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, <b>Jun14157</b>, 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 <i>K</i>_i and cellular EC₅₀. Using this platform, we validated hits from virtual screening and identified two novel inhibitors, <b>Jun15716</b> and <b>Jun15799</b>. This FP assay offers a robust and scalable tool for the mechanistic characterization and high-throughput screening of 2C-targeting antivirals.