Structural insights into the antiviral efficacy of AG7404 against human rhinovirus 3C proteases

Human rhinoviruses (hRVs) are major causative agents of the common cold and contribute to lower respiratory tract infections. Although extensive research efforts continue to explore potential therapeutic interventions, no clinically approved antiviral treatment currently exists for hRV infections. This study provides the structural basis of hRV 3C protease inhibition in several serotypes by AG7404, a modified rupintrivir derivative. AG7404 showed potent antiviral activity against hRV-B14, hRV-A16 and hRV-A21 with EC₅₀ values of 0.108, 0.191 and 0.187 µM, respectively, and directly inhibited purified hRV-B14 3C protease with an IC₅₀ of 0.046 µM. The 2.11 Å crystal structure of hRV-B14 3C protease in complex with AG7404 revealed covalent binding to the catalytic Cys146 and occupation of the substrate-binding pockets by the ligand. Comparative structural analyses incorporating ligand-free hRV-B14 3C protease structures as well as rupintrivir-bound 3C protease structures from other hRV serotypes revealed significant conformational variability of the βcII-βdII region. Molecular dynamics simulations of the structural models of hRV-A16 and hRV-A21 3C proteases showed that the binding interactions of AG7404, including critical water-mediated networks, are conserved across serotypes despite sequence variations. These findings offer structural insights into the binding mode of AG7404 and establish a foundation for the rational design of broad-spectrum antivirals targeting hRV 3C proteases.