丹酚酸B及其镁盐通过阻止病毒融合抑制SARS-CoV-2感染靶细胞  

Salvianolic acid B and its magnesium salt inhibit SARS-CoV-2 infection of Vero-E6 cells by blocking spike protein-mediated membrane fusion

作　　者：杨婵 程晨 王进绅 陈珂 詹剑 潘晓彦 许鑫锋 徐伟[1] 刘叔文[1,6] YANG Chan;CHENG Chen;WANG Jinshen;CHEN Ke;ZHAN Jian;PAN Xiaoyan;XU Xinfeng;XU Wei;LIU Shuwen(Guangdong Provincial Key Laboratory of New Drug Screening,School of Pharmaceutical Sciences,Southern Medical University,Guangzhou 510515,China;Shenzhen Bay Laboratory,Shenzhen 518107,China;Shenzhen Graduate School,Peking University,Shenzhen 518055,China;Griffith University,Gold Coast 4222,Australia;CAS Key Laboratory of Special Pathogens and Biosafety,Wuhan Institute of Virology,Chinese Academy of Sciences,Wuhan 430071,China;State Key Laboratory of Organ Failure Research,Guangdong Provincial Institute of Nephrology,Southern Medical University,Guangzhou 510515,China)

机构地区：[1]广东省新药筛选重点实验室,南方医科大学药学院,广东广州510515 [2]深圳湾实验室,广东深圳518107 [3]北京大学深圳研究生院,广东深圳518055 [4]澳大利亚格里菲斯大学,澳大利亚黄金海岸4222 [5]中国科学院武汉病毒所,湖北武汉430071 [6]器官衰竭防治国家重点实验室//广东省肾脏病研究所,广东广州510515

出　　处：《南方医科大学学报》2021年第4期475-482,共8页Journal of Southern Medical University

基　　金：广东省“新药创制”科技重大专项项目(2019B020202002);国家中医药管理局中医药循证能力建设项目(ZZ13-035-02,2019XZZXLG04);深圳湾实验室重点项目(S201101001)。

摘　　要：目的探究中药单体丹酚酸B(Sal-B)及其镁盐体外抑制SARS-CoV-2感染靶细胞的效果及作用机制。方法利用感染性SARS-CoV-2及SARS-CoV-2假病毒体外细胞感染模型,检测Sal-B及其镁盐制剂注射用丹参多酚酸盐(ZDDY)的抗SARSCoV-2活性;利用分子对接技术与分子动力学模拟技术,寻找Sal-B的抗病毒作用靶点;利用圆二色谱技术,检测六螺旋束(6-HB)的α-螺旋构象;利用SARS-CoV-2 S蛋白介导的细胞-细胞融合体系,检测Sal-B是否作用于新冠病毒入侵宿主的膜融合过程;利用流式细胞术,检测Sal-B是否作用于新冠病毒受体结合区RBD蛋白。结果Sal-B与ZDDY在非洲绿猴肾细胞(Vero-E6)感染模型上抑制SARS-CoV-2的半数有效浓度EC50分为55.47μmol/L,36.07μg/mL;Sal-B与ZDDY抑制SARS-CoV-2的进入阶段,抑制SARS-CoV-2假病毒活性的IC_(50)分别为1.69μmol/L,24.81μg/mL;Sal-B可与SARS-CoV-2 S2亚基的8个氨基酸位点结合,亲和力为-8.2 kcal/moL,Sal-B在分子对接预测位点与SARS-CoV-2 S2亚基稳定结合;Sal-B干扰SARS-CoV-2 HR1与HR2形成6-HB,显著降低6-HB的α-螺旋百分比(P<0.05);Sal-B浓度依赖性抑制SARS-CoV-2 S蛋白介导的细胞-细胞膜融合,IC_(50)为3.33μmol/L;Sal-B不影响SARS-CoV-2 RBD蛋白与受体ACE2结合。结论Sal-B及其镁盐制剂ZDDY体外可有效抑制SARS-CoV-2感染靶细胞,Sal-B通过抑制病毒膜融合过程发挥抗SARS-CoV-2活性。Objective The investigate the inhibitory effects of the traditional Chinese medicine(TCM)monomer salvianolic acid B(Sal-B)and its magnesium salt Salvia Miltiorrhiza Polyphenolate Injection(ZDDY)against SARS-CoV-2 infection in vitro and explore the molecular mechanism.Methods The anti-SARS-CoV-2 activity of Sal-B and ZDDY was assessed using the authentic and pseudotyped SARS-CoV-2 infection assay.The antiviral targets of Sal-B were identified by molecular docking and molecular dynamics simulation.Circular dichroism spectroscopy was used to examine the structural characteristics of HR1 and HR2 regions of SARS-CoV-2 S protein,and the S protein-mediated cell-cell fusion assay was used to evaluate the effect of Sal-B on virus-cell membrane fusion.Flow cytometry was carried out to analyze the effect of Sal-B on the binding of SARS-CoV-2 RBD to hACE2 receptor.Results The median effective concentrations(EC50)of Sal-B and ZDDY against SARSCoV-2 infection in Vero-E6 cells were 55.47μmol/L and 36.07μg/mL,respectively.Both Sal-B and ZDDY successfully inhibited the entry of SARS-CoV-2 pseudovirus into the cells that stably expressed human ACE2(ACE2/293T),with half maximal inhibitory concentrations(IC_(50))of 1.69μmol/L and 24.81μg/mL,respectively.Sal-B showed a binding affinity of-8.2 kcal/mol to the 6-helix bundle(6-HB)of SARS-CoV-2 S protein.Molecular dynamics simulation showed stable binding between Sal-B and the 6-HB of SARS-CoV-2 S protein at the predicted binding site.Sal-B disturbed the formation of the secondary structure of 6-HB in HR1P/HR2P mixture,resulting in a significantly loweredα-helicity(P<0.05).Sal-B dose-dependently inhibited SARS-CoV-2 S protein-mediated cell-cell fusion,with an IC_(50)of 3.33μmol/L.Sal-B showed no effect on RBD-Fc protein binding to the ACE2 receptor.Conclusion Sal-B and its magnesium salt ZDDY can inhibit the entry of SARS-CoV-2 in Vero-E6 cells in vitro by blocking SARS-CoV-2 spike protein-mediated virus-cell membrane fusion.

关 键 词：SARS-CoV-2 棘突蛋白 抗病毒药物 进入抑制剂 中药 

分 类 号：R285.5[医药卫生—中药学]