ABSTRACT
Viral protease is an attractive target for antiviral therapeutics, but current viral protease inhibitor screening methods still need to be improved. Here, we systematically investigated the sites that may accommodate exogenous short peptides within Enhanced Green Fluorescent Protein (EGFP)and constructed a series of recombinant green fluorescent proteins (rGFPs). Meanwhile, a cell-based, simple and reliable assay system named DIFF-rGFP was developed relying on the co-expression of rGFP and the protease for protease inhibitor screening with the example of 3CLpro, in which the fluorescence intensity increases with the action of the inhibitor. The DIFF-rGFP assay avoided the requirement of a higher biosafety lab and can be performed in a high-throughput manner. For proof of concept, we demonstrated this method to discover novel inhibitors against SARS-CoV-2. We believe the proposed method, in combination with available drug libraries, may accelerate the identification of novel antivirals.