School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China.
Anal Chem. 2020 May 19;92(10):7020-7027. doi: 10.1021/acs.analchem.0c00263. Epub 2020 May 7.
The drug-target protein interaction is the basis of drug screening and precise therapy in modern clinical medicine. How to acquire the information about the drug-target protein interaction in single living cell is a great challenge due to the shortage of efficient methods. Here we propose a new strategy for in situ studying the drug-target protein interaction in single living cells based on the competition of candidate drugs to the fluorescent probe-target complexes and fluorescence correlation spectroscopy (FCS) with a microfluidic chip. In this study, we used ABL kinase (target) as a model and synthesized a fluorescent probe (Cy3-dasatinib) with an affinity to the target using ABL inhibitor dasatinib as a precursor. We systematically investigated the association of the probe with targets and the dissociation of the drug-target complexes in the presence of candidate drug. We presented a new parameter IC (τ) to assess the inhibitory effect of drugs on the basis of the changes in the characteristic diffusion time (τ) and the binding ratio () of fluorescent probes during the drug competition process in living cells. We found a remarkable difference of IC (τ) values in living cells and in solutions, suggesting it is quite necessary to evaluate the drug-target interactions in living cells. Compared with current methods, our approach can be used to in situ and real-time study the drug-target interaction in living cells, and it may become a promising and universal tool for in situ drug research at molecular level.
药物-靶标蛋白相互作用是现代临床医学中药物筛选和精准治疗的基础。由于缺乏有效的方法，如何在单个活细胞中获取药物-靶标蛋白相互作用的信息是一个巨大的挑战。在这里，我们提出了一种基于候选药物与荧光探针-靶复合物竞争和带有微流控芯片的荧光相关光谱（FCS）的原位研究单个活细胞中药物-靶标蛋白相互作用的新策略。在这项研究中，我们以 ABL 激酶（靶标）为模型，使用 ABL 抑制剂 dasatinib 作为前体合成了一种对靶标具有亲和力的荧光探针（Cy3-dasatinib）。我们系统地研究了探针与靶标的结合以及在候选药物存在下药物-靶复合物的解离。我们提出了一个新的参数 IC（τ），基于荧光探针在活细胞中药物竞争过程中特征扩散时间（τ）和结合比（）的变化，评估药物对其的抑制作用。我们发现活细胞和溶液中 IC（τ）值有显著差异，这表明在活细胞中评估药物-靶标相互作用是非常必要的。与目前的方法相比，我们的方法可以用于原位和实时研究活细胞中的药物-靶标相互作用，它可能成为一种有前途的、通用的原位分子水平药物研究工具。
