Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent responsible for the worldwide coronavirus disease 2019 (COVID-19) outbreak. Investigation has confirmed that polysaccharide heparan sulfate can bind to the spike protein and block SARS-CoV-2 infection. Theoretically, similar structure of nature polysaccharides may also have the impact on the virus. Indeed, some marine polysaccharide has been reported to inhibit SARS-Cov-2 infection in vitro, however the convinced targets and mechanism are still vague. By high throughput screening to target 3CLpro enzyme, a key enzyme that plays a pivotal role in the viral replication and transcription using nature polysaccharides library, we discover the mixture polysaccharide 375 from seaweed Ecklonia kurome Okam completely block 3Clpro enzymatic activity (IC₅₀, 0.48 µM). Further, the homogeneous polysaccharide 37502 from the 375 may bind to 3CLpro molecule well (kD value : 4.23 × 10⁻⁶). Very interestingly, 37502 also can potently disturb spike protein binding to ACE2 receptor (EC₅₀, 2.01 µM). Importantly, polysaccharide 375 shows good anti-SARS-CoV-2 infection activity in cell culture with EC₅₀ values of 27 nM (99.9% inhibiting rate at the concentration of 20 µg/mL), low toxicity (LD₅₀: 136 mg/Kg on mice). By DEAE ion-exchange chromatography, 37501, 37502 and 37503 polysaccharides are purified from native 375. Bioactivity test show that 37501 and 37503 may impede SARS-Cov-2 infection and virus replication, however their individual impact on the virus is significantly less that of 375. Surprisingly, polysaccharide 37502 has no inhibition effect on SARS-Cov-2. The structure study based on monosaccharide composition, methylation, NMR spectrum analysis suggest that 375 contains guluronic acid, mannuronic acid, mannose, rhamnose, glucouronic acid, galacturonic acid, glucose, galactose, xylose and fucose with ratio of 1.86 : 9.56 : 6.81 : 1.69 : 1.00 : 1.75 : 1.19 : 11.06 : 4.31 : 23.06. However, polysaccharide 37502 is an aginate which composed of mannuronic acid (89.3 %) and guluronic acid (10.7 %), with the molecular weight (Mw) of 27.9 kDa. These results imply that mixture polysaccharides 375 works better than the individual polysaccharide on SARS-Cov-2 may be the cocktail-like polysaccharide synergistic function through targeting multiple key molecules implicated in the virus infection and replication. The results also suggest that 375 may be a potential drug candidate against SARS-CoV-2.

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夜来香Ecklonia kurome鸡尾酒样靶向多糖的结构表征及其抗SARS-CoV-2活性

严重急性呼吸系统综合症冠状病毒2（SARS-CoV-2）是导致2019年全球冠状病毒病（COVID-19）爆发的病原体。研究已经证实，多糖硫酸乙酰肝素可以结合刺突蛋白并阻断SARS-CoV-2感染。从理论上讲，天然多糖的相似结构也可能对病毒产生影响。实际上，已经报道了一些海洋多糖在体外抑制SARS-Cov-2感染，但是确信的靶标和机制仍然不清楚。通过高通量筛选使用天然多糖文库靶向3CLpro酶（一种在病毒复制和转录中起关键作用的关键酶），我们从海藻Ecklonia kurome中发现了混合多糖375Okam完全阻断了3Clpro的酶活性（IC _50为0.48 µM）。此外，来自375的均质多糖37502可以很好地结合3CLpro分子（kD值：4.23×10 ^-6）。非常有趣的是，37502也可以有效地干扰刺突蛋白结合ACE2受体（EC ₅₀，2.01μM）。重要的是，多糖375在细胞培养中显示出良好的抗SARS-CoV-2感染活性，EC ₅₀值为27 nM（浓度为20 µg / mL时抑制率为99.9％），低毒性（LD ₅₀：在小鼠上为136mg / Kg）。通过DEAE离子交换色谱法，从天然375中纯化了37501、37502和37503多糖。生物活性测试表明37501和37503可能会阻止SARS-Cov-2感染和病毒复制，但是它们对病毒的单独影响却明显小于375.令人惊讶的是，多糖37502对SARS-Cov-2没有抑制作用。基于单糖组成，甲基化，NMR谱分析的结构研究表明，375含有古龙糖醛酸，甘露糖醛酸，甘露糖，鼠李糖，葡糖醛酸，半乳糖醛酸，葡萄糖，半乳糖，木糖和岩藻糖，比例为1.86：9.56：6.81：1.69 ：1.00：1.75：1.19：11.06：4.31：23.06。然而，多糖37502是一种由甘露糖醛酸（89.3％）和古洛糖醛酸（10.7％）组成的海藻酸酯，分子量为（Mw）为27.9 kDa。这些结果暗示混合多糖375比SARS-Cov-2上的单个多糖更好地起作用，其可能是通过靶向与病毒感染和复制有关的多个关键分子而具有的鸡尾酒样多糖协同功能。结果还表明375可能是抗SARS-CoV-2的潜在候选药物。