Identification of fangchinoline as a broad-spectrum enterovirus inhibitor through reporter virus based high-content screening

Qiu-Yan Zhang; Jia-Qi Li; Qi Li; Yang Zhang; Zhe-Rui Zhang; Xiao-Dan Li; Hong-Qing Zhang; Cheng-Lin Deng; Feng-Xia Yang; Yi Xu; Bo Zhang

doi:10.1016/j.virs.2024.02.006

April 2024

. doi: 10.1016/j.virs.2024.02.006

Citation: Qiu-Yan Zhang, Jia-Qi Li, Qi Li, Yang Zhang, Zhe-Rui Zhang, Xiao-Dan Li, Hong-Qing Zhang, Cheng-Lin Deng, Feng-Xia Yang, Yi Xu, Bo Zhang. Identification of fangchinoline as a broad-spectrum enterovirus inhibitor through reporter virus based high-content screening .VIROLOGICA SINICA, 2024, 39(2) : 301-308. http://dx.doi.org/10.1016/j.virs.2024.02.006

基于报告病毒的高内涵抗病毒药物筛选系统明确防己诺林碱可作为广谱的抗肠道病毒抑制剂

张秋艳 ^a,b ,
李嘉琪 ^b ,
李琪 ^b ,
张扬 ^c ,
张哲瑞 ^b ,
李晓丹 ^d ,
张宏庆 ^b ,
邓成林 ^b ,
杨峰霞 ^a ,
徐翼 ^a,, ,
张波 ^a,b,,

广州市妇女儿童医疗中心;中国科学院武汉病毒研究所

通讯作者： 徐翼, xuyi70@163.com; 张波, zhangbo@wh.iov.cn
收稿日期： 2023-07-05
录用日期： 2024-02-26

摘要

手足口病（HFMD）是一种常见的儿科疾病，主要由肠道病毒引起，肠道病毒是重要的人类病原体。目前，还没有可用的抗病毒药物治疗肠道病毒感染。本研究利用EV-A71-eGFP报告病毒开发了一种优良的高内涵抗病毒筛选系统。基于该系统，我们筛选了包含1042种天然化合物的药物库，以鉴定潜在的EV-A71抑制剂。防己诺林碱（FAN）是一种双苄基异喹啉类生物碱，对引起手足口病的EV-A71、CV-A10、CV-B3和CV-A16等多种肠道病毒具有抑制作用。进一步的研究表明，FAN靶向肠道病毒生命周期的早期阶段。通过进一步筛选抗FAN的EV-A71耐药病毒株，我们证明VP1的两个突变（E145G和V258I）可使病毒对FAN产生抗性，推测VP1蛋白可能是FAN的潜在靶点。我们的研究表明，FAN是一种有效的EV-A71抑制剂，具有进一步成为一种广谱抗肠道病毒药物的潜力。
- 肠道病毒
- , 高内涵抗病毒药物筛选系统
- , 防己诺林碱

Identification of fangchinoline as a broad-spectrum enterovirus inhibitor through reporter virus based high-content screening

Qiu-Yan Zhang ^a,b ,
Jia-Qi Li ^b ,
Qi Li ^b ,
Yang Zhang ^c ,
Zhe-Rui Zhang ^b ,
Xiao-Dan Li ^d ,
Hong-Qing Zhang ^b ,
Cheng-Lin Deng ^b ,
Feng-Xia Yang ^a ,
Yi Xu ^a,, ,
Bo Zhang ^a,b,,

a. The Joint Center of Translational Precision Medicine, Department of Infections and Diseases, Guangzhou Institute of Pediatrics, Guangzhou Women and Children's Medical Center, 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 Science and Technology of China, Department of Life Sciences and Medicine, Hefei, 230026, China;
d. Hunan Normal University, School of Medicine, Changsha, 410081, China

Corresponding author: Yi Xu, xuyi70@163.com Bo Zhang, zhangbo@wh.iov.cn
Received Date: 05 July 2023
Accepted Date: 26 February 2024

