Methotrexate inhibition of SARS-CoV-2 entry, infection and inflammation revealed by bioinformatics approach and a hamster model

Yun Ti Chen, Yu Hsiu Chang, Nikhil Pathak, Shey Cherng Tzou, Yong Chun Luo, Yen Chao Hsu, Tian Neng Li, Jung Yu Lee, Yi Cyun Chen, Yu Wei Huang, Hsin Ju Yang, Nung Yu Hsu, Hui Ping Tsai, Tein Yao Chang, Shu Chen Hsu, Ping Cheng Liu, Yuan Fan Chin, Wen Chin Lin, Chuen Mi Yang, Hsueh Ling WuChia Ying Lee, Hui Ling Hsu, Yi Chun Liu, Jhih Wei Chu, Lily Hui Ching Wang, Jann Yuan Wang, Chih Heng Huang, Chi Hung Lin, Po Shiuan Hsieh, Yan Hwa Wu Lee, Yi Jen Hung*, Jinn Moon Yang*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


Background: Drug repurposing is a fast and effective way to develop drugs for an emerging disease such as COVID-19. The main challenges of effective drug repurposing are the discoveries of the right therapeutic targets and the right drugs for combating the disease. Methods: Here, we present a systematic repurposing approach, combining Homopharma and hierarchal systems biology networks (HiSBiN), to predict 327 therapeutic targets and 21,233 drug-target interactions of 1,592 FDA drugs for COVID-19. Among these multi-target drugs, eight candidates (along with pimozide and valsartan) were tested and methotrexate was identified to affect 14 therapeutic targets suppressing SARS-CoV-2 entry, viral replication, and COVID-19 pathologies. Through the use of in vitro (EC50 = 0.4 μM) and in vivo models, we show that methotrexate is able to inhibit COVID-19 via multiple mechanisms. Results: Our in vitro studies illustrate that methotrexate can suppress SARS-CoV-2 entry and replication by targeting furin and DHFR of the host, respectively. Additionally, methotrexate inhibits all four SARS-CoV-2 variants of concern. In a Syrian hamster model for COVID-19, methotrexate reduced virus replication, inflammation in the infected lungs. By analysis of transcriptomic analysis of collected samples from hamster lung, we uncovered that neutrophil infiltration and the pathways of innate immune response, adaptive immune response and thrombosis are modulated in the treated animals. Conclusions: We demonstrate that this systematic repurposing approach is potentially useful to identify pharmaceutical targets, multi-target drugs and regulated pathways for a complex disease. Our findings indicate that methotrexate is established as a promising drug against SARS-CoV-2 variants and can be used to treat lung damage and inflammation in COVID-19, warranting future evaluation in clinical trials.

Original languageEnglish
Article number1080897
JournalFrontiers in Immunology
StatePublished - 21 Dec 2022


  • drug repurposing
  • inflammation
  • methotrexate
  • multi-target drugs
  • SARS-CoV-2


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