San Li^1,2, Hui-Ming Dang^1,2, Shan Xu², Lei Liu¹ , Yi-gang Tong^1,2

Abstract

Purpose: To identify anti-respiratory syncytial virus (RSV-A) candidates from the pool of approved drugs through drug repositioning and preliminary elucidation of their mechanisms of action. Methods: Human laryngeal epithelial carcinoma cells (HEp-2) were infected with RSV-A (GenBank: PQ594188). The cytopathic effect (CPE) was used to conduct an initial screening of 1213 compounds from the drug library. For the selected candidate drug, emvododstat, a dihydroorotate dehydrogenase (DHODH) inhibitor, further analysis was performed with Cell Counting Kit-8 (CCK-8) assay to determine the viral copy number using absolute quantification and to measure the half-maximal effective concentration (EC50), half-maximal cytotoxic concentration (CC50), and selectivity index (SI = CC50/EC50). A time-of-addition assay (TOA) was conducted to determine the phase of the antiviral action, and the potential mechanism involved was determined, in addition to the DHODH inhibitory properties of the drug. Results: Emvododstat exhibited potent anti-RSV-A activity with an EC50 of 5.23 nmol/L and SI > 19,120 thereby outperforming Ribavirin (EC50 = 14.5 μmol/L, SI = 52). The TOA assays revealed that Emvododstat primarily acted during the post-entry phase, with minimal inhibition of viral entry. Conclusion: This study has demonstrated, for the first time, that Emvododstat exerts nanomolar inhibitory activity and high selectivity against RSV-A viruses. This finding highlights the potential of drug repositioning strategies in antiviral drug development. Although its mechanism of action has not been fully clarified, the high selectivity and low cytotoxicity of Emvododstat provide an important basis for its clinical application