In vitro and in silico Antiviral Activity of Biflavonoids From Garcinia madruno Against Mpox Virus

Aim Although current human transmission of monkeypox virus (MPXV) remains low, therapeutic options are limited. Biflavonoids, known for their antiviral properties, were evaluated in vitro and in silico against MPXV. Procedures Three biflavonoids; Morelloflavone (Mo), Volkensiflavone (Vo), and Fukugiside (Fu); isolated from Garcinia madruno, were tested at concentrations of 1.6, 3.1, 6.3, and 12.5 µM against an MPXV strain isolated in Medellín, Colombia (2022). Combined, pre-, co-, and post-infection treatment strategies were assessed in Vero-E6 cells at a multiplicity of infection (MOI) of 2. Cytotoxicity, plaque reduction assays, and molecular docking analyses were performed. Results and Discussion None of the biflavonoids exhibited cytotoxicity at concentrations up to 100 µM. All reduced MPXV infection by >70.9% across all concentrations under combined treatment strategies. In individual strategies at 6.3 µM, Mo, Vo, and Fu inhibited viral attachment by 59.4%, 79.3%, and 77.8%, respectively. Vo and Fu reduced the infection at co-treatment by 93.7% and 95.1%, respectively. Mo and Fu inhibited infection at post-entry steps by 64.4% and 56.7%, respectively. Molecular docking analyses revealed strong binding affinities (<–7.7 kcal/mol) between all biflavonoids and the MPXV proteins Methyltransferase and Poxin. Mo showed the most favorable binding energy (–8.4 kcal/mol), while Fu formed up to 12 hydrogen bonds with two catalytic residues of Methyltransferase. These findings indicate that biflavonoids could inhibit MPXV via distinct mechanisms: Mo affects both attachment and post-entry steps, while Vo and Fu show a possible direct activity against this virus and reduce attachment. Further studies are needed to validate their effects and clarify their therapeutic potential.