S5092 inhibit ASFV infection by targeting the pS273R protease activity in vitro

African Swine Fever (ASF) is a highly infectious viral disease caused by African swine fever virus (ASFV). Currently, there is no effective vaccine, and it is urgent to discover antiviral agents to deal with the epidemic. ASFV pS273R is the only known cysteine protease that catalyzes the maturation of the polyprotein precursors pp220 and pp62 through proteolytic cleavage, a critical step in ASFV assembly. In this study, we performed a high-throughput screening of an FDA-approved drug library to identify several compounds that target the active site of the ASFV pS273R enzyme via molecular docking. We initially identified 778 molecules based on the "Drug-like Five Principles", with S5092 emerging as the most promising candidate. S5092 is a prodrug that can be metabolized into Fenbendazole (FBZ) and Oxfendazole (OFZ). In exogenous cleavage experiments, S5092, FBZ, and OFZ significantly inhibited the cleavage of the viral polyprotein pp220 and pp62, as well as the host antiviral protein gasdermin D (GSDMD) by pS273R, indicating that these compounds inhibit the enzymatic activity of pS273R. Importantly, S5092 and its metabolites reduced the level of ASFV infection, as evidenced by the detection of viral genomic copies and viral titer. Collectively, our findings indicate that S5092 suppresses ASFV infection by inhibiting the enzyme activity of pS273R, a multifunction protein, positioning it as a novel and specific drug candidate for the prevention and control of ASF.