Computational Characterization of Hispidin as a Multi‐Target Inhibitor of Monkeypox Virus: Docking, MD Simulation, and DFT Analysis

ABSTRACT Monkeypox is a re‐emerging viral disease with limited therapeutic options. In this study, we investigated the antiviral potential of Hispidin, a fungal‐derived polyphenol, against monkeypox virus using a comprehensive in silico workflow. Three essential viral proteins were examined: Vaccinia Virus Thymidylate Kinase (VTK) (2v54), viral profilin‐like protein (VPP) (4qwo), and viral RNA polymerase (VRP) (6bed). Molecular docking performed with AutoDock Vina (UCSF Chimera) identified VTK as the only target with consistently strong predicted affinity, whereas interactions with VPP and VRP were weaker and less stable. Molecular dynamics simulations (100 ns, Desmond) further demonstrated that the Hispidin‐VTK complex remained structurally stable, with low protein‐ligand RMSD (∼1.75‐2.25 Å) and persistent key contacts, while the VPP and VRP complexes exhibited higher mobility and solvent exposure. MM/GBSA‐MM/PBSA and per‐residue decomposition analyses likewise confirmed the VTK‐Hispidin complex as the strongest binder, supported by pronounced stabilizing residue contributions. Toxicity prediction using ProTox 3.0 classified Hispidin as GHS Class IV (LD 50 ≈ 1000 mg/kg), indicating low acute toxicity, and cardioToxCSM predicted no major cardiotoxic liabilities. PASS analysis suggested diverse biological activities (Pa > 0.5), including antiviral and antioxidant potential. ADMET profiling (preADMET) supported favorable pharmacokinetics with good intestinal absorption and moderate BBB penetration. DFT calculations (B3LYP/6‐311G++(d,p)) predicted a planar, electron‐rich structure and a moderate HOMO‐LUMO gap (3.09 eV), consistent with redox stability. Overall, the findings provide strong computational evidence only for VTK inhibition, while results for VPP and VRP remain preliminary. Hispidin shows a favorable toxicity and ADMET profile, supporting its further experimental evaluation as a potential anti‐monkeypox therapeutic candidate.