Untargeted metabolomics and in silico analysis identifies potential DNA gyrase inhibitors from an endophytic fungus Alternaria alternata P02PL2

Abstract The escalating threat of multidrug-resistant (MDR) bacterial pathogens underscores the urgent need for novel antimicrobial agents. Here, we combined a one-strain many compounds (OSMAC) and untargeted metabolomics approaches to investigate the bioactivities and the chemical space of the endophytic fungus Alternaria alternata P02PL2, isolated from a medicinal plant, Sclerocarya birrea . The antibacterial activities were assessed against contemporary clinical isolates, MDR Pseudomonas aeruginosa 5625574, methicillin-resistant Staphylococcus aureus (MRSA) 5627679, and a collection culture of S. aureus ATCC 25923 for reference. We first tested for bioactivities across eight growth media to optimize the production of bioactive secondary metabolites. The bioactive fungal extract fractions exhibited minimum inhibitory concentrations (MICs) ranging from 0.25 to 0.5 mg/mL. This was followed by an untargeted metabolomics analysis, which identified 41 secondary metabolites. Virtual screening, molecular docking, and 200-ns molecular dynamics simulations targeting S. aureus and P. aeruginosa DNA gyrase revealed lead compounds with superior binding affinities (e.g., fluorescein −9.2 kcal/mol for P. aeruginosa ; pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro −8.8 kcal/mol for S. aureus ) compared to reference ligands. Binding free energy calculations highlighted strong affinities, with 5,7-dihydroxy-2-(4-hydroxyphenyl)-6,8-bis(3,4,5-trihydroxyoxan-2-yl)chromen-4-one (−42.16 kcal/mol) and fluorescein (−41.42 kcal/mol) outperforming the P. aeruginosa reference ligand evo (−19.80 kcal/mol) and 80s (−48.74 kcal/mol) leading for S. aureus . Per-residue energy decomposition and quantum chemical analyses further elucidated key interactions and reactivity profiles. These results position A. alternata P02PL2 as a promising source of DNA gyrase inhibitors to combat antimicrobial resistance. Key points • A. alternata extracts inhibits S. aureus and Pseudomonas with MIC of 0.25-0.5 mg/mL • Inhibitors included Diketopiperazine (S. aureus), C-glycosyl flavone (Pseudomonas) • In silico analyses suggest the metabolites putatively inhibit bacterial DNA gyrase