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

作　　者：张成玮 张文开 李宝虎 马梦君 孙俊 张思琪 葛玲 马树敏 ZHANG Chengwei;ZHANG Wenkai;LI Baohu;MA Mengjun;SUN Jun;ZHANG Siqi;GE Ling;MA Shumin(Key Laboratory of Marine Drugs,Ministry of Education,School of Medicine and Pharmacy,Ocean University of China,Qingdao 266003,China;PROTAC Biopharmaceutical Technology Co.,Ltd,Qingdao 266042,China)

机构地区：[1]中国海洋大学海洋药物教育部重点实验室,医药学院,山东青岛266003 [2]青岛普泰科生物医药科技有限公司,山东青岛266042

出　　处：《中国海洋药物》2024年第4期42-50,共9页Chinese Journal of Marine Drugs

基　　金：国家重点研发计划青年科学家项目(2022YFC2804400);国家自然科学基金项目(22107095);山东省自然科学基金项目(ZR2021YQ53);2019年度创新人才引进项目(LMDBCXRC201901)资助。

摘　　要：目的 构建稳定表达HaloTag蛋白的细胞筛选模型,用于蛋白水解靶向嵌合体(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FlagHalo-NLS(细胞核定位)蛋白的HEK-293T细胞株;采用Western Blotting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0.3μmol/L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。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 0.3μM.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.

关 键 词：HALOTAG 蛋白水解靶向嵌合体 稳转细胞株 活性筛选 

分 类 号：R965[医药卫生—药理学]