Qingao Xue ,  Ze Liang ,  Yi Zhang ,  Fei Wang ,  Fulian Wang ,  Lili Liu ,  Guang Yang ,  Lei Yan

Bioorganic Chemistry

DOI:10.1016/j.bioorg.2026.109627

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.

Highlights

• A DNA-encoded library (DEL) screening strategy was established to rapidly identify small-molecule binders targeting the conserved heptad repeat 1 (HR1) domain of the SARS-CoV-2 spike protein, enabling efficient mining of repurposed drug candidates from a ∼ 4 billion-compound chemical space.
• Four clinically approved drugs (alvimopan, olmesartan, rabeprazole sulfide, and omeprazole) were validated as HR1-targeting agents, sharing biaryl/heteroaryl cores and hydrogen-bond acceptor groups that mediate specific interactions with HR1 (binding affinities ranging from micromolar to millimolar).
• Two distinct inhibitory mechanisms were delineated: classical competitive occupancy of the HR1 hydrophobic groove (olmesartan, rabeprazole sulfide) and a novel ‘molecular wedge’ mode disrupting the trimeric HR1 interface (alvimopan), providing complementary strategies for targeting viral fusion.
• Olmesartan and rabeprazole sulfide exhibited potent inhibition of SARS-CoV-2-mediated cell-cell fusion, with efficacy comparable to the positive control Salsingle bondC, validating their potential as lead compounds for anti-COVID-19 therapeutics.
• This study establishes a robust pipeline integrating DEL screening, biophysical validation, molecular docking, and functional assays, offering valuable chemical scaffolds and mechanistic insights for developing broad-spectrum coronaviral fusion inhibitors.