Oridonin inhibits SARS-CoV-2 replication by targeting viral proteinase and polymerase

Zherui Zhang; Hongqing Zhang; Yanan Zhang; Qiuyan Zhang; Qiaojie Liu; Yanyan Hu; Xiaoling Chen; Jing Wang; Yujia Shi; Chenglin Deng; Peng Gong; Bo Zhang; Xiaodan Li; Bing Zhu; Hanqing Ye

doi:10.1016/j.virs.2023.04.008

June 2023

. doi: 10.1016/j.virs.2023.04.008

Citation: Zherui Zhang, Hongqing Zhang, Yanan Zhang, Qiuyan Zhang, Qiaojie Liu, Yanyan Hu, Xiaoling Chen, Jing Wang, Yujia Shi, Chenglin Deng, Peng Gong, Bo Zhang, Xiaodan Li, Bing Zhu, Hanqing Ye. Oridonin inhibits SARS-CoV-2 replication by targeting viral proteinase and polymerase .VIROLOGICA SINICA, 2023, 38(3) : 470-479. http://dx.doi.org/10.1016/j.virs.2023.04.008

冬凌草甲素通过靶向新型冠状病毒蛋白酶和聚合酶抑制病毒复制

张哲瑞 ^a,b ,
张宏庆 ^b,c ,
张亚南 ^a,b ,
张秋艳 ^b ,
刘巧洁 ^b ,
胡艳艳 ^b,c ,
陈晓玲 ^b,c ,
王静 ^b,c ,
石宇佳 ^d ,
邓成林 ^b ,
龚鹏 ^b,c ,
张波 ^a,b,c,, ,
李晓丹 ^d,, ,
朱冰 ^a,, ,
叶寒青 ^b,,

a. 广州医科大学附属广州市妇女儿童医疗中心, 儿科研究所, 病毒实验室;
b. 中国科学院生物安全大科学研究中心, 武汉病毒学研究所, 特殊病原体与生物安全实验室;
c. 中国科学院大学;
d. 湖南师范大学, 医学院

通讯作者： 张波, zhangbo@wh.iov.cn; 李晓丹, lxd@live.cn; 朱冰, zhubing@gzhmu.edu.cn; 叶寒青, yehq@wh.iov.cn
收稿日期： 2022-12-18
录用日期： 2023-04-27

摘要

新型冠状病毒肺炎（COVID-19）自2019年12月在我国暴发以来，已成为全球公共卫生危机。目前临床上能够有效治疗该疾病的药物较少。新型冠状病毒（SARS-CoV-2）的主蛋白酶（M^pro）、木瓜样蛋白酶（PLpro）和聚合酶（RdRp）是参与病毒复制的主要病毒蛋白，常作为抗冠状病毒药物开发的靶点。在本研究中，我们在细胞水平测试了冬凌草甲素对SARS-CoV-2的抗病毒活性。时相加药和复制子实验证明冬凌草甲素能够有效抑制病毒进入细胞后基因组的复制。抗病毒机制研究表明冬凌草甲素对病毒复制的抑制依赖于α，β-不饱和羰基的氧化活性。酶活实验发现冬凌草甲素能够抑制SARS-CoV-2 M^pro的活性，而且在高浓度下对PLpro介导的去泛素化和RdRp催化的RNA延伸活性也有一定的抑制。此外冬凌草甲素还展现出对蝙蝠SARS-like CoV和SARS-CoV-2 omicron变异株（BA.1和BA.2）的抑制活性，这突出了其作为泛冠状病毒抗病毒药物的潜力。
- 新型冠状病毒
- , 冬凌草甲素
- , 抗病毒
- , 蛋白酶抑制剂

Oridonin inhibits SARS-CoV-2 replication by targeting viral proteinase and polymerase

