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. doi: 10.1016/j.virs.2022.04.002
Citation: Yuan Zhang, Chunjie Li, Xianliang Ke, Dan Luo, Yan Liu, Quanjiao Chen, Hanzhong Wang, Xiaohui Song, Zhenhua Zheng. Development of a biosensor assessing SARS-CoV-2 main protease proteolytic activity in living cells for antiviral drugs screening [J].VIROLOGICA SINICA, 2022, 37(3) : 459-461.  http://dx.doi.org/10.1016/j.virs.2022.04.002

用于抗病毒药物筛选的SARS-CoV-2主要蛋白酶蛋白水解活性生物传感器的研制

  • SARS-CoV-2引起的新型冠状病毒疫情在世界范围内迅速爆发,病毒的M蛋白酶(也称作3CL蛋白酶)在病毒的复制及致病过程中至关重要,因此,被认为是一种极具吸引力的SARS-CoV-2抗病毒药物研发靶点。但至今为止,还没有被批准的M蛋白酶抑制剂用于新冠病毒的治疗。本研究建立了一种灵敏的、基因编码的M蛋白酶活性检测传感器。此传感器由白介素-1β前体-冠状病毒M蛋白酶切割位点以及Gaussia荧光素酶(GLuc)三部分组成,我们将其命名为i-MS-GLuc。在传感器中,由于白介素-1β前体的易聚集性,GLuc的催化活性处于被抑制状态,而新冠病毒的M蛋白酶可以识别传感器中的切割位点,并对传感器进行切割,从而释放及激活GLuc,使其荧光素酶活性大幅度上升。这种传感器灵敏度高、操作简单、反应在活细胞内进行。这些特性使其成为活细胞内检测病毒蛋白酶活性的有利工具,并可以以此建立抗病毒药物的高通量筛选平台。

Development of a biosensor assessing SARS-CoV-2 main protease proteolytic activity in living cells for antiviral drugs screening

  • Highlights:
    The biosensor reported in our study can monitor SARS-CoV-2 Mpro activity in living cells instead of in vitro solutions.
    The biosensor reported in our study is sensitive and easy to operate.
    It is suitable for high-throughput screening.
    It has the potential to be used in small animal models.

  • 加载中
    1. Bartok, E., Bauernfeind, F., Khaminets, M.G., Jakobs, C., Monks, B., Fitzgerald, K.A., Latz, E., Hornung, V., 2013. iGLuc:a luciferase-based inflammasome and protease activity reporter. Nat. Methods 10, 147-154.

    2. Bowlby, M.R., Case, J.F., 1991. Flash kinetics and spatial patterns of bioluminescence in the copepod gaussia-princeps. Mar. Biol. 110, 329-336.

    3. Froggatt, H.M., Heaton, B.E., Heaton, N.S., 2020. Development of a fluorescence-based, high-throughput SARS-CoV-2 3CL(pro) reporter assay. J. Virol. 94, e01265, 20.

    4. Jin, Z., Du, X., Xu, Y., Deng, Y., Liu, M., Zhao, Y., Zhang, B., Li, X., Zhang, L., Peng, C., Duan, Y., Yu, J., Wang, L., Yang, K., Liu, F., Jiang, R., Yang, X., You, T., Liu, X., Yang, X., Bai, F., Liu, H., Liu, X., Guddat, L.W., Xu, W., Xiao, G., Qin, C., Shi, Z., Jiang, H., Rao, Z., Yang, H., 2020. Structure of M(pro) from SARS-CoV-2 and discovery of its inhibitors. Nature 582, 289-293.

    5. Pillaiyar, T., Manickam, M., Namasivayam, V., Hayashi, Y., Jung, S.H., 2016. An overview of severe acute respiratory syndrome-coronavirus (SARS-CoV) 3CL protease inhibitors:peptidomimetics and small molecule chemotherapy. J. Med. Chem. 59, 6595-6628.

    6. Rawson, J.M.O., Duchon, A., Nikolaitchik, O.A., Pathak, V.K., Hu, W.S., 2021. Development of a cell-based luciferase complementation assay for identification of SARS-CoV-2 3CL(pro) inhibitors. Viruses 13, 173.

    7. Wu, F., Zhao, S., Yu, B., Chen, Y.M., Wang, W., Song, Z.G., Hu, Y., Tao, Z.W., Tian, J.H., Pei, Y.Y., Yuan, M.L., Zhang, Y.L., Dai, F.H., Liu, Y., Wang, Q.M., Zheng, J.J., Xu, L., Holmes, E.C., Zhang, Y.Z., 2020. A new coronavirus associated with human respiratory disease in China. Nature 579, 265-269.

    8. Zhou, P., Yang, X.L., Wang, X.G., Hu, B., Zhang, L., Zhang, W., Si, H.R., Zhu, Y., Li, B., Huang, C.L., Chen, H.D., Chen, J., Luo, Y., Guo, H., Jiang, R.D., Liu, M.Q., Chen, Y., Shen, X.R., Wang, X., Zheng, X.S., Zhao, K., Chen, Q.J., Deng, F., Liu, L.L., Yan, B., Zhan, F.X., Wang, Y.Y., Xiao, G.F., Shi, Z.L., 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273.

    9. Zhu, W., Xu, M., Chen, C.Z., Guo, H., Shen, M., Hu, X., Shinn, P., Klumpp-Thomas, C., Michael, S.G., Zheng, W., 2020. Identification of SARS-CoV-2 3CL protease inhibitors by a quantitative high-throughput screening. ACS Pharmacol Transl Sci 3, 1008-1016.

  • 加载中
  • 10.1016j.virs.2022.04.002-ESM.docx

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    Development of a biosensor assessing SARS-CoV-2 main protease proteolytic activity in living cells for antiviral drugs screening

      Corresponding author: Xiaohui Song, songxiaohui@zgwhfe.com
      Corresponding author: Zhenhua Zheng, zhengzh@wh.iov.cn
    • a Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China;
    • b Department of Gastroenterology, Wuhan Children's Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, 430015, China;
    • c Department of Obstetrics, Wuhan Children's Hospital, Wuhan Maternal and Child Healthcare Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, 430015, China;
    • d University of the Chinese Academy of Sciences, Beijing, 100049, China

    Abstract: Highlights:
    The biosensor reported in our study can monitor SARS-CoV-2 Mpro activity in living cells instead of in vitro solutions.
    The biosensor reported in our study is sensitive and easy to operate.
    It is suitable for high-throughput screening.
    It has the potential to be used in small animal models.

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