Virtual Screening and Molecular Dynamics Simulation Targeting the ATP Domain of African Swine Fever Virus Type II DNA Topoisomerase.

African Swine Fever Virus (ASFV) Topo II ATPase domain, resistant to conventional inhibitors (e.g., ICRF-187) due to M18/W19 steric clashes, was targeted via hierarchical virtual screening (Schrödinger) of the Chembridge library combined with MM/GBSA calculations. Five ligands (10012949, 40242484, 46712145, 15880207, and 33688815) showed high affinity, with 46712145 adopting symmetrical π-π stacking, hydrogen bonds, and alkyl interactions to bypass steric hindrance. Molecular dynamics simulations (100 ns) revealed ligand-induced flexibility, evidenced by elevated RMSD/Rg values versus the free protein. DCCM analysis highlighted enhanced anti-correlated motions between GHKL motifs and sensor domains in chain B/C, suggesting stabilization of a non-catalytic conformation to inhibit ATP hydrolysis. Free energy landscape (FEL) analysis showed 46712145 occupying a broad, shallow energy basin, enabling conformational adaptability, contrasting the narrow deep well of the free protein. This study proposes a symmetric ligand design strategy and conformational capture mechanism to block ATPase activity. Compound 46712145 demonstrates stable binding and dynamic regulation, providing a novel lead scaffold for anti-ASFV drug development. These findings establish a structural framework for combating ASFV through targeted ATPase inhibition.