Identification of Licoflavone C as a cap-dependent endonuclease inhibitor against severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus with a high fatality rate. Currently no approved drugs or vaccines are available against it. Sharing a common replication mechanism with negative-stranded, segmented viruses (NSVs), SFTSV utilizes a cap-dependent endonuclease (CEN) domain of the L segment to execute the cap-snatching process upon genome transcription initiation. Given the crucial role of CEN in the life cycle of NSVs, it is considered a promising target for discovery of antiviral agents against SFTSV. In this study, we established a high-throughput FRET-based enzymatic screening system to discover inhibitors of SFTSV CEN from a chemical library containing 3467 natural compounds. Finally, three compounds, i.e., Licoflavone C, 3,4-dicaffeoylquinic acid, and oleanolic acid displayed exceptional antiviral effects and minimal cytotoxicity. Licoflavone C (EC₅₀ = 1.85 μM) was selected for further investigation. Administration of Licoflavone C (20 mg/kg, i.v.) significantly reduced tissue viral loads in SFTSV-challenged mouse model. We demonstrated that Licoflavone C did not directly bind to the active pocket of SFTSV CEN but disrupted its active conformation, resulting in substrate non-competitive inhibition. Licoflavone C also exhibited broad-spectrum inhibition on several NSV CENs (HRTV, GTV, and LCMV) besides SFTSV. Furthermore, 15 analogs of Licoflavone C sharing a typical flavonoid structure were verified for targeting SFTSV CEN and exhibiting antiviral activities. In conclusion, Licoflavone C is a promising inhibitor of SFTSV, offering insights into targeting CEN with flavonoids in drug discovery.