Fangchinoline demonstrates efficacy against TGEV through pAPN binding, as determined by preliminary toxicity evaluation

Abstract

Through structure-based virtual screening, this study identified the botanical compound fangchinoline (FAN) as an inhibitor of the porcine aminopeptidase N (pAPN) receptor and evaluated its anti-transmissible gastroenteritis virus (TGEV) activity, mechanism, and safety. FAN exhibited potent antiviral efficacy against TGEV in PK-15 and IPEC-J2 cells (EC₅₀ = 2.862 μM and 3.670 μM, respectively), significantly reducing viral RNA copies and N protein expression while suppressing the upregulation of the inflammatory cytokines IL-6 and TNF-α. It also showed broad-spectrum potential by inhibiting PEDV (EC₅₀ = 2.243 μM). Mechanistic studies using a cellular thermal shift assay (CETSA) and drug affinity-responsive target stability (DARTS) confirmed that FAN can bind to pAPN, affecting its thermal and enzyme stability. Time-of-addition assays indicated that FAN interferes with both the early (attachment and internalization) and late replication phases of TGEV. In acute toxicity tests, FAN showed low toxicity (LD₅₀ in mice: 2060.23 mg/kg for males, 1481.93 mg/kg for females) and no mutagenic potential in the Ames test. Favorable pharmacokinetic parameters were also observed in rats. In conclusion, FAN exerts its anti-TGEV effect by targeting the critical pAPN receptor. Its plant origin, potent and broad-spectrum antiviral activity, elucidated mechanism, and promising safety profile support its further development as a lead compound for anti-coronavirus strategies.

Keywords

Fangchinoline / APN / TGEV / PEDV / Acute toxicity test / Ames test

References

[1] Antas M, Olech M. First report of transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) in pigs from Poland. BMC Veterinary Research, 2024, 20(1): Article ID: 517

[2] Chan EWC, Wong SK, Chan HT. An overview on the chemistry, pharmacology and anticancer properties of tetrandrine and fangchinoline (alkaloids) from Stephania tetrandra roots. Journal of Integrative Medicine, 2021, 19(4): 311-316

[3] Chen Y, Zhang Y, Wang X, Zhou J, Ma L, Li J, Yang L, Ouyang H, Yuan H, Pang D. Transmissible gastroenteritis virus: An update review and perspective. Viruses, 2023

[4] Doyle LP, Hutchings LM. A transmissible gastroenteritis in pigs. Journal of the American Veterinary Medical Association, 1946, 108(829): 257-259

[5] Hu WW, Zhang S, Shen YM, Yang Q. Epidermal growth factor receptor is a co-factor for transmissible gastroenteritis virus entry. Virology, 2018, 521: 33-43

[6] Jang J, Yoon SH, Lee W, Yu J, Yoon J, Shim S, Kim H. Time-calibrated phylogenomics of the porcine epidemic diarrhea virus: Genome-wide insights into the spatiotemporal dynamics. Genes & GenomiCs, 2018, 40(8): 825-834

[7] Kim DE, Min JS, Jang MS, Lee JY, Shin YS, Song JH, Kim HR, Kim S, Jin YH, Kwon S. Natural bis-benzylisoquinoline alkaloids-Tetrandrine, Fangchinoline, and Cepharanthine, inhibit human coronavirus OC43 infection of MRC-5 human lung cells. Biomolecules, 2019

[8] Lendeckel U, Karimi F, Al Abdulla R, Wolke C. The role of the ectopeptidase APN/CD13 in cancer. Biomedicines, 2023

[9] Li Y, Wang J, Liu Y, Luo X, Lei W, Xie L. Antiviral and virucidal effects of curcumin on transmissible gastroenteritis virus in vitro. Journal of General Virology, 2020, 101(10): 1079-1084

[10] Li J, Zhou J, Zhang T, Wu H, Li F, Qi C, Fan L, Yuan X, Wang W, Guo R, et al.. Effective inhibition of PDCoV infection in chimeric APN gene-edited neonatal pigs. Journal of Virology, 2024, 98(8): Article ID: e0061124

[11] Liu Q, Wang HY. Porcine enteric coronaviruses: An updated overview of the pathogenesis, prevalence, and diagnosis. Veterinary Research Communications, 2021, 45(2-3): 75-86

[12] Lu CY, Amin MA, Fox DA. CD13/Aminopeptidase N is a potential therapeutic target for inflammatory disorders. Journal of Immunology, 2020, 204(1): 3-11

[13] Mina-Osorio P. The moonlighting enzyme CD13: Old and new functions to target. Trends in Molecular Medicine, 2008, 14(8): 361-371

