Targeting SARS-CoV-2 Main Protease: A Bacteria-Based Colorimetric Assay for Screening Natural Antiviral Inhibitors

SARS-CoV-2 main protease (M^pro) is essential for viral polyprotein processing and represents a prime target for antiviral drug discovery. However, most available screening strategies rely on computational predictions or cell-free biochemical approaches that provide limited functional context and often require specialized instrumentation, while mammalian cell-based models remain costly and require high biosafety levels. Accordingly, there remains a shortage of simple, rapid, and biosafe functional screening tools suitable for early-stage prioritization of potential M^pro inhibitors, particularly those derived from natural sources and in urgent situations such as the COVID-19 pandemic. In this study, a bacterial colorimetric reporter assay was developed that directly links SARS-CoV-2 M^pro activity to β-galactosidase function in <i>Escherichia coli</i>. To the best of our knowledge, the developed assay represents the first bacterial colorimetric model for functional detection of SARS-CoV-2 M^pro inhibition based on a phenotypic readout. The assay enables the rapid visual detection of protease inhibition on X-gal-containing medium and provides a cost-effective and biosafe platform for prioritizing candidate inhibitors, under standard laboratory conditions, prior to further validation. Due to its bacterial expression context, this assay is intended for functional screening to provide the most promising candidate compounds and/or extracts for subsequent biochemical or mammalian cell-based validation; it is not intended to determine quantitative potency or to replace further validation approaches. It should be noted that the selective compound uptake in <i>E. coli</i> restricts the range of chemical compositions that can be evaluated using this method. Therefore, proof-of-concept application was demonstrated using pomegranate juice, a representative natural inhibitor source, rather than most currently known specific M^pro inhibitors. In addition, other plant-derived preparations, including rhubarb, grape, and red/black currant juices, were tested demonstrating the assay's applicability to diverse natural matrices.