Luca Dellafiora

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.

Poly- and perfluoroalkyl substances (PFAS) are pollutants disrupting lipid metabolism and cell membrane integrity with unclear molecular mechanisms. Molecular docking and molecular dynamics (MD) simulations were used to investigate whether the perfluorinated analogue of palmitic acid (PFHxDA, parametrized through SwissParam) could enter membrane lipid metabolism and get embedded in cell membranes, possibly interfering with their properties. The possible role of PFHxDA as substrate for enzymes involved in the biosynthesis of membrane lipids was studied by investigating its interaction with two enzymes belonging to the phosphatidylcholine biosynthetic pathway, Acyl-CoA synthetase 5 and Choline Phosphotransferase 1, through 200 ns MD simulations. PFHxDA and metabolic derivatives displayed binding and dynamic behaviors comparable to native substrates, suggesting they might be either substrates or inhibitors. Concerning membranes, PFHxDA-containing lipids showed altered bilayer observables such as cholesterol packing, area per lipid, membrane thickness, and membrane density during 300 ns MD simulations, possibly affecting membrane fluidity and organization. Although theoretical, these results provide hypothesis-generating yet compelling mechanistic insights into PFAS-mediated disruption of lipid metabolism and membrane integrity, prioritizing specific experimental follow-up.

The health benefits of soy and soy derived foods are often attributed to their isoflavone (ISFs) content, known to include phytoestrogens. However, EFSA has concluded that there is insufficient scientific evidence to establish a causal relationship between ISFs consumption and the proposed health claims. Although phase-II metabolites of ISFs are often considered as detoxified products with reduced estrogenic effects, recent studies indicate that ISFs metabolites may contribute and even enhance the biological activity of ISFs. However, current knowledge on phase-II metabolites of ISFs is limited, even though representing the major ISFs metabolites circulating in the bloodstreams in humans. This study investigates the role of phase-II metabolism in the biological activity of ISFs, focusing on estrogenicity and cytotoxicity in estrogen-sensitive Ishikawa cells (expressing estrogen receptors (ER) α and β). ISFs concentrations from 0.001 to 10 µM, with 1:10 dilution steps, were tested. Cleavage to parent compounds during incubation was also evaluated using high-performance liquid chromatography tandem mass spectrometry. The results showed that phase-II metabolites, particularly sulfates, can still exhibit estrogenic activity, challenging the general assumption that phase-II metabolism always leads to the inactivation of ISFs. In particular, at 10 µM daidzein-4'-sulfate exhibited a comparable estrogenic activity to daidzein at the same concentration, and genistein-7-sulfate exhibited estrogenic potential at higher concentrations. Molecular dynamics simulations revealed that genistein and its conjugates can stably interact with ERβ. This study highlights the importance of phase-II metabolism for ISFs activity and suggests that the sulfates formed should be examined more closely in further investigations.

Serum albumin carries several ligands in the circulation. Previous studies demonstrated that certain flavonoid aglycones and their sulfate metabolites can influence the albumin binding of Site I ligand drugs. To get a deeper insight, the effects of chrysin, quercetin, luteolin, myricetin, and their sulfate derivatives on the albumin binding of warfarin (Site I) and biliverdin (heme site) were examined. Ultracentrifugation experiments were carried out using human serum albumin (HSA), bovine serum albumin (BSA), human serum, and fetal bovine serum (FBS). In addition, fluorescence spectroscopic and modeling studies (warfarin–HSA) as well as circular dichroism-based measurements (biliverdin–HSA) were also performed. Chrysin-7-O-sulfate and quercetin-3′-O-sulfate considerably displaced warfarin from HSA, while warfarin–BSA interaction was strongly disturbed by luteolin-3′-O-sulfate and quercetin-3′-O-sulfate. Quercetin-3′-O-sulfate, luteolin-3′-O-sulfate, and chrysin-7-O-sulfate caused the marked displacement of biliverdin from HSA. In contrast, quercetin-3′-O-sulfate and luteolin-3′-O-sulfate significantly increased the stability of biliverdin–BSA complex. Flavonoid sulfates did not affect the free fraction of warfarin and biliverdin in spiked human serum samples. However, in the presence of 10% FBS (as in cell culture media), high flavonoid levels elevated the free fraction of warfarin, while certain flavonoids considerably decreased the free fraction of biliverdin. Our results demonstrate the complex modulation of warfarin–albumin and biliverdin–albumin interactions by flavonoids. It likely does not influence the albumin binding of Site I and heme site ligands in the human circulation, but flavonoids can strongly affect ligand–BSA interactions in cell culture media.

Campylobacter jejuni is a widespread foodborne pathogen causing campylobacteriosis, a disease leading to diarrhea, fever, and gastroenteritis, able to adapt to many niches. Here, we present a hybrid in silico/in vitro study investigating the modulation of C. jejuni chorismate synthase by peptides. This enzyme belongs to the shikimate pathway, and it is an interesting target for selective growth modulation, being crucial for bacteria but not present in animals. To account for the identification of “natural” modulators, a library of 400 dipeptides is screened in silico through docking and molecular dynamics simulations to identify possible inhibiting sequences. The dipeptide glutamate‐aspartate (ED) