子：
2.9
通讯作者：
Hamachi, Itaru
中科院分区：
文献类型：
--
作者：
Ueda, Tsuyoshi;Tamura, Tomonori;Hamachi, Itaru
由于其在调节细胞信号转导中的关键作用，分子伴侣热休克蛋白90（Hsp 90）已成为各种疾病的新的治疗靶点，包括癌症，炎症和神经系统疾病。然而，缺乏方法，使我们能够直接评估小分子配体的细胞内Hsp 90的结合，使抑制剂的发展更加困难。在这里，我们报告了一个简单的基于细胞的检测系统的Hsp 90抑制剂在活细胞环境中。在该策略中，通过竞争性抑制配体导向的N-酰基-N-烷基磺酰胺（LDNASA）化学介导的Hsp 90标记来评价感兴趣配体的结合活性。使用几种已知的Hsp 90抑制剂，我们证明了我们的方法可以很容易地检测活细胞中Hsp 90的配体结合事件。我们的系统适用于高通量的配体筛选，我们发现了一个新的小分子候选人，结合到N-末端ATP结合域的热休克蛋白90。这些结果证明了使用基于竞争性LDNAA的方法来直接评估活细胞中的配体活性并从化学文库中鉴定有效的候选药物。
Because of its critical roles in regulating cellular signal transduction, the molecular chaperone heat-shock protein 90 (Hsp90) has become a novel therapeutic target for various diseases, including cancer, inflammation, and neurological diseases. However, the lack of methods that allow us to directly evaluate the binding of small molecule ligands to intracellular Hsp90 makes the inhibitor development more difficult. Here, we report a simple cell-based assay system for the Hsp90 inhibitor in live-cell environments. In this strategy, the binding activity of ligands of interest is evaluated by competitive inhibition of ligand-directed N-acyl-N-alkyl sulfonamide (LDNASA) chemistry-mediated Hsp90 labeling. Using several known Hsp90 inhibitors, we demonstrated that our method could easily detect the ligand-binding event of Hsp90 in live cells. Our system is applicable to high-throughput ligand screening, and we discovered a new small molecule candidate that binds to the N-terminal ATP binding domain of Hsp90. These results demonstrate the use of the competitive LDNASA-based approach to directly evaluate ligand activity in live cells and identify potent drug candidates from chemical libraries.