Target fishing in the "kinome": integrated in silico/in vitro discovery of novel kinase inhibitory activities of alternariol.

Alternariol (AOH), an emerging mycotoxin produced by Alternaria species, is a frequent contaminant of plant-derived foods and a major contributor to chronic dietary exposure to Alternaria toxins. Despite evidence of its cytotoxic, genotoxic, endocrine-disrupting, and immunomodulatory effects, its molecular mechanisms of toxicity remain incompletely understood, limiting mechanistically informed risk assessment. As evidence suggests that AOH acts as a multi-kinase inhibitor, this study aimed to expand its kinase target spectrum through integrated computational and experimental approaches. Ligand-based screening of structurally characterized kinase-ligand complexes, followed by molecular docking, molecular dynamics simulations, and free-energy calculations, identified phosphoinositide 3-kinase (PI3K) as a promising target. Functional validation was performed in Caco-2 and HT-29 human intestinal cells exposed to AOH (1-20 μM) for up to 5 h. PI3K activity was assessed by quantifying phosphorylation of its downstream effector Akt by Western blot, while AOH metabolism was characterized by liquid chromatography-tandem mass spectrometry. AOH induced a concentration-dependent reduction in phosphorylated Akt in Caco-2 cells at concentrations ≥ 10 μM without affecting total Akt, whereas no inhibition was observed in HT-29 cells. Immunofluorescence microscopy revealed decreased global protein phosphorylation in Caco-2 cells but increased phosphorylation in HT-29 cells. These contrasting responses were associated with differences in phase II metabolism, with extensive glucuronidation and sulfation occurring only in HT-29 cells. Computational analyses indicated that these metabolites exhibit reduced fitting into the ATP-binding pocket of PI3K. Overall, the findings identify PI3K as a novel molecular target of AOH and demonstrate that cellular metabolism critically determines its kinase-inhibitory activity.