Metabolomics profiling of Calocybe indica for identification of potential ligands that bind to bacterial DNA Gyrase B.

The increasing prevalence of antimicrobial resistance has intensified the search for novel antibacterial agents from natural sources. In the present study, the antibacterial potential of Calocybe indica KUFC144 was investigated through metabolite profiling and computational analysis. Tissue culture was established from fruiting bodies and molecularly identified based on ITS and 28 S rRNA gene sequences, confirming its identity as C. indica. The methanolic mycelial extract exhibited antibacterial activity against both Bacillus subtilis and Klebsiella pneumoniae, with inhibition zones of 10.33 ± 0.43 mm and 13.78 ± 0.07 mm, respectively. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) further demonstrated greater antibacterial efficacy against K. pneumoniae. Liquid chromatography tandem mass spectrometry analysis identified 33 metabolites belonging to diverse chemical classes, including phenylethanoid glycosides, flavonoids, limonoids, thiophene derivatives, lipids and organic acids. Based on identification confidence, relative abundance, structural diversity, and reported antimicrobial or pharmacological activities, six metabolites: forsythiaside, leonoside A, myricoside, salannin, (10)-gingerol and 5-(4-hydroxybut-1-ynyl)-2,2'-bithiophene, were prioritized for molecular docking against the ATPase domain of Escherichia coli DNA gyrase B (PDB ID: 1EI1). Among the tested ligands, forsythiaside exhibited the highest Vina score (predicted binding affinity of - 9.7 kcal/mol), followed by leonoside A (- 9.0 kcal/mol) and myricoside (- 8.7 kcal/mol), with extensive hydrogen-bonding and hydrophobic interactions within the active site. STRING analysis further placed GyrB within a functionally interconnected network associated with DNA topology, replication, repair and transcription. ADME profiling revealed distinct pharmacokinetic characteristics among the metabolites, with (10)-gingerol and 5-(4-hydroxybut-1-ynyl)-2,2'-bithiophene showing comparatively favourable predicted gastrointestinal absorption and drug-likeness. In contrast, the highly polar glycosidic metabolites exhibited low predicted gastrointestinal absorption. Normal mode analysis indicated favourable structural stability and flexibility of the protein-ligand complexes. Collectively, these findings demonstrate that C. indica KUFC144 is a rich source of antibacterial metabolites and highlight forsythiaside as a promising GyrB-targeting lead based on the Vina score (predicted binding affinity). At the same time, the ADME analysis provides additional information for prioritizing the identified metabolites. The study provides a foundation for future biochemical validation and the development of mushroom-derived antibacterial agents.