基于细胞热漂移测定(CETSA)技术鉴定活细胞内药物靶标的标准操作流程

A standard operating procedure for identification of drug targets in living cells based on cell thermal shift assay (CETSA)

摘要:

【目的】细胞热漂移测定(cell thermal shift assay, CETSA)技术是一种检测细胞内药物(配体)和蛋白质(靶标)相互作用的技术，原理是当蛋白质结合药物后，其热稳定性会发生变化，通过测定这种变化去鉴定药物和蛋白之间的相互作用。本研究以治疗多发性骨髓瘤的靶向药帕比司他(panobinostat)为例，建立基于蛋白印迹杂交(Western blotting)和CETSA技术的药物靶蛋白鉴定的标准操作流程。【方法】首先用药物panobinostat处理培养的K562细胞，然后加热处理细胞、裂解细胞及提取可溶性蛋白，以及用抗靶蛋白的抗体经Western blotting定量可溶性蛋白。【结果】经Western blotting定量及曲线拟合，成功得到3个蛋白——组蛋白去乙酰化酶(histone deacetylase, HDAC1)、人突触蛋白(human syntaxin-4, STX4)以及四三肽重复结构域(tetratricopeptide repeat domain 38, TTC38)随温度变化的热熔解曲线和恒定温度条件下的药物剂量反应曲线。【结论】HDAC1、STX4及TTC38为药物panobinostat在K562细胞中的靶蛋白。本研究建立了基于CETSA和Western blotting技术，鉴定活细胞中药物靶标蛋白的标准操作流程。实验可在2-3 d内完成。

Abstract:

[Objective] Cell thermal shift assay (CETSA) is a biophysical technique allowing the direct study of drug (ligand) binding to proteins (targets) in cells and tissues by measuring variation in the proteins’ thermal stability upon ligand binding. In this paper, taking panobinostat, a drug targeting multiple myeloma, as an example, we developed a standard operating procedure for the identification of drug targets in K562 cells with Western blotting and CETSA. [Methods] Experimental procedures included treating cells with the drug, heat treatment of cells, cell lysis, total soluble protein extraction and quantitation by Western blotting with specific antibodies. [Results] Through quantification by Western blotting and curve fitting, we obtained the CETSA melting curve and isothermal dose-response curve of histone deacetylase (HDAC1), syntaxin-4 (STX4), and tetratricopeptide repeat protein 38 (TTC38) in K562 cells, respectively.[Conclusion] HDAC1, STX4, and TTC38 were the targets of panobinostat in K562 cells. A standard operating procedure for identifying the target proteins in living cells was established by CESTA and Western blotting. The experiment can be completed in 2-3 d.