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.
Objective: 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.
Objective: The median effective concentrations (EC50) of Sal-B and ZDDY against SARSCoV-2 infection in Vero-E6 cells were 55.47 μmol/L and 36.07 μg/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 (IC50) of 1.69 μmol/L and 24.81 μg/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 lowered α-helicity (P < 0.05). Sal-B dose-dependently inhibited SARS-CoV-2 S protein-mediated cell-cell fusion, with an IC50 of 3.33 μmol/L. Sal-B showed no effect on RBD-Fc protein binding to the ACE2 receptor.
Objective: 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.
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（VeroE6）感染模型上抑制SARS-CoV-2的半数有效浓度EC50分为55.47 μmol/L，36.07 μg/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IC50分别为1.69 μmol/L，24.81 μg/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，IC50为3.33 μmol/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。
Keywords: anti-viral drugs; entry inhibitors; severe acute respiratory syndrome coronavirus 2; spike protein; traditional Chinese medicine.