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 mu M) 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 mu M 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.
Target fishing in the “kinome”: integrated in silico/in vitro discovery of novel kinase inhibitory activities of alternariol / Crudo, F., Pedroni, L., Perugino, F., Call, F., Borsos, E., Rieger, J., Del Favero, G., Marko, D., Dall'Asta, C., Dellafiora, L.. - In: ARCHIVES OF TOXICOLOGY. - ISSN 0340-5761. - (2026). [10.1007/s00204-026-04608-4]
Target fishing in the “kinome”: integrated in silico/in vitro discovery of novel kinase inhibitory activities of alternariol
Crudo F.;Pedroni L.;Marko D.;Dall'Asta C.;Dellafiora L.
2026-01-01
Abstract
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 mu M) 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 mu M 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
