A Computational-Experimental Strategy Identifies K-7174 against African Swine Fever

African swine fever (ASF), caused by the African swine fever virus (ASFV), poses a catastrophic threat to global swine production due to the absence of effective vaccines or therapeutics. Targeting the virally encoded RNA polymerase (RNAP), which is essential for viral transcription, offers a promising antiviral strategy. Capitalizing on our previously resolved high-resolution structure of ASFV RNAP, we conducted a rigorous structure-based virtual screening of two compound libraries (T001 and LF1000) to identify selective inhibitors. From 100 candidates validated in vitro, nine compounds identified at the genome level by qPCR demonstrated >98% inhibition of ASFV replication in porcine alveolar macrophages (PAMs). The lead compound, K-7174—a repurposed agent known to modulate proteasome and GATA activity—exhibited potent, dose-dependent antiviral efficacy (IC₅₀ = 2.502 µM), minimal cytotoxicity, and high binding affinity for ASFV RNAP. Molecular docking simulations indicated potential interactions with key catalytic residues, including Glu1051 and Ala456, proposing a mechanism that may involve active-site disruption. However, definitive confirmation of this binding pose requires further experimental validation through high-resolution structural methods such as cryo-EM. Our study not only nominates K-7174 as a promising anti-ASFV candidate but also establishes an integrated computational–experimental framework for accelerating the discovery of antiviral agents targeting viral polymerases.