Production of a genetically encoded biosensor for evaluating enterovirus 71 3C protease inhibitors

Enterovirus 71 (EV-71) is a major causative agent of hand, foot, and mouth disease (HFMD), which mainly affects infants and children. However, there are no effective clinical drugs for the treatment of HFMD. The 3C protease (3Cpro) is an ideal drug target for antivirals, as this enzyme plays an indispensable role in virus replication. Considering the limitations of the peptide substrates used in the fluorescence resonance energy transfer (FRET) assay, there is an urgent need to design improved 3Cpro biosensors for assay development. In this study, we developed a genetically encoded biosensor based on a dimerization-dependent red fluorescent protein (ddRFP) system for evaluating 3Cpro inhibitors. The 3Cpro biosensor has many beneficial properties, such as economical bioproduction, a simple dual-mode readout, and a high emission wavelength. Using the 3Cpro biosensor, rupintrivir was identified as a competitive 3Cpro inhibitor <i>in vitro</i>. Our research highlights a promising avenue for producing 3Cpro biosensors from <i>E. coli</i> cells. The 3Cpro biosensor provides a reliable biochemical tool for the rapid assessment of antivirals against enterovirus infections.