基于双靶点表面等离子体共振传感器的中药抗新冠病毒活性成分筛选研究
Screening of anti-SARS-CoV-2 ligands from Chinese herbs based on a dual-target surface plasmon resonance biosensor
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摘要: 新型冠状病毒感染(coronavirus disease-2019, COVID-19) 给全球公共卫生防控和临床诊疗系统带来了巨大的挑战, 开发有效的抗病毒药物是当前药学研究的一项重要任务。中医药在抗击新冠疫情中发挥了重要作用, 中药中含有的化学成分众多、结构类型多样, 是寻找抗新型冠状病毒(severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) 活性先导化合物的天然宝库。本研究根据中药多成分、多靶点的作用特征, 针对新冠病毒入侵细胞过程中两种关键蛋白S蛋白受体结合域(S protein receptor binding domain, SRBD) 和血管紧张素转换酶2 (angiotensin converting enzyme 2, ACE2), 构建了一种新型的双靶点表面等离子体共振(surface plasmon resonance, SPR) 传感器, 对6种中药的活性成分进行筛选鉴定, 最终从葛根中发现黄豆苷能够与SRBD和ACE2结合。通过SPR亲和力实验测定黄豆苷与ACE2结合的KD为5.18 μmol·L-1; 竞争性ELISA结合实验表明, 黄豆苷能够抑制SRBD与ACE2的结合, 20 μmol·L-1黄豆苷的抑制率为38.6%; 分子对接实验进一步证实黄豆苷在SRBD-ACE2复合物结合区域附近有最佳结合。本研究表明, 双靶点SPR筛选系统的结果准、效率高, 特别适合复杂药物体系的筛选和中药药效物质的研究, 为探究中药活性成分抗新冠病毒作用机制研究提供药效物质基础, 并为抗新冠病毒药物开发提供先导化合物来源。Abstract: The epidemic of COVID-19 has brought great challenges to the global public health prevention and control system combined with clinical diagnosis and treatment system, and it makes the development of effective antiviral drugs an important task in current pharmaceutical research. Traditional Chinese medicine (TCM) has played an important role in the prevention and control of COVID-19. Due to its numerous chemical components and various structural types, TCM becomes a natural library for searching for lead compounds against SARS-CoV-2. In this study, a novel dual-target surface plasmon resonance (SPR) biosensor was developed for S protein receptor binding domain (SRBD) and angiotensin converting enzyme 2 (ACE2) which are two key proteins in the process of SARS-CoV-2 invading cells according to characteristics of synergistic effects of multiple components and comprehensive regulation of multiple targets of TCM. The SPR biosensor was applied to screen and identify active components from six TCMs, and daidzin from Puerariae Lobatae Radix was identified to bind with SRBD and ACE2. The affinity constant (KD) of daidzin and ACE2 was 5.18 μmol·L-1 through the SPR affinity assay. Competitive ELISA assay showed that daidzin could inhibit the binding of SRBD and ACE2, and the inhibition rate of daidzin (20 μmol·L-1) was 38.6%. Molecular docking experiments further confirmed that daidzin had the best binding near the binding region of SRBD-ACE2 complex. This study shows that the dual-target SPR screening system is accurate and efficient, and is particularly suitable for screening of complex drug systems and effective substances study of TCM. It provides a material basis for exploring the mechanism of TCM active constituents against SARS-CoV-2, and provides a source of lead compounds for the development of anti-SARS-CoV-2 drugs.
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