Yang C, Cheng C, Wang J, Chen K, Zhan J, Pan X, Xu X, Xu W, Liu S
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China.
Shenzhen Bay Laboratory, Shenzhen 518107, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2021 Apr 20;41(4):475-482. doi: 10.12122/j.issn.1673-4254.2021.04.01.
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 and explore the molecular mechanism.
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.
The median effective concentrations (EC) 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 (IC) 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 ( < 0.05). Sal-B dose-dependently inhibited SARS-CoV-2 S protein-mediated cell-cell fusion, with an IC of 3.33 μmol/L. Sal-B showed no effect on RBD-Fc protein binding to the ACE2 receptor.
Sal-B and its magnesium salt ZDDY can inhibit the entry of SARS-CoV-2 in Vero-E6 cells by blocking SARS-CoV-2 spike protein-mediated virus-cell membrane fusion.
研究中药单体丹酚酸B（Sal-B）及其镁盐注射用丹参多酚酸盐（ZDDY）对严重急性呼吸综合征冠状病毒2（SARS-CoV-2）感染的抑制作用，并探讨其分子机制。
采用真实型和假病毒型SARS-CoV-2感染试验评估Sal-B和ZDDY的抗SARS-CoV-2活性。通过分子对接和分子动力学模拟确定Sal-B的抗病毒靶点。利用圆二色光谱检测SARS-CoV-2 S蛋白HR1和HR2区域的结构特征，采用S蛋白介导的细胞-细胞融合试验评估Sal-B对病毒-细胞膜融合的影响。通过流式细胞术分析Sal-B对SARS-CoV-2受体结合域（RBD）与人类血管紧张素转换酶2（hACE2）受体结合的影响。
Sal-B和ZDDY对Vero-E6细胞中SARS-CoV-2感染的半数有效浓度（EC）分别为55.47 μmol/L和36.07 μg/mL。Sal-B和ZDDY均成功抑制了SARS-CoV-2假病毒进入稳定表达人ACE2（ACE2/293T）的细胞，半数最大抑制浓度（IC）分别为1.69 μmol/L和24.81 μg/mL。Sal-B对SARS-CoV-2 S蛋白的6螺旋束（6-HB）的结合亲和力为-8.2 kcal/mol。分子动力学模拟显示Sal-B与SARS-CoV-2 S蛋白的6-HB在预测结合位点处稳定结合。Sal-B干扰了HR1P/HR2P混合物中6-HB二级结构的形成，导致α螺旋度显著降低（P<0.05）。Sal-B剂量依赖性地抑制SARS-CoV-2 S蛋白介导的细胞-细胞融合，IC为3.33 μmol/L。Sal-B对RBD-Fc蛋白与ACE2受体的结合无影响。
Sal-B及其镁盐ZDDY可通过阻断SARS-CoV-2刺突蛋白介导的病毒-细胞膜融合来抑制SARS-CoV-2在Vero-E6细胞中的进入。
