Small molecules targeting SARS-CoV-2 nucleocapsid reveal a conserved RNA-binding vulnerability across human coronaviruses

SARS-CoV, MERS-CoV, and SARS-CoV-2 exemplify the persistent threat posed by coronaviruses, with their capacity for zoonotic spill over, rapid transmission, and severe disease resulting in substantial morbidity, mortality, and socio-economic burden. The repeated emergence of highly pathogenic coronaviruses highlights the urgent need for broad-spectrum therapeutic strategies. The nucleocapsid (N) protein, essential for RNA binding, genome packaging, and viral replication, remains a highly conserved yet underexplored antiviral target. Sequence alignment revealed 90% identity between SARS-CoV-2 and SARS-CoV N proteins, and 48.1% identity between SARS-CoV-2 and MERS-CoV N proteins. In our earlier work, we identified two small molecules, ceftazidime and sennoside A, that bind the N-terminal domain of the SARS-CoV-2 N protein and inhibit nucleic acid binding, and identified their binding sites using NMR chemical shift perturbation assays. Here, we observed that several residues involved in inhibitor binding are conserved across betacoronaviruses, suggesting a shared druggable vulnerability. We have purified recombinant N proteins from SARS-CoV, MERS-CoV, and SARS-CoV-2, and demonstrated by electrophoretic mobility shift assays that both compounds significantly reduced RNA binding. Their inhibitory concentrations (IC₅₀) were determined using fluorescence polarization. The docking analyses indicated that both inhibitors target the RNA-binding pocket of the N-NTD, consistent with a conserved mechanism of action. Collectively, our findings reveal a conserved RNA-binding vulnerability in coronavirus N proteins and highlights the pan-coronavirus therapeutic potential of these inhibitors.