[14] Niu Z, Zhang S, Xu S, Wang J, Wang S, Hu X, Zhang L, Ren L, Zhang J, Liu X, et al.. Porcine epidemic diarrhea virus replication in human intestinal cells reveals potential susceptibility to cross-species infection. Viruses, 2023

[15] Sadhu S, Dandotiya J, Dalal R, Khatri R, Mykytyn AZ, Batra A, Kaur M, Chandwaskar R, Singh V, Kamboj A, et al.. Fangchinoline inhibits SARS-CoV-2 and MERS-CoV entry. Antiviral Research, 2023

[16] Shim JS, Kim JH, Cho HY, Yum YN, Kim SH, Park HJ, Shim BS, Choi SH, Kwon HJ. Irreversible inhibition of CD13/aminopeptidase N by the antiangiogenic agent curcumin. Chemistry & Biology, 2003, 10(8): 695-704

[17] Sun D, Shi H, Chen J, Guo D, Liu Q, He X, Bao J, Wang Y, Qiu H, Feng L. Virus-binding activity of the truncated C subunit of porcine aminopeptidase N expressed in Escherichia coli. Biotechnology Letters, 2012, 34(3): 533-539

[18] Sun Y, Liu Z, Shen S, Zhang M, Liu L, Ghonaim AH, Li Y, Zhang S, Li W. Inhibition of porcine deltacoronavirus entry and replication by cepharanthine. Virus Research, 2024, 340 Article ID: 199303

[19] Verhulst E, Garnier D, De Meester I, Bauvois B. Validating cell surface proteases as drug targets for cancer therapy: What do we know, and where do we go?. Cancers, 2022

[20] Wan Z, Lu Y, Liao Q, Wu Y, Chen X. Fangchinoline inhibits human immunodeficiency virus type 1 replication by interfering with gp160 proteolytic processing. PLoS ONE, 2012, 7(6): Article ID: e39225

[21] Wang X, Niu Z, Jia Y, Cui M, Han L, Zhang Y, Liu Z, Bi D, Liu S. Ubenimex inhibits cell proliferation, migration and invasion by inhibiting the expression of APN and inducing autophagic cell death in prostate cancer cells. Oncology Reports, 2016, 35(4): 2121-2130

[22] Wang M, Zhang XM, Fu X, Zhang P, Hu WJ, Yang BY, Kuang HX. Alkaloids in genus stephania (Menispermaceae): A comprehensive review of its ethnopharmacology, phytochemistry, pharmacology and toxicology. Journal of Ethnopharmacology, 2022, 293 Article ID: 115248

[23] Whitworth KM, Rowland RRR, Petrovan V, Sheahan M, Cino-Ozuna AG, Fang Y, Hesse R, Mileham A, Samuel MS, Wells KD, et al.. Resistance to coronavirus infection in amino peptidase N-deficient pigs. Transgenic Research, 2019, 28(1): 21-32

[24] Wickström M, Larsson R, Nygren P, Gullbo J. Aminopeptidase N (CD13) as a target for cancer chemotherapy. Cancer Science, 2011, 102(3): 501-508

[25] Xu K, Zhou Y, Mu Y, Liu Z, Hou S, Xiong Y, Fang L, Ge C, Wei Y, Zhang X, et al.. CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance. eLife, 2020, 9 Article ID: e57132

[26] Yin JC, Glende J, Schwegmann-Wessels C, Enjuanes L, Herrler G, Ren XF. Cholesterol is important for a postadsorption step in the entry process of transmissible gastroenteritis virus. Antiviral Research, 2010, 88(3): 311-316

[27] Zhang J, Wu Z, Yang H. Aminopeptidase N knockout pigs are not resistant to Porcine Epidemic Diarrhea Virus infection. Virologica Sinica, 2019, 34(5): 592-595

[28] Zhang Y, Qi D, Gao Y, Liang C, Zhang Y, Ma Z, Liu Y, Peng H, Zhang Y, Qin H, et al.. History of uses, phytochemistry, pharmacological activities, quality control and toxicity of the root of Stephania tetrandra S. Moore: A review. Journal of Ethnopharmacology, 2020, 260 Article ID: 112995

[29] Zhang W, Shen H, Wang M, Fan X, Wang S, Wuri N, Zhang B, He H, Zhang C, Liu Z, et al.. Fangchinoline inhibits the PEDV replication in intestinal epithelial cells via autophagic flux suppression. Frontiers in Microbiology, 2023, 14 Article ID: 1164851

[30] Zheng Y, Feng J, Song Y, Yu Y, Ling M, Zhang M, Xie H, Li W, Xu W. Design and synthesis of APN and 3CLpro dual-target inhibitors based on STSBPT with anticoronavirus activity. Journal of Medical Virology, 2024, 96(3): Article ID: e29512

Funding

National Key Research and Development Program of China(2023YFD1800801)

RIGHTS & PERMISSIONS

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