A robust FRET-based biochemical assay for the discovery of chikungunya virus nsP2 protease inhibitors.

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, is a significant global health concern, yet no approved antiviral drugs exist. The nsP2 protease, which processes viral polyproteins into functional units for virus replication, represents an attractive antiviral drug target. Therefore, targeting the nsP2 protease is a valuable strategy for developing antivirals against CHIKV infections. In this study, we developed an integrated platform for the discovery of nsP2 protease inhibitors, comprising recombinant protein production and an optimized fluorescence resonance energy transfer (FRET)-based biochemical assay. Using a robust FRET assay, we identified punicalagin as a potential noncompetitive inhibitor of the nsP2 protease in vitro. Our systematic optimization workflow provides a valuable methodology for the rapid screening of antiviral drugs.