基于细胞的TMPRSS2抑制剂高通量筛选模型建立与应用  

Establishment and application of a cell-based high-throughput screening model for TMPRSS2 inhibitors

作　　者：尤宝庆 周雯雯 李妍[1] 张晶[1] 司书毅[1] YOU Bao-qing;ZHOU Wen-wen;LI Yan;ZHANG Jing;SI Shu-yi(Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China)

机构地区：[1]中国医学科学院、北京协和医学院,医药生物技术研究所,北京100050

出　　处：《药学学报》2024年第12期3273-3281,共9页Acta Pharmaceutica Sinica

基　　金：中国医学科学院医学与健康科技创新工程重大协同创新项目(2021-I2M-1-054)。

摘　　要：跨膜丝氨酸蛋白酶2 (transmembrane serine protease 2,TMPRSS2)是人体中广泛存在的细胞表面蛋白,参与严重急性呼吸综合征冠状病毒2 (severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)等多种病毒的感染和前列腺癌细胞侵袭、肿瘤生长和转移过程等。本研究使用Boc-Gln-Ala-Arg-AMC作为表征TMPRSS2切割活性的底物,在过表达TMPRSS2的Vero E6细胞(Vero E6/TMPRSS2)中建立了TMPRSS2抑制剂细胞筛选模型,通过对国家新药(微生物)筛选实验室天然与合成化合物纯品库进行高通量筛选,得到了7个具有TMPRSS2抑制活性的低毒化合物。表面等离子共振(surface plasmon resonance,SPR)检测证明所得抑制剂均可与TMPRSS2发生中等强度的结合,且呈现浓度依赖性;细胞-细胞融合实验表明,所得抑制剂可通过抑制TMPRSS2切割SARS-CoV-2 S蛋白,抑制SARS-CoV-2 S蛋白介导的细胞-细胞融合的发生,呈现浓度依赖性;假病毒活性评价结果显示,小分子抑制剂对野生型SARS-CoV-2假病毒感染Opti-HEK-293T-ACE2受体细胞表现出不同程度的抑制活性。本研究成功建立了细胞模型用于TMPRSS2抑制剂的高通量筛选,并初步证实筛选所得的抑制剂具有体外抗TMPRSS2活性的作用,为抗SARS-CoV-2的新药研发提供了新结构骨架。Transmembrane serine protease 2(TMPRSS2) is a cell surface protease widely present in the human body.It is involved in the infection of various viruses such as severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),and in the cell invasion,tumor growth and metastasis processes of prostate cancer.This study used Boc-Gln-Ala-Arg-AMC as the fluorescent substrate to determine the cleavage activity of TMPRSS2 towards SARS-CoV-2 S protein.Then cell-based screening model for TMPRSS2 inhibitors was established in Vero E6 cells overexpressing TMPRSS2(Vero E6/TMPRSS2).Seven compounds exhibiting TMPRSS2 inhibitory activities with low toxicity were obtained through high-throughput screening(HTS) from natural and synthetic compound pure product library of National Center for Screening Novel Microbial Drugs.Surface plasmon resonance(SPR) has shown that the obtained inhibitors could bind to TMPRSS2 with moderate affinity in a dose dependent manner.Cell-cell fusion experiments have shown that the obtained inhibitors can inhibit the occurrence of S protein mediated cell-cell fusion by inhibiting TMPRSS2 cleavage of SARS-CoV-2 S protein in a concentration dependent manner.Preliminary pseudovirus experiment showed that the inhibitors may reduce the pseudovirus infection into Opti-HEK-293T-ACE2 cells to varying degrees.In a word,this study successfully established a cell-based HTS model for TMPRSS2 inhibitor and preliminarily confirmed that the seven screened inhibitors possessed in vitro anti-TMPRSS2 activities,providing new structural scaffolds for the development of new drugs against SARS-CoV-2.

关 键 词：严重急性呼吸综合征冠状病毒2 跨膜丝氨酸蛋白酶2 高通量筛选模型 表面等离子共振 细胞-细胞融合 

分 类 号：R966[医药卫生—药理学]