The SARS-CoV-2 M^pro Dimer-Based Screening System: A Synthetic Biology Tool for Identifying Compounds with...

The COVID-19 endemic remains a global concern. The search for effective antiviral candidates is still needed to reduce disease risk. However, the availability of high biosafety level laboratory facilities for drug screening is limited in number. To address this issue, a screening system that could be utilized at lower biosafety levels remains essential. This study aimed to develop a novel SARS-CoV-2 main protease (M^pro) dimer-based screening system (DBSS) utilizing synthetic biology in Escherichia coli BL21(DE3). We linked the SARS-CoV-2 M^pro with the DNA-binding domain of AraC regulatory protein, which regulates the reporter gene expression. Protein modeling and molecular docking showed that saquinavir could bind to AraC-M^pro both in its monomer and dimer forms. The constructed DBSS assay indicated the screening system could detect saquinavir inhibitory activity at a concentration range of 4-10 μg/mL compared to the untreated control (P ≤ 0.05). The Vero E6 cell assay validated the DBSS result that saquinavir at 4-10 μg/mL exhibited antiviral activity against SARS-CoV-2. Our DBSS could be used for preliminary screening of numerous drug candidates that possess a dimerization inhibitor activity of SARS-CoV-2 M^pro and also minimize the use of a high biosafety level laboratory.