In silico identification of isosilybin a targeting squalene epoxidase as an antifungal adjuvant with in vitro validation against azole-resistant Candida and emerging yeasts

The rising incidence of antifungal resistance in Candida species and emerging non-albicans yeasts has markedly reduced the efficacy of azole antifungal therapy, highlighting the need for resistance modifying strategies. Natural products represent a valuable reservoir of bioactive scaffolds, and computational prioritization facilitates the systematic identification of pharmacologically relevant lead molecules. In this study, we employed an integrated in silico-in vitro antimicrobial pharmacology approach to identify Isosilybin A as a potential ERG1-targeting antifungal adjuvant and to assess its biological relevance using Isosilybin A enriched plant extract. A comprehensive virtual screening of 17,967 phytochemicals from the Indian Medicinal Plants, Phytochemistry And Therapeutics (IMPPAT) database was performed against an AlphaFold-predicted structure of squalene epoxidase (ERG1) from Candida albicans , followed by drug-likeness and ADMET profiling. Seven shortlisted phytochemicals were subjected to 100 ns molecular dynamics simulations. Among these, Isosilybin A demonstrated persistent interactions with key active-site residues of ERG1 and exhibited stable dynamic behavior throughout the simulation. Favorable binding free energy estimated using Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) method positioning it as the foremost mechanistically prioritized candidate. To experimentally validate this computational prioritization, Isosilybin A enriched Silybum marianum extract was evaluated against azole-resistant Candida species and emerging multidrug-resistant yeast isolates. While the extract alone exhibited no detectable antifungal activity, its combination with fluconazole consistently restored azole susceptibility across all tested isolates. Growth inhibition was observed at a minimum inhibitory concentration of 0.78 mg/mL extract combined with 0.78 μg/mL fluconazole, whereas fluconazole monotherapy remained ineffective. Collectively, these findings establish Isosilybin A as a computationally identified ERG1-interacting antifungal adjuvant, with extract-based in vitro validation supporting its role in restoring fluconazole efficacy against resistant Candida and emerging yeasts. Further studies employing purified compounds are warranted to conclusively delineate the pharmacological contribution of Isosilybin A.