Structure- and Dynamics-Driven Discovery of Small Molecule Inhibitors Targeting a Conserved Pocket near the Fusion...

The continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has challenged the durability of vaccine-mediated protection, reinforcing the need for broad-spectrum antiviral agents. Herein, we performed a structure- and dynamics-driven virtual screening to identify novel small-molecule inhibitors of SARS-CoV-2 cell entry by targeting a conserved pocket within the S2 subunit of the spike glycoprotein domain. Extensive molecular dynamics simulations of the S2 domain revealed a druggable binding pocket close to the fusion peptide site. Consensus pharmacophore-guided virtual screening of ∼900,000 compounds yielded 11 candidates selected for biological evaluation. The selected compounds reduced SARS-CoV-2 infection in vitro at nanomolar concentrations against different variants with an increased selectivity index. Moreover, viral-cell entry and mechanistic experiments support the selective inhibition of the early steps of viral fusion. Finally, in silico evolutionary analyses showed good conservation of the pharmacophore region among different SARS-related coronaviruses. Our results provide a basis for further optimization studies on novel antifusion SARS-CoV-2 inhibitors with potential cross-clade antiviral activity.