In vitro enzymatic assays invalidate dihydromyricetin as a potential inhibitor against enterovirus A71 3C protease: beware of fluorescence quenching artifacts.

Enterovirus A71 (EV-A71) is an important pathogen that causes hand, foot, and mouth disease (HFMD) outbreaks worldwide. Although vaccines against EV-A71 have been approved in China, there are currently no efficacious antivirals for HFMD treatment. The 3C protease (3Cpro) of EV-A71, an essential enzyme involved in viral replication, represents an attractive target for antiviral development. Despite being proposed as a promising 3Cpro inhibitor through structure-based virtual screening, 3Cpro inhibition by dihydromyricetin (DHM) remains to be rigorously investigated. In this study, our quantitative enzymatic assays, including a fluorescence resonance energy transfer (FRET)-based approach and a dimeric RFP cleavage assay (DRCA), invalidated DHM as a potential 3Cpro inhibitor in vitro. Our study revealed that fluorescence quenching, a common screening artifact, should be carefully considered when natural products are identified as 3Cpro inhibitors. Candidate hit compounds from primary screening require rigorous pharmacological assessments before being reported as new findings.