Zherui Zhang ^a,b ,
Hongqing Zhang ^b,c ,
Yanan Zhang ^a,b ,
Qiuyan Zhang ^b ,
Qiaojie Liu ^b ,
Yanyan Hu ^b,c ,
Xiaoling Chen ^b,c ,
Jing Wang ^b,c ,
Yujia Shi ^d ,
Chenglin Deng ^b ,
Peng Gong ^b,c ,
Bo Zhang ^a,b,c,, ,
Xiaodan Li ^d,, ,
Bing Zhu ^a,, ,
Hanqing Ye ^b,,

a. Virus Laboratory, Institute of Pediatrics, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, 510623, China;
b. Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, 430071, China;
c. University of Chinese Academy of Sciences, Beijing, 100049, China;
d. Hunan Normal University, School of Medicine, Changsha, 410081, China

Corresponding author: Bo Zhang, zhangbo@wh.iov.cn Xiaodan Li, lxd@live.cn Bing Zhu, zhubing@gzhmu.edu.cn Hanqing Ye, yehq@wh.iov.cn
Received Date: 18 December 2022
Accepted Date: 27 April 2023

Abstract

COVID-19 has become a global public health crisis since its outbreak in China in December 2019. Currently there are few clinically effective drugs to combat SARS-CoV-2 infection. The main protein (M^pro), papain-like protease (PL^pro) and RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 are involved in the viral replication, and might be prospective targets for anti-coronavirus drug development. Here, we investigated the antiviral activity of oridonin, a natural small-molecule compound, against SARS-CoV-2 infection in vitro. The time-of-addition analysis showed that oridonin efficiently inhibited SARS-CoV-2 infection by interfering with the genome replication at the post-entry stage. Mechanistically, the inhibition of viral replication by oridonin depends on the oxidation activity of α, β-unsaturated carbonyl. Further experiments showed that oridonin not only effectively inhibited SARS-CoV-2 M^pro activity, but also had some inhibitory effects on PL^pro-mediated deubiquitinating and viral polymerase-catalyzed RNA elongation activities at high concentrations. In particular, oridonin could inhibit the bat SARS-like CoV and the newly emerged SARS-CoV-2 omicron variants (BA.1 and BA.2), which highlights its potential as a pan-coronavirus antiviral agent. Overall, our data provide strong evidence that oridonin is an efficient antiviral agent against SARS-CoV-2 infection.
- SARS-CoV-2
- , Oridonin
- , Antiviral
- , Protease inhibitor

