Development of a Bacterial Colorimetric Reporter System for Functional Screening of SARS-Cov-2 Main Protease Inhibitors Using Plant Preparations (juices): A Proof-of-Concept Study

Abstract

SARS-CoV-2 main protease (Mpro) is essential for viral polyprotein processing and represents a prime target for antiviral drug discovery. However, most available screening strategies rely on biochemical and computational approaches that lack the biological context of living cells, or costly mammalian-cell based models. Therefore, there remains a shortage of simple and biosafe cellular models enabling rapid, functional screening of potential Mpro inhibitors, particularly those derived from natural sources and in urgent situations such as the COVID-19 pandemic. In this study, a bacterial colorimetric reporter system was developed that directly links SARS-CoV-2 Mpro activity to {beta}-galactosidase function in Escherichia coli. To the best of our knowledge, the developed system represents the first bacterial colorimetric model for direct monitoring of SARS-CoV-2 Mpro inhibition in living cells. The system enables real-time visual detection of protease inhibition on X-gal-containing medium and provides a cost-effective, biologically relevant, biosafe alternative to existing screening assays. Functional validation was performed using pomegranate juice as a representative natural inhibitor source. The system provides a simple, scalable, and biosafe platform for the primary screening of antiviral candidates, including phytochemicals, under standard laboratory conditions.