Identification of Inhibitors of Tubulin Polymerization Using a CRISPR-Edited Cell Line with Endogenous Fluorescent Tagging of β-Tubulin and Histone H1.
Identification of Inhibitors of Tubulin Polymerization Using a CRISPR-Edited Cell Line with Endogenous Fluorescent Tagging of β-Tubulin and Histone H1.
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DOI：
10.3390/biom13020249
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发表时间：
2023-01-29
期刊：
影响因子：
5.5
通讯作者：
中科院分区：
文献类型：
--
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微管蛋白是一种在维持细胞结构和促进细胞分裂方面发挥关键作用的蛋白质。抑制微管蛋白聚合已被证明是抑制癌细胞增殖的有效策略。过去，鉴定可以抑制微管蛋白聚合的化合物需要使用纯化的微管蛋白或固定细胞的免疫荧光进行体外测定。这项研究提出了一种使用 CRISPR 编辑的细胞系来识别微管蛋白聚合抑制剂的新方法，该细胞系表达荧光标记的 β-微管蛋白和核蛋白，从而能够通过高内涵成像分析 (HCI) 实现微管蛋白聚合动力学的可视化。用已知的微管蛋白聚合抑制剂、秋水仙碱和长春新碱处理细胞，并使用HCl确认所产生的表明微管蛋白聚合抑制的表型变化。此外，还筛选了 429 种激酶抑制剂文库，鉴定出三种抑制微管蛋白聚合的化合物（ON-01910、HMN-214 和 KX2-391）。活细胞追踪分析证实，化合物治疗会导致微管蛋白快速解聚。这些发现表明，带有荧光标记内源性β-微管蛋白的CRISPR编辑细胞可用于筛选包含不同化学家族的大型化合物库，以鉴定新型微管蛋白聚合抑制剂。
Tubulin is a protein that plays a critical role in maintaining cellular structure and facilitating cell division. Inhibiting tubulin polymerization has been shown to be an effective strategy for inhibiting the proliferation of cancer cells. In the past, identifying compounds that could inhibit tubulin polymerization has required the use of in vitro assays utilizing purified tubulin or immunofluorescence of fixed cells. This study presents a novel approach for identifying tubulin polymerization inhibitors using a CRISPR-edited cell line that expresses fluorescently tagged β-tubulin and a nuclear protein, enabling the visualization of tubulin polymerization dynamics via high-content imaging analysis (HCI). The cells were treated with known tubulin polymerization inhibitors, colchicine, and vincristine, and the resulting phenotypic changes indicative of tubulin polymerization inhibition were confirmed using HCI. Furthermore, a library of 429 kinase inhibitors was screened, resulting in the identification of three compounds (ON-01910, HMN-214, and KX2-391) that inhibit tubulin polymerization. Live cell tracking analysis confirmed that compound treatment leads to rapid tubulin depolymerization. These findings suggest that CRISPR-edited cells with fluorescently tagged endogenous β-tubulin can be utilized to screen large compound libraries containing diverse chemical families for the identification of novel tubulin polymerization inhibitors.
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