References
1. Amporndanai, K., Meng, X., Shang, W., Jin, Z., Rogers, M., Zhao, Y., Rao, Z., Liu, Z.J., Yang, H., Zhang, L., O'neill, P.M., Samar Hasnain, S., 2021. Inhibition mechanism of SARS-CoV-2 main protease by ebselen and its derivatives. Nat Commun. 12, 3061.
2. Chopra, B., Dhingra, A.K., 2021. Natural products:A lead for drug discovery and development. Phytother Res. 35, 4660-4702.
3. Cuadrado, A., Pajares, M., Benito, C., Jimenez-Villegas, J., Escoll, M., Fernandez-Gines, R., Garcia Yague, A.J., Lastra, D., Manda, G., Rojo, A.I., Dinkova-Kostova, A.T., 2020. Can Activation of NRF2 Be a Strategy against COVID-19? Trends Pharmacol Sci. 41, 598-610.
4. Dai, W., Zhang, B., Jiang, X.M., Su, H., Li, J., Zhao, Y., Xie, X., Jin, Z., Peng, J., Liu, F., Li, C., Li, Y., Bai, F., Wang, H., Cheng, X., Cen, X., Hu, S., Yang, X., Wang, J., Liu, X., Xiao, G., Jiang, H., Rao, Z., Zhang, L.K., Xu, Y., Yang, H., Liu, H., 2020. Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease. Science 368, 1331-1335.
5. Freitas, B.T., Durie, I.A., Murray, J., Longo, J.E., Miller, H.C., Crich, D., Hogan, R.J., Tripp, R.A., Pegan, S.D., 2020. Characterization and Noncovalent Inhibition of the Deubiquitinase and deISGylase Activity of SARS-CoV-2 Papain-Like Protease. ACS Infect Dis, 6, 2099-2109.
6. Fu, L., Ye, F., Feng, Y., Yu, F., Wang, Q., Wu, Y., Zhao, C., Sun, H., Huang, B., Niu, P., Song, H., Shi, Y., Li, X., Tan, W., Qi, J., Gao, G.F., 2020. Both Boceprevir and GC376 efficaciously inhibit SARS-CoV-2 by targeting its main protease. Nat Commun. 11, 4417.
7. Fujita, E., Nagao, Y., Kaneko, K., Nakazawa, S., Kuroda, H., 1976. The antitumor and antibacterial activity of the Isodon diterpenoids. Chem Pharm Bull. (Tokyo) 24, 2118-2127.
8. Gyebi, G.A., Ogunro, O.B., Adegunloye, A.P., Ogunyemi, O.M., Afolabi, S.O., 2021. Potential inhibitors of coronavirus 3-chymotrypsin-like protease (3CL(pro)):an in silico screening of alkaloids and terpenoids from African medicinal plants. J Biomol Struct Dyn. 39, 3396-3408.
9. He, H., Jiang, H., Chen, Y., Ye, J., Wang, A., Wang, C., Liu, Q., Liang, G., Deng, X., Jiang, W., Zhou, R., 2018. Oridonin is a covalent NLRP3 inhibitor with strong anti-inflammasome activity. Nat Commun. 9, 2550.
10. Huang, H., Weng, H., Dong, B., Zhao, P., Zhou, H., Qu, L., 2017. Oridonin Triggers Chaperon-mediated Proteasomal Degradation of BCR-ABL in Leukemia. Sci Rep. 7, 41525.
11. 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.
12. Kumar, S.B., Krishna, S., Pradeep, S., Mathews, D.E., Pattabiraman, R., Murahari, M., Murthy, T.P.K., 2021. Screening of natural compounds from Cyperus rotundus Linn against SARS-CoV-2 main protease (M(pro)):An integrated computational approach. Comput Biol Med. 134, 104524.
13. Littler, D.R., Liu, M., Mcauley, J.L., Lowery, S.A., Illing, P.T., Gully, B.S., Purcell, A.W., Chandrashekaran, I.R., Perlman, S., Purcell, D.F.J., Quinn, R.J., Rossjohn, J., 2021. A natural product compound inhibits coronaviral replication in vitro by binding to the conserved Nsp9 SARS-CoV-2 protein. J Biol Chem 297. 101362.
14. Ma, C., Hu, Y., Townsend, J.A., Lagarias, P.I., Marty, M.T., Kolocouris, A.,Wang, J., 2020a. Ebselen, Disulfiram, Carmofur, PX-12, Tideglusib, and Shikonin Are Nonspecific Promiscuous SARS-CoV-2 Main Protease Inhibitors. ACS Pharmacol Transl Sci. 3, 1265-1277.
15. Ma, C., Sacco, M.D., Hurst, B., Townsend, J.A., Hu, Y., Szeto, T., Zhang, X., Tarbet, B., Marty, M.T., Chen, Y.,Wang, J., 2020b. Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease. Cell Res. 30, 678-692.
