Abstract

Enteroviruses (EVs) are significant human pathogens, and the development of orally available, broad-spectrum antiviral agents remains an urgent need. The viral protein 2C, a conserved nonstructural protein with helicase activity, is a promising target for antiviral intervention. Although the peptide 2CL was previously identified as a 2C inhibitor, its cell-penetrating peptide motif and extended core sequence may limit its binding efficiency and pharmacological properties. Here, we report the optimization of 2CL to develop a novel peptidomimetic, 2CA-1, with a more compact structure and absence of a cell-penetrating motif. Besides its potent cellular antiviral activity, achieved by precise docking into the 2C binding pocket to inhibit helicase function, 2CA-1 exhibited excellent oral bioavailability in a murine model, significantly reducing viral loads and showing broad efficacy against multiple enteroviruses including CV-A6, CV-A16, CV-B3, Echo11, EV-D68 and rhinovirus. This study not only presents 2CA-1 as an optimized 2C-targeted antiviral candidate but also highlights its potential as an orally available and broad-spectrum therapeutic against EVs.