Identification of four compounds as potential inhibitory agents for SARS-CoV-2 spike protein

The COVID-19 pandemic, driven by SARS-CoV-2, continues to present global health challenges due to viral mutations and immune escape mechanisms. While current therapies primarily target viral entry and replication proteins, therapeutic agents that directly inhibit the Spike receptor-binding domain (S-RBD) are lacking. This study aimed to utilize virtual screening and biological methods to identify potential therapeutic compounds with anti-SARS-CoV-2 activity. We conducted virtual screening and molecular docking of 3,014 compounds obtained from customized drug library to identify candidates with high affinity for S-RBD. The biological activity of these candidates was assessed using cellular thermal shift assays, surface plasmon resonance, immunoprecipitation, immunofluorescent analysis, and pseudoviral invasion tests against three pseudoviral variants: 2019-nCoV, Delta, and Omicron. We identified that DOTAP chloride (K _D = 49.94 µM), cefotiam hexetil hydrochloride (K _D = 142.26 µM), melittin (K _D = 34.98 µM) and teicoplanin (K _D = 73.58 µM) exerted antiviral activity. These compounds demonstrated potent binding to S-RBD through hydrogen bonding and π-interactions. They effectively inhibited S-RBD/hACE2 binding and pseudoviral entry with different efficacy: DOTAP chloride (25 µM) inhibited pseudoviral entry of both the 2019-nCoV and Omicron strains, while cefotiam hexetil hydrochloride, melittin and teicoplanin showed broader spectrum activity across the variants. Our findings suggest that DOTAP chloride, cefotiam hexetil hydrochloride, melittin and teicoplanin could serve as potential therapeutic candidates for COVID-19 treatment. Their multimodal mechanisms—combining direct S-RBD binding, host receptor modulation, and electrostatic interference—indicate potential for overcoming challenges posed by viral evolution.