Abstract: bioRxiv preprint doi: https://doi.org/10.1101/2021.11.18.469078; this version posted November 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Metformin Suppresses SARS-CoV-2 in Cell Culture 2 3 Haripriya Parthasarathy1, Dixit Tandel1,2, and Krishnan H. Harshan1,2,€ 4 5 1 CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India 500007 6 2 Academy for Scientific and Innovative Research (AcSIR), Ghaziabad-201002, India 7 8 € Correspondence: hkrishnan@ccmb.res.in 9 10 Keywords 11 SARS-CoV-2, COVID-19, anti-viral activity, Metformin, AMPK, Diabetes 12 13 14 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.11.18.469078; this version posted November 19, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 15 ABSTRACT 16 People with diabetes are reported to have a higher risk of experiencing severe 17 COVID-19 complications. Metformin, a first-line medication for type 2 diabetes, has 18 antiviral properties. Some studies have indicated its prognostic potential in COVID- 19 19. Here, we report that metformin significantly inhibits SARS-CoV-2 growth in cell 20 culture models. SARS-CoV-2 infection of gut epithelial cell line, Caco2, resulted in 21 higher phosphorylation of AMPK. Metformin reduced viral titers in the infected cells 22 by nearly 99%, and by about 90% when cells were treated prior to infection. 23 Metformin pre-treatment resulted in further phosphorylation of AMPK and caused a 24 ten-fold reduction of viral titers indicating its potential in preventing naïve infections. 25 Confirming the positive impact of AMPK activation, another AMPK activator AICAR 26 substantially inhibited of viral titers and, AMPK inhibitor Compound C, augmented it 27 considerably. Metformin treatment post-SARS-CoV-2 infection resulted in nearly 28 hundred-fold reduction of viral titers, indicating that the antiviral potency of the drug 29 is far higher in infected cells, while still being able to reduce fresh infection. 30 Metformin displayed SARS-CoV-2 TCID50 and TCID90 at 3.5 and 8.9 mM, 31 respectively. In conclusion, our study demonstrates that metformin is very effective in 32 limiting the replication of SARS-CoV-2 in cell culture and thus possibly could offer 33 double benefits to diabetic COVID-19 patients by lowering both blood glucose levels 34 and viral load.