Identification of Four Compounds with S-RBD-Binding and Pseudovirus Entry-Inhibitory Activity.

COVID-19, caused by SARS-CoV-2, remains a global health challenge because of viral evolution and immune escape. Although current therapies primarily target viral entry and replication, agents that directly interfere with the Spike receptor-binding domain (S-RBD) remain limited. Here, we combined virtual screening with biological validation to identify compounds capable of interfering with S-RBD function. A total of 3014 compounds from a customized drug library were screened by molecular docking. Candidate binding and functional activity were subsequently evaluated using cellular thermal shift assays, surface plasmon resonance, immunoprecipitation, immunofluorescence, and pseudovirus-entry assays against 2019-nCoV, Delta, and Omicron pseudoviruses. DOTAP chloride (KD = 49.94 μM), cefotiam hexetil hydrochloride (KD = 142.26 μM), melittin (KD = 34.98 μM), and teicoplanin (KD = 73.58 μM) showed detectable binding to the S-RBD and inhibited Spike-mediated pseudovirus entry. Molecular docking suggested that these interactions were mediated by potential binding modes involving hydrogen bonds and π-interactions. The compounds interfered with S-RBD/hACE2 colocalisation and inhibited pseudovirus entry with variant-dependent efficacy. DOTAP chloride (25 μM) inhibited entry of the 2019-nCoV and Omicron pseudoviruses, whereas the other three compounds showed activity across the tested variants. These findings identify four compounds with RBD-binding and entry-inhibition properties for further development as entry inhibitors.