Metabolomics-Guided Discovery of Anticancer Metabolites from Marine Sponge-Associated Bacillus safensis: In Vitro and In Silico Evaluation

Marine-derived bacteria are valuable sources of novel anticancer agents, particularly those associated with sponges in tropical ecosystems. This study aimed to investigate the cytotoxic and metabolomic profile of Bacillus safensis P.039.5, isolated from marine sponge Petrosia nigricans collected near Pari Island, Indonesia. Cytotoxic screening against MCF-7 breast cancer cells revealed significant inhibition, with an IC50 value of 186.63 μg/mL and a high selectivity index compared to doxorubicin. Metabolite profiling using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS/MS) identified 28 putative compounds, dominated by peptides and alkaloids. Among these, militarinone A, maculosin, and verpacamide A showed the strongest predicted binding affinities in molecular docking studies against five breast cancer-related targets: ER-α, HER2, EGFR, CDK2, and topoisomerase IIα. Militarinone A exhibited the highest binding affinity (-11.11 kcal/mol), exceeding native ligands and reference drugs. To our knowledge, this is the first report of militarinone A detection in B. safensis P.039.5, demonstrating the utility of a metabolomics-guided approach to prioritize bioactive compounds from underexplored marine bacteria. These findings highlight the pharmacological potential of B. safensis as a sustainable source of multi-target anticancer leads.