16. Mandal, A., Jha, A.K., Hazra, B., 2021. Plant Products as Inhibitors of Coronavirus 3CL Protease. Front Pharmacol. 12, 583387.
17. Mccarthy, M.W., 2023. VV116 as a potential treatment for COVID-19. Expert Opin Pharmacother. 24, 675-678.
18. Newman, D.J., Cragg, G.M., 2016. Natural Products as Sources of New Drugs from 1981 to 2014. J Nat Prod. 79, 629-661.
19. Olagnier, D., Farahani, E., Thyrsted, J., Blay-Cadanet, J., Herengt, A., Idorn, M., Hait, A., Hernaez, B., Knudsen, A., Iversen, M.B., Schilling, M., Jorgensen, S.E., Thomsen, M., Reinert, L.S., Lappe, M., Hoang, H.D., Gilchrist, V.H., Hansen, A.L., Ottosen, R., Nielsen, C.G., Moller, C., Van Der Horst, D., Peri, S., Balachandran, S., Huang, J., Jakobsen, M., Svenningsen, E.B., Poulsen, T.B., Bartsch, L., Thielke, A.L., Luo, Y., Alain, T., Rehwinkel, J., Alcami, A., Hiscott, J., Mogensen, T.H., Paludan, S.R., Holm, C.K., 2020. SARS-CoV2-mediated suppression of NRF2-signaling reveals potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate. Nat Commun. 11, 4938.
20. Owen, D.R., Allerton, C.M.N., Anderson, A.S., Aschenbrenner, L., Avery, M., Berritt, S., Boras, B., Cardin, R.D., Carlo, A., Coffman, K.J., Dantonio, A., Di, L., Eng, H., Ferre, R., Gajiwala, K.S., Gibson, S.A., Greasley, S.E., Hurst, B.L., Kadar, E.P., Kalgutkar, A.S., Lee, J.C., Lee, J., Liu, W., Mason, S.W., Noell, S., Novak, J.J., Obach, R.S., Ogilvie, K., Patel, N.C., Pettersson, M., Rai, D.K., Reese, M.R., Sammons, M.F., Sathish, J.G., Singh, R.S.P., Steppan, C.M., Stewart, A.E., Tuttle, J.B., Updyke, L., Verhoest, P.R., Wei, L., Yang, Q., Zhu, Y., 2021. An oral SARS-CoV-2 M(pro) inhibitor clinical candidate for the treatment of COVID-19. Science 374, 1586-1593.
21. Ratajczak, M.Z., Kucia, M., 2020. SARS-CoV-2 infection and overactivation of Nlrp3 inflammasome as a trigger of cytokine "storm" and risk factor for damage of hematopoietic stem cells. Leukemia 34, 1726-1729.
22. Sacco, M.D., Ma, C., Lagarias, P., Gao, A., Townsend, J.A., Meng, X., Dube, P., Zhang, X., Hu, Y., Kitamura, N., Hurst, B., Tarbet, B., Marty, M.T., Kolocouris, A., Xiang, Y., Chen, Y., Wang, J., 2020. Structure and inhibition of the SARS-CoV-2 main protease reveal strategy for developing dual inhibitors against M(pro) and cathepsin L. Sci Adv. 6, eabe0751.
23. Saravolatz, L.D., Depcinski, S., Sharma, M., 2023. Molnupiravir and Nirmatrelvir-Ritonavir:Oral Coronavirus Disease 2019 Antiviral Drugs. Clin Infect Dis. 76, 165-171.
24. Shah, A., 2020. Novel Coronavirus-Induced NLRP3 Inflammasome Activation:A Potential Drug Target in the Treatment of COVID-19. Front Immunol. 11, 1021.
25. Shang, W., Dai, W., Yao, C., Xu, L., Tao, X., Su, H., Li, J., Xie, X., Xu, Y., Hu, M., Xie, D., Jiang, H., Zhang, L., Liu, H., 2022. In vitro and in vivo evaluation of the main protease inhibitor FB2001 against SARS-CoV-2. Antiviral Res. 208, 105450.
26. Sun, Q., Ye, F., Liang, H., Liu, H., Li, C., Lu, R., Huang, B., Zhao, L., Tan, W., Lai, L., 2021. Bardoxolone and bardoxolone methyl, two Nrf2 activators in clinical trials, inhibit SARS-CoV-2 replication and its 3C-like protease. Signal Transduct Target Ther. 6, 212.
27. Unoh, Y., Uehara, S., Nakahara, K., Nobori, H., Yamatsu, Y., Yamamoto, S., Maruyama, Y., Taoda, Y., Kasamatsu, K., Suto, T., Kouki, K., Nakahashi, A., Kawashima, S., Sanaki, T., Toba, S., Uemura, K., Mizutare, T., Ando, S., Sasaki, M., Orba, Y., Sawa, H., Sato, A., Sato, T., Kato, T., Tachibana, Y., 2022. Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19. J Med Chem. 65, 6499-6512.
