Abstract

The membrane fusion process mediated by the SARS-CoV-2 spike protein is a key therapeutic target. Its heptad repeat 1 (HR1) domain forms a conserved trimeric groove critical for forming the fusion-competent six-helix bundle with HR2. We used DNA-encoded library screening to identify small molecules binding HR1. Hits including Rabeprazole-related compound E (Rab RCE), Omeprazole, Alvimopan, and Olmesartan were characterized. Biophysical assays confirmed binding, while computational simulations revealed distinct interaction modes, with Alvimopan showing high predicted affinity. Cell-cell fusion assays demonstrated potent inhibitory activity for Olmesartan and Rab RCE. Notably, Rabeprazole and Rab RCE showed partial antiviral activity against SARS-CoV-2 variants and HCoV-OC43, rescuing virus-induced apoptosis. Mechanistically, Rabeprazole competitively occupies the HR2-binding groove on HR1, blocking fusion. Our findings identify HR1-targeting molecules like Rabeprazole as promising leads for broad-spectrum coronaviral fusion inhibitors.