Park Da Kyeong, Lee Sang-Hee, Kweon Hee-Seok, Lee Zee-Won, Lee Kyung-Bok
Center for Bio-Imaging & Translational Research and Bioimaging Data Curation Center, Korea Basic Science Institute (KBSI), Cheongju 28119, Republic of Korea.
bHLBIO, Cheongju 28119, Republic of Korea.
Int J Mol Sci. 2025 Jun 13;26(12):5705. doi: 10.3390/ijms26125705.
Interactions between chemical drugs and their target proteins are fundamental to drug screening and precision therapy in modern clinical medicine. However, elucidating these interactions within living cells remains challenging due to the limited availability of efficient detection methods. Despite substantial efforts, technical limitations still impede the identification of direct interactors. In this study, we present a simple method to detect the binding between a chemical drug and its target proteins in live cells. This approach utilizes a self-labeling protein (SLP) tag system, specifically HaloTag which is a modified haloalkane dehalogenase, combined with spatially localized expression of the SLP. To implement this system, dasatinib was conjugated to a HaloTag ligand, and the HaloTag protein was expressed in specific intracellular compartments, such as endosomes or F-actin structures. Upon treatment of cells with the HaloTag ligand-conjugated dasatinib, green fluorescent protein (GFP)-fused cytoplasmic dasatinib target proteins were observed to co-localize with the HaloTag at these subcellular structures, thereby indicating direct drug-target binding. This method provides a good spatial resolution with a high signal-to-noise ratio and low false-positive signals across a high background and false-positive/false-negative signals from endogenous proteins and/or non-specific binding. In this context, we believe that our method is a useful platform for visualizing the binding between chemical drugs and their cytoplasmic targets within living systems.
化学药物与其靶蛋白之间的相互作用是现代临床医学中药物筛选和精准治疗的基础。然而，由于高效检测方法的可用性有限，在活细胞内阐明这些相互作用仍然具有挑战性。尽管付出了巨大努力，但技术限制仍然阻碍了直接相互作用分子的鉴定。在本研究中，我们提出了一种在活细胞中检测化学药物与其靶蛋白之间结合的简单方法。该方法利用了一种自标记蛋白（SLP）标签系统，特别是HaloTag，它是一种经过修饰的卤代烷脱卤酶，并结合了SLP的空间定位表达。为了实现这个系统，将达沙替尼与HaloTag配体偶联，并在特定的细胞内区室中表达HaloTag蛋白，如内体或F-肌动蛋白结构。在用与HaloTag配体偶联的达沙替尼处理细胞后，观察到绿色荧光蛋白（GFP）融合的细胞质达沙替尼靶蛋白与HaloTag在这些亚细胞结构中共定位，从而表明药物与靶标的直接结合。该方法具有良好的空间分辨率，在高背景下具有高信噪比和低假阳性信号，以及来自内源性蛋白质和/或非特异性结合的假阳性/假阴性信号。在这种情况下，我们相信我们的方法是一个用于可视化活系统中化学药物与其细胞质靶标之间结合的有用平台。
