近日,皖南医学院陈云雨副教授团队联合中国医学科学院-北京协和医学院医药生物技术研究所司书毅教授团队在Cell & Bioscience杂志发表了最新研究成果,报道了一种新型基于荧光偏振(fluorescence polarization)技术和生物素-亲和素反应(biotin-avidin system)原理的Mpro小分子抑制剂高通量筛选方法,这尚属国内外首次报道。
目前,由SARS-CoV-2感染导致的COVID-19席卷全球,已成为全球公共卫生关注的焦点。截至2021年12月2日,COVID-19确诊病例达2.6亿人,死亡病例达521万人。积极开发新型广谱抗冠状病毒药物刻不容缓。鉴于半胱氨酸蛋白酶Mpro进化保守且人体缺乏其同源蛋白酶,这使Mpro成为广谱抗冠状病毒药物开发的理想靶标之一。积极开发简便、快速、灵敏、经济的新型Mpro小分子抑制剂高通量筛选模型具有重要意义。
研究团队以荧光探针FITC-S-Biotin(FITC-AVLQSGFRKK-Biotin)作为新冠病毒Mpro水解底物,成功建立了Mpro小分子抑制剂三明治样荧光偏振高通量筛选模型。活性化合物可以抑制Mpro对FITC-S-Biotin的水解作用,故此FITC-S-Biotin可与亲和素结合,分子量增大,旋转速度变慢,在荧光偏振筛选模型中表现较高的mP值;非活性化合物由于不能抑制Mpro对FITC-S-Biotin的水解作用,将切割FITC-S-Biotin产生分子量较小的FITC-AVLQ片段,旋转速度加快,在荧光偏振筛选模型中则表现较低的mP值。
Figure: Optimization of the newly developed sandwich-like FP screening assay.
应用本筛选模型对天然产物化合物库进行高通量筛选,成功筛选到新型竞争性抑制剂二鹅掌菜酚(dieckol)具有良好的抑制活性,其IC50值为4.5±0.4 μM。表面等离子共振实验结果表明,二鹅掌菜酚与Mpro具有较高的亲和力,KD值为0.22 μM。分子对接结果表明,二鹅掌菜酚能与Mpro活性中心形成牢固的氢键,证实了其竞争性结合模式。
Figure: The inhibition mechanism of dieckol.
与常规的FRET筛选方法相比,在本荧光偏振筛选模型中,底物的单孔使用量仅为20 nM,为FRET筛选模型中底物单孔使用量的1/500,且操作简便,每轮筛选时间仅为1 h,展现了良好的稳定性、灵敏性和经济性,对其他致病性病毒蛋白酶抑制剂的高效筛选与发现也具有重要的借鉴和参考价值。
此外,由于荧光偏振检测中FITC的发射光为535 nm,更好地避免了天然产物自身荧光对筛选模型可靠性的干扰。2021年7月1日,针对本筛选方法的建立与应用,研究团队正式提交了国家发明专利申请,目前专利内容已正式公布(申请公布号:CN113699212A)。
皖南医学院2020级药理学硕士研究生闫干干和中国医学科学院-北京协和医学院医药生物技术研究所2019级博士研究生李东升为论文第一作者,中国医学科学院-北京协和医学院医药生物技术研究所张晶副教授、司书毅教授和皖南医学院陈云雨副教授为论文通讯作者。此外,中国医学科学院-北京协和医学院药物研究所天然药物活性物质与功能国家重点实验室林媛副教授在分子对接研究中给予了帮助和支持。
附文献信息:
Title: Development of a simple and miniaturized sandwich-like fluorescence polarization assay for rapid screening of SARS-CoV-2 main protease inhibitors.
DOI: https://doi.org/10.1186/s13578-021-00720-3
Abstract: Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible and has caused a pandemic named coronavirus disease 2019 (COVID-19), which has quickly spread worldwide. Although several therapeutic agents have been evaluated or approved for the treatment of COVID-19 patients, efficacious antiviral agents are still lacking. An attractive therapeutic target for SARS-CoV-2 is the main protease (Mpro), as this highly conserved enzyme plays a key role in viral polyprotein processing and genomic RNA replication. Therefore, the identification of efficacious antiviral agents against SARS-CoV-2 Mpro using a rapid, miniaturized and economical high-throughput screening (HTS) assay is of the highest importance at the present. Results In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel and step-by-step sandwich-like FP screening assay to quickly identify SARS-CoV-2 Mpro inhibitors from a natural product library. Using this screening assay, dieckol, a natural phlorotannin component extracted from a Chinese traditional medicine Ecklonia cava, was identified as a novel competitive inhibitor against SARS-CoV-2 Mpro in vitro with an IC50 value of 4.5 ± 0.4 µM. Additionally, dieckol exhibited a high affinity with SARS-CoV-2 Mpro using surface plasmon resonance (SPR) analysis and could bind to the catalytic sites of Mpro through hydrogen-bond interactions in the predicted docking model. Conclusions This innovative sandwich-like FP screening assay enables the rapid discovery of antiviral agents targeting viral proteases, and dieckol will be an excellent lead compound for generating more potent and selective antiviral agents targeting SARS-CoV-2 Mpro.
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