| 张成玮,张文开,李宝虎,等.以HaloTag为靶点的蛋白降解药物细胞筛选模型的建立及应用[J].中国海洋药物,2024,43(4):42-50. |
| 以HaloTag为靶点的蛋白降解药物细胞筛选模型的建立及应用 |
| Construction and Application of Protein Degrader Cell Screening Based on HaloTag |
| 投稿时间：2023-03-24 修订日期：2023-04-20 |
| DOI：10.13400/j.cnki.cjmd.2024.04.008 |
| 中文关键词: HaloTag PROTAC 稳转细胞株 活性筛选 |
| English Keywords:HaloTag PROTAC Stable cell line Activity screening |
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| 中文摘要: |
| 目的 构建稳定表达HaloTag蛋白的细胞筛选模型，用于PROTAC降解剂的活性筛选和E3泛素连接酶配体的开发。方法 采用慢病毒技术分别构建稳定表达HA-Halo（细胞质定位）和Flag-Halo-NLS（细胞核定位）蛋白的HEK-293T细胞株；采用Western blot检测HaloTag降解剂VHL-HP3靶向降解HA-Halo和 Flag-Halo-NLS蛋白的水平。结果 成功构建了稳定表达HA-Halo和Flag-Halo-NLS蛋白的HEK-293T的细胞模型；化合物VHL-HP3能显著降解HA-Halo和Flag-Halo-NLS蛋白的水平，且在0.3 μM时均表现出显著的降解活性。结论 稳定表达HaloTag蛋白的细胞株，可作为以HaloTag蛋白为靶点的PROTAC降解剂活性筛选的模型细胞，为海洋药物来源的E3泛素连接酶配体的开发提供了高通量的筛选方法。 |
| English Summary: |
| Objective The cell screening model for stable expression of HaloTag protein was constructed for the activity screening of PROTAC degraders and the development of E3 ubiquitin ligase ligands. Methods Lentivirus technology was used to construct HEK-293T cell lines stably expressing HA-Halo and Flag-Halo-NLS proteins, and Western blot was used to detect the levels of HA-Halo and Flag-Halo-NLS proteins targeted by HaloTag degrader VHL-HP3. Results Cell models of HEK-293T stably expressing HA-Halo and Flag-Halo-NLS proteins were successfully constructed; compound VHL-HP3 significantly degraded the levels of HA-Halo and Flag-Halo-NLS proteins and both showed significant degradation activity at 0.3 μM. Conclusion Stably expressing HaloTag protein cell lines can be used as model cells for screening the activity of PROTAC degraders targeting HaloTag protein, providing a high-throughput screening method for the development of E3 ubiquitin ligase ligands of marine drug origin. |
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