Abstract: Front. Biosci. (Landmark Ed) 2023; 28(1): 8 https://doi.org/10.31083/j.fbl2801008 Original Research Inhibition of SARS-CoV-2 Mpro with Vitamin C, L-Arginine and a Vitamin C/L-Arginine Combination Ivana Đukić1 , Nevena Kaličanin2 , Milan Sencanski3 , Snezana B. Pajovic4 , Jelena Milicevic3 , Jelena Prljic3 , Slobodan Paessler5,6 , Radivoje Prodanović1 , Sanja Glisic3, * 1 University of Belgrade-Faculty of Chemistry, 11000 Belgrade, Serbia 2 University of Belgrade-Institute of Chemistry, Technology and Metallurgy, 11000 Belgrade, Serbia 3 Laboratory of Bioinformatics and Computational Chemistry, Institute of Nuclear Sciences Vinca, National Institute of the Republic of Serbia, University of Belgrade, 11001 Belgrade, Serbia 4 Department of Molecular Biology and Endocrinology, VINCA Institute of Nuclear Sciences, National Institute of the Republic of Serbia, University of Belgrade, 11001 Belgrade, Serbia 5 Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USA 6 Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555, USA *Correspondence: sanja@vinca.rs (Sanja Glisic) Academic Editor: Paramjit S. Tappia Submitted: 20 October 2022 Revised: 7 December 2022 Accepted: 23 December 2022 Published: 13 January 2023 Abstract Background: Drug resistance is a critical problem in health care that affects therapy outcomes and requires new approaches to drug design. SARS-CoV-2 Mpro mutations are of concern as they can potentially reduce therapeutic efficacy. Viral infections are amongst the many disorders for which nutraceuticals have been employed as an adjunct therapy. The aim of this study was to examine the potential in vitro activity of L-arginine and vitamin C against SARS-CoV-2 Mpro . Methods: The Mpro inhibition assay was developed by cloning, expression, purification, and characterization of Mpro . Selected compounds were then screened for protease inhibition. Results: Larginine was found to be active against SARS-CoV-2 Mpro , while a vitamin C/L-arginine combination had a synergistic antiviral action against Mpro . These findings confirm the results of our previous in silico repurposing study that showed L-arginine and vitamin C were potential Mpro inhibitors. Moreover, they suggest a possible molecular mechanism to explain the beneficial effect of arginine in COVID patients. Conclusions: The findings of the current study are important because they help to identify COVID-19 treatments that are efficient, inexpensive, and have a favorable safety profile. The results of this study also suggest a possible adjuvant nutritional strategy for COVID-19 that could be used in conjunction with pharmacological agents. Keywords: anti SARS-CoV-2; Mpro ; COVID-19; arginine; vitamin C/arginine combination; Mpro candidate inhibitors