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Phomoxanthone A, a bioactive xanthone dimer isolated from the endophytic fungus Phomopsis sp., is a mitochondrial toxin weakening cellular respiration and electron transport chain activity by a fast breakup of the mitochondrial assembly. Here, a multi-disciplinary strategy has been developed and applied for identifying phomoxanthone A target(s) to fully address its mechanism of action, based on drug affinity response target stability and targeted limited proteolysis. Both approaches point to the identification of carbamoyl-phosphate synthase 1 as a major phomoxanthone A target in mitochondria cell lysates, giving also detailed insights into the ligand/target interaction sites by molecular docking and assessing an interesting phomoxanthone A stimulating activity on carbamoyl-phosphate synthase 1. Thus, phomoxanthone A can be regarded as an inspiring molecule for the development of new leads in counteracting hyperammonemia states.

Carbamoyl-Phosphate Synthase 1 as a Novel Target of Phomoxanthone A, a Bioactive Fungal Metabolite