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

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. 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 uM) and in vivo models, we show that methotrexate is able to inhibit COVID-19 via multiple mechanisms. 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. 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. Overall design: Male Syrian golden hamsters, aged 7-9 weeks old were obtained from Taiwan National Laboratory Animal Center. Hamsters were randomly allocated to study groups for antiviral evalution. 100 µL of PBS containing 1x104 pfu of SARS-CoV-2 was intranasal instilled under intraperitoneal anesthesia with Zoletil 50 (4mg/kg)/ Xylazine (0.5mg/kg) at day 0, and the mock-infected hamsters were challenged with 100 µL of PBS. Treatment groups were intraperitoneally given either MTX (2?mg/kg/day) or PBS (vehicle controls) for five consecutive days. At day-4 postinfection, all the hamsters were euthanized, and the lung tissues were for RNA extraction.