28. Vuong, W., Khan, M.B., Fischer, C., Arutyunova, E., Lamer, T., Shields, J., Saffran, H.A., Mckay, R.T., Van Belkum, M.J., Joyce, M.A., Young, H.S., Tyrrell, D.L., Vederas, J.C., Lemieux, M.J., 2020. Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication. Nat Commun. 11, 4282.
29. Wang, M., Cao, R., Zhang, L., Yang, X., Liu, J., Xu, M., Shi, Z., Hu, Z., Zhong, W., Xiao, G., 2020. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 30, 269-271.
30. Wang, Q., Wu, J., Wang, H., Gao, Y., Liu, Q., Mu, A., Ji, W., Yan, L., Zhu, Y., Zhu, C., Fang, X., Yang, X., Huang, Y., Gao, H., Liu, F., Ge, J., Sun, Q., Yang, X., Xu, W., Liu, Z., Yang, H., Lou, Z., Jiang, B., Guddat, L.W., Gong, P.,Rao, Z., 2020. Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase. Cell 182, 417-428 e413.
31. 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.
32. Yang, H., Huang, J., Gao, Y., Wen, Z., Peng, L., Ci, X., 2020. Oridonin attenuates carrageenan-induced pleurisy via activation of the KEAP-1/Nrf2 pathway and inhibition of the TXNIP/NLRP3 and NF-kappaB pathway in mice. Inflammopharmacology 28, 513-523.
33. Yang, H., Lv, H., Li, H., Ci, X., Peng, L., 2019. Oridonin protects LPS-induced acute lung injury by modulating Nrf2-mediated oxidative stress and Nrf2-independent NLRP3 and NF-kappaB pathways. Cell Commun Signal. 17, 62.
34. Ye, Y.C., Wang, H.J., Xu, L., Liu, W.W., Liu, B.B., Tashiro, S., Onodera, S., Ikejima, T., 2012. Oridonin induces apoptosis and autophagy in murine fibrosarcoma L929 cells partly via NO-ERK-p53 positive-feedback loop signaling pathway. Acta Pharmacol Sin. 33, 1055-1061.
35. Zhang, J.L., Li, Y.H., Wang, L.L., Liu, H.Q., Lu, S.Y., Liu, Y., Li, K., Liu, B., Li, S.Y., Shao, F.M., Wang, K., Sheng, N., Li, R., Cui, J.J., Sun, P.C., Ma, C.X., Zhu, B., Wang, Z., Wan, Y.H., Yu, S.S., Che, Y., Wang, C.Y., Wang, C., Zhang, Q., Zhao, L.M., Peng, X.Z., Cheng, Z., Chang, J.B., Jiang, J.D., 2021. Azvudine is a thymus-homing anti-SARS-CoV-2 drug effective in treating COVID-19 patients. Signal Transduct Target Ther. 6, 414.
36. Zhang, Q.Y., Deng, C.L., Liu, J., Li, J.Q., Zhang, H.Q., Li, N., Zhang, Y.N., Li, X.D., Zhang, B., Xu, Y., Ye, H.Q., 2021. SARS-CoV-2 replicon for high-throughput antiviral screening. J Gen Virol. 102, 001583.
37. Zhang, Y., Li, C., Ke, X., Luo, D., Liu, Y., Chen, Q., Wang, H., Song, X., Zheng, Z., 2022. Development of a biosensor assessing SARS-CoV-2 main protease proteolytic activity in living cells for antiviral drugs screening. Virol Sin. 37, 459-461.
38. Zhang, Z.R., Zhang, Y.N., Li, X.D., Zhang, H.Q., Xiao, S.Q., Deng, F., Yuan, Z.M., Ye, H.Q., Zhang, B., 2020. A cell-based large-scale screening of natural compounds for inhibitors of SARS-CoV-2. Signal Transduct Target Ther. 5, 218.
39. Zhang, Z.R., Zhang, Y.N., Zhang, H.Q., Zhang, Q.Y., Li, N., Li, Q., Deng, C.L., Zhang, B., Li, X.D., Ye, H.Q., 2022. Berbamine hydrochloride potently inhibits SARS-CoV-2 infection by blocking S protein-mediated membrane fusion. PLoS Negl Trop Dis. 16, e0010363.
40. Zhong, B., Peng, W., Du, S., Chen, B., Feng, Y., Hu, X., Lai, Q., Liu, S., Zhou, Z.W., Fang, P., Wu, Y., Gao, F., Zhou, H.,Sun, L., 2022. Oridonin Inhibits SARS-CoV-2 by Targeting Its 3C-Like Protease. Small Sci, 2, 2100124.
41. 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.
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