In silico Evaluation of Metabolites Identified by LC-HRMS from the Ethyl Acetate Extract of Pometia pinnata Peel as Human Thymidylate Synthase Inhibitors

Cancer remains a major global health challenge, highlighting the need for new anticancer agents with improved selectivity and safety. Human thymidylate synthase (hTS) is a key enzyme in DNA biosynthesis and an established molecular target in cancer therapy. This study aimed to characterize metabolites present in the ethyl acetate extract (EAE) of Pometia pinnata fruit peels and explore their structural compatibility with the hTS active site using an integrated liquid chromatography-high-resolution mass spectrometry (LC–HRMS) and in silico approach. Ethyl acetate extract of P. pinnata peels underwent phytochemical analysis, metabolite profiling, ADMET prediction, and molecular docking to explore interactions with the hTS active site. Untargeted LC–HRMS analysis followed by annotation refinement resulted in the putative identification of 18 metabolites, predominantly fatty acid derivatives, fatty acid amides, and selected phenolic and aromatic compounds (protocatechuic acid, vanillin, fraxetin, pinostrobin, and 4-indolecarbaldehyde). Early-stage in silico evaluation indicated that most metabolites exhibited physicochemical and ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) profiles generally consistent with oral exposure. Molecular docking was employed as a hypothesis-generating tool to examine plausible binding orientations within the hTS active site. Several metabolites showed spatial overlap and interaction patterns comparable to the reference ligand Tomudex. Among them, pinostrobin displayed the most favourable docking score (-8.8 kcal/mol), approaching that of Tomudex (-9.0 kcal/mol). However, docking scores were used only for relative comparison within the same protocol. The study’s findings provide a chemically consistent metabolite profile of P. pinnata fruit peel and support the prioritization of selected metabolites for further experimental validation.