Development of a Cell-Based Recombinant Green Fluorescent Protein Assay System for Generalized Discovery of Viral Protease Inhibitors

Viral proteases are critical targets for Antiviral drug development, but current screening methods for Protease Inhibitors often require high biosafety levels or lack cell-based relevance. Here, we developed a novel cell-based assay system utilizing recombinant green fluorescent protein (GFP) technology, designated as DIFF-recombinant GFP (DIFF-rGFP), for potentially high-throughput screening of viral Protease Inhibitors. By systematically investigating potential insertion sites within the green fluorescent protein (GFP), we constructed a series of recombinant green fluorescent proteins (rGFPs) that accommodate exogenous protease cleavage sequences. Using the 3-Chymotrypsin like protease (3CL^pro) of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) as a model, we demonstrated that the DIFF-rGFP assay relies on the coexpression of rGFP and the protease, with fluorescence intensity increasing upon inhibitor action. This assay eliminates the need for high biosafety laboratories and is performed at the cellular level. For proof of concept, we validated this method using two well-characterized SARS-CoV-2 3CL^pro inhibitors, GC376 and ensitrelvir, to demonstrate its applicability for inhibitor screening. Our results indicate that the DIFF-rGFP assay is a safe, efficient, and reliable platform for identifying viral Protease Inhibitors with potential applications in accelerating Antiviral drug discovery.

antiviral drugs; protease; recombinant GFP; screening method.