Broad-spectrum coronavirus inhibition by RSV fusion inhibitors targeting six-helix bundle formation.

Waning vaccine-derived immunity and the emergence of new SARS-CoV-2 subvariants continue to challenge global public health, emphasizing the need for novel inhibitors against SARS-CoV-2 infection. Molecular inhibitors targeting virus entry are promising candidates as they can disrupt viral replication at the earliest stage. In this study, we utilized a fluorescence-based SARS-CoV-2 pseudovirus screening platform to identify entry inhibitors from a repurposed library of bioactive compounds. Ziresovir and TMC353121, both small molecules known as respiratory syncytial virus (RSV) fusion inhibitors, were identified as hits that inhibited SARS-CoV-2 pseudovirus entry. In vitro and in silico analyses, together with site-directed mutagenesis, demonstrated that these molecules interfere with the formation of the six-helix bundle (6HB) in the SARS-CoV-2 spike (S) protein, thereby inhibiting membrane fusion. Additionally, both compounds exhibited inhibitory effects against SARS-CoV pseudovirus, wild-type SARS-CoV-2, and several other coronaviruses. These findings identify Ziresovir and TMC353121 as mechanistically validated scaffolds that target conserved fusion mechanisms across different viral families. While further optimization is required to enhance their potency against infectious SARS-CoV-2, this study provides a strategic foundation and chemical templates for the development of broad-spectrum fusion inhibitors to combat current and future coronavirus threats.