Design of a Fluorescence Polarization Probe for Enterovirus 2C Proteins

Enteroviruses (EVs), such as EV-D68,
EV-A71, and CVB3,
cause significant
human disease; yet, no antivirals are currently approved. The highly
conserved 2C protein, an essential AAA+ ATPase and helicase, is a
prime antiviral target; however, it lacks suitable assays for inhibitor
screening. Here, we report a fluorescence polarization (FP) assay
using a rationally designed probe, Jun14157, which binds
a conserved allosteric site in 2C with high affinity. This assay enables
the quantitative assessment of binding to diverse 2C inhibitors with
high signal-to-background ratios, DMSO tolerance, and a strong correlation
between FP Ki and cellular EC50. Using this platform, we validated hits from virtual screening and
identified two novel inhibitors, Jun15716 and Jun15799. This FP assay offers a robust and scalable tool for the mechanistic
characterization and high-throughput screening of 2C-targeting antivirals.