Abstract

Hand, foot, and mouth disease (HFMD) is a common pediatric illness mainly caused by enteroviruses, which are important human pathogens. Currently, there are no available antiviral agents for the therapy of enterovirus infection. In this study, an excellent high-content antiviral screening system utilizing the EV-A71-eGFP reporter virus was developed. Using this screening system, we screened a drug library containing 1042 natural compounds to identify potential EV-A71 inhibitors. Fangchinoline (FAN), a bis-benzylisoquinoline alkaloid, exhibits potential inhibitory effects against various enteroviruses that cause HFMD, such as EV-A71, CV-A10, CV-B3 and CV-A16. Further investigations revealed that FAN targets the early stage of the enterovirus life cycle. Through the selection of FAN-resistant EV-A71 viruses, we demonstrated that the VP1 protein could be a potential target of FAN, as two mutations in VP1 (E145G and V258I) resulted in viral resistance to FAN. Our research suggests that FAN is an efficient inhibitor of EV-A71 and has the potential to be a broad-spectrum antiviral drug against human enteroviruses.
- Hand, foot, and mouth disease (HFMD)
- , Enterovirus
- , EV-A71
- , High-content screening
- , Fangchinoline (FAN)

References
1. Arita, M., Wakita, T., Shimizu, H., 2008. Characterization of pharmacologically active compounds that inhibit poliovirus and enterovirus 71 infectivity. J Gen Virol, 89, 2518-2530.
2. Bajaber, N., Ramanathan, B., 2021. Methods in Screening Antiviral Drugs Against Enterovirus 71. Methods Mol Biol, 2296, 167-184.
3. Bello, A.M., Roshorm, Y.M., 2022. Recent progress and advances towards developing enterovirus 71 vaccines for effective protection against human hand, foot and mouth disease (HFMD). Biologicals, 79, 1-9.
4. Bian, L., Gao, F., Mao, Q., Sun, S., Wu, X., Liu, S., Yang, X., Liang, Z., 2019. Hand, foot, and mouth disease associated with coxsackievirus A10:more serious than it seems. Expert Rev Anti Infect Ther, 17, 233-242.
5. Chopra, B., Dhingra, A.K., 2021. Natural products:A lead for drug discovery and development. Phytother Res, 10.1002/ptr.7099.
6. Deng, C., Li, X., Liu, S., Xu, L., Ye, H., Qin, C.F., Zhang, B., 2015. Development and characterization of a clinical strain of Coxsackievirus A16 and an eGFP infectious clone. Virol Sin, 30, 269-276.
7. Deng, C.L., Yeo, H., Ye, H.Q., Liu, S.Q., Shang, B.D., Gong, P., Alonso, S., Shi, P.Y., Zhang, B., 2014. Inhibition of enterovirus 71 by adenosine analog NITD008. J Virol, 88, 11915-11923.
8. Dong, S., Yu, R., Wang, X., Chen, B., Si, F., Zhou, J., Xie, C., Li, Z., Zhang, D., 2022. Bis-Benzylisoquinoline Alkaloids Inhibit Porcine Epidemic Diarrhea Virus In Vitro and In Vivo. Viruses 14, 1231.
9. Elshabrawy, H.A., Fan, J., Haddad, C.S., Ratia, K., Broder, C.C., Caffrey, M., Prabhakar, B.S., 2014. Identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and Ebola, Hendra, and Nipah viruses by using a novel high-throughput screening assay. J Virol, 88, 4353-4365.
10. Fan, H., He, S.T., Han, P., Hong, B., Liu, K., Li, M., Wang, S., Tong, Y., 2022. Cepharanthine:A Promising Old Drug against SARS-CoV-2. Adv Biol (Weinh), 6, e2200148.
11. Guo, B., Su, J., Zhang, T., Wang, K., Li, X., 2015. Fangchinoline as a kinase inhibitor targets FAK and suppresses FAK-mediated signaling pathway in A549. J Drug Target, 23, 266-274.
12. Huang, Y., Zhou, Y., Lu, H., Yang, H., Feng, Q., Dai, Y., Chen, L., Yu, S., Yao, X., Zhang, H., Jiang, M., Wang, Y., Han, N., Hu, G., He, Y., 2015. Characterization of severe hand, foot, and mouth disease in Shenzhen, China, 2009-2013. J Med Virol. 87, 1471-1479.
