Punicalin targets RSV glycoprotein and fusion protein at non-overlapping sites to prevent viral escape

Respiratory syncytial virus (RSV) causes substantial respiratory disease, while treatment is limited by drug resistance. Molecular docking of a 3,880-compound natural-product library identifies punicalin (PUN) as a dual-target RSV entry inhibitor. PUN inhibits the evaluated RSV-A and RSV-B strains, including a clinical isolate, and retains activity against strains resistant to ziresovir, ribavirin, nirsevimab, and palivizumab. After 20 passages under PUN selection, the half-maximal inhibitory concentration (IC₅₀) increases 3.88-fold, remaining below the predefined >10-fold resistance threshold. Mechanistic analyses suggest that PUN interacts with the G protein cystine-noose region involving N178, N179, and W183 to inhibit attachment and with an F protein region involving V243, T245, and P246 to stabilize prefusion F and inhibit membrane fusion. These regions do not overlap known resistance hotspots. In RSV-infected mice, nebulized PUN provides sustained respiratory exposure, reduces lung viral loads and pathology, and attenuates inflammatory responses.