13. Kataoka, C., Suzuki, T., Kotani, O., Iwata-Yoshikawa, N., Nagata, N., Ami, Y., Wakita, T., Nishimura, Y., Shimizu, H., 2015. The Role of VP1 Amino Acid Residue 145 of Enterovirus 71 in Viral Fitness and Pathogenesis in a Cynomolgus Monkey Model. PLoS Pathog, 11, e1005033.
14. Kim, D.E., Min, J.S., Jang, M.S., Lee, J.Y., Shin, Y.S., Song, J.H., Kim, H.R., Kim, S., Jin, Y.H., Kwon, S., 2019. Natural Bis-Benzylisoquinoline Alkaloids-Tetrandrine, Fangchinoline, and Cepharanthine, Inhibit Human Coronavirus OC43 Infection of MRC-5 Human Lung Cells. Biomolecules 9, 696.
15. Kinobe, R., Wiyatno, A., Artika, I.M., Safari, D., 2022. Insight into the Enterovirus A71:A review. Rev Med Virol, 32, e2361.
16. Kobayashi, K., Sudaka, Y., Takashino, A., Imura, A., Fujii, K., Koike, S., 2018. Amino Acid Variation at VP1-145 of Enterovirus 71 Determines Attachment Receptor Usage and Neurovirulence in Human Scavenger Receptor B2 Transgenic Mice. J Virol. 92, e00681-18.
17. Li, J.Q., Deng, C.L., Gu, D., Li, X., Shi, L., He, J., Zhang, Q.Y., Zhang, B., Ye, H.Q., 2018. Development of a replicon cell line-based high throughput antiviral assay for screening inhibitors of Zika virus. Antiviral Res, 150, 148-154.
18. Li, Q., Maddox, C., Rasmussen, L., Hobrath, J.V., White, L.E., 2009. Assay development and high-throughput antiviral drug screening against Bluetongue virus. Antiviral Res, 83, 267-273.
19. Liu, T., Liu, X., Li, W., 2016. Tetrandrine, a Chinese plant-derived alkaloid, is a potential candidate for cancer chemotherapy. Oncotarget, 7, 40800-40815.
20. Lu, W.W., Kung, F.Y., Deng, P.A., Lin, Y.C., Lin, C.W., Kung, S.H., 2017. Development of a fluorescence resonance energy transfer-based intracellular assay to identify novel enterovirus 71 antivirals. Arch Virol, 162, 713-720.
21. Martins Lima, A., Bragina, M.E., Burri, O., Bortoli Chapalay, J., Costa-Fraga, F.P., Chambon, M., Fraga-Silva, R.A., Stergiopulos, N., 2019. An optimized and validated 384-well plate assay to test platelet function in a high-throughput screening format. Platelets, 30, 563-571.
22. Newman, D.J., Cragg, G.M., 2016. Natural Products as Sources of New Drugs from 1981 to 2014. J Nat Prod, 79, 629-661.
23. Nishimura, Y., Lee, H., Hafenstein, S., Kataoka, C., Wakita, T., Bergelson, J.M., Shimizu, H., 2013. Enterovirus 71 binding to PSGL-1 on leukocytes:VP1-145 acts as a molecular switch to control receptor interaction. PLoS Pathog, 9, e1003511.
24. Saguil, A., Kane, S.F., Lauters, R., Mercado, M.G., 2019. Hand-Foot-and-Mouth Disease:Rapid Evidence Review. Am Fam Physician, 100, 408-414.
25. Sakurai, Y., Kolokoltsov, A.A., Chen, C.C., Tidwell, M.W., Bauta, W.E., Klugbauer, N., Grimm, C., Wahl-Schott, C., Biel, M., Davey, R.A., 2015. Ebola virus. Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment. Science, 347, 995-998.
26. Shan, L., Tong, L., Hang, L., Fan, H., 2019. Fangchinoline supplementation attenuates inflammatory markers in experimental rheumatoid arthritis-induced rats. Biomed Pharmacother, 111, 142-150.
27. Shang, B., Deng, C., Ye, H., Xu, W., Yuan, Z., Shi, P.Y., Zhang, B., 2013. Development and characterization of a stable eGFP enterovirus 71 for antiviral screening. Antiviral Research, 97, 198-205.
28. Sharma, V., Goessling, L.S., Brar, A.K., Eghtesady, P., 2023. Multiple subtypes of coxsackievirus group B can cause congenital heart disease. Birth Defects Res, 115, 171-178.
29. Ulferts, R., De Boer, S.M., Van Der Linden, L., Bauer, L., Lyoo, H.R., Mate, M.J., Lichiere, J., Canard, B., Lelieveld, D., Omta, W., Egan, D., Coutard, B., Van Kuppeveld, F.J., 2016. Screening of a Library of FDA-Approved Drugs Identifies Several Enterovirus Replication Inhibitors That Target Viral Protein 2C. Antimicrob Agents Chemother, 60, 2627-2638.
30. Wan, Z., Lu, Y., Liao, Q., Wu, Y., Chen, X., 2012. Fangchinoline inhibits human immunodeficiency virus type 1 replication by interfering with gp160 proteolytic processing. PLoS One, 7, e39225.
31. Wang, C.D., Huang, J.G., Gao, X., Li, Y., Zhou, S.Y., Yan, X., Zou, A., Chang, J.L., Wang, Y.S., Yang, G.X., He, G.Y., 2010. Fangchinoline induced G1/S arrest by modulating expression of p27, PCNA, and cyclin D in human prostate carcinoma cancer PC3 cells and tumor xenograft. Biosci Biotechnol Biochem, 74, 488-493.
32. Wang, J., Liu, J., Fang, F., Wu, J., Ji, T., Yang, Y., Liu, L., Li, C., Zhang, W., Zhang, X.,Teng, Z., 2022. Genomic surveillance of coxsackievirus A10 reveals genetic features and recent appearance of genogroup D in Shanghai, China, 2016-2020. Virol Sin, 37, 177-186.
33. Xi, J., Ma, C., Wei, Z., Yin, B., Zhao, S., Quan, W., Yang, J., Yuan, J., Qiang, B., Ye, F., Peng, X., 2021. A single mutation in the cis-acting replication element identified within the EV-A71 2C-coding region causes defects in virus production in cell culture. Emerg Microbes Infect. 10, 1988-1999.
34. Xing, Z., Zhang, Y., Zhang, X., Yang, Y., Ma, Y., Pang, D., 2013. Fangchinoline induces G1 arrest in breast cancer cells through cell-cycle regulation. Phytother Res, 27, 1790-1794.
35. Yin, Z., Chen, Y.L., Schul, W., Wang, Q.Y., Gu, F., Duraiswamy, J., Kondreddi, R.R., Niyomrattanakit, P., Lakshminarayana, S.B., Goh, A., Xu, H.Y., Liu, W., Liu, B., Lim, J.Y., Ng, C.Y., Qing, M., Lim, C.C., Yip, A., Wang, G., Chan, W.L., Tan, H.P., Lin, K., Zhang, B., Zou, G., Bernard, K.A., Garrett, C., Beltz, K., Dong, M., Weaver, M., He, H., Pichota, A., Dartois, V., Keller, T.H.,Shi, P.Y., 2009. An adenosine nucleoside inhibitor of dengue virus. Proc Natl Acad Sci U S A, 106, 20435-20439.
36. Zhang, M., Wang, H., Tang, J., He, Y., Xiong, T., Li, W., Qu, Y., Mu, D., 2021. Clinical characteristics of severe neonatal enterovirus infection:a systematic review. BMC Pediatr, 21, 127.
37. Zhang, Q., MacDonald, N. E., Smith, J. C., Cai, K., Yu, H., Li, H., Lei, C., 2014. Severe enterovirus type 71 nervous system infections in children in the Shanghai region of China:clinical manifestations and implications for prevention and care. Pediatr Infect Dis J. 33, 482-487.
38. 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.
39. Zhang, X., Zhang, Y., Li, H., Liu, L., 2022. Hand-Foot-and-Mouth Disease-Associated Enterovirus and the Development of Multivalent HFMD Vaccines. Int J Mol Sci. 24, 169.
40. Zhang, Y., Ke, X., Zheng, C., Liu, Y., Xie, L., Zheng, Z., Wang, H., 2017. Development of a luciferase-based biosensor to assess enterovirus 71 3C protease activity in living cells. Sci Rep, 7, 10385.
41. Zhang, Z., Yang, E., Hu, C., Cheng, H., Chen, C.Y., Huang, D., Wang, R., Zhao, Y., Rong, L., Vignuzzi, M., Shen, H., Shen, L., Chen, Z.W., 2017. Cell-Based High-Throughput Screening Assay Identifies 2',2'-Difluoro-2'-deoxycytidine Gemcitabine as a Potential Antipoliovirus Agent. ACS Infect Dis, 3, 45-53.
42. 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.
Proportional views
Electronic Supplementary Material

10.1016j.virs.2024.02.006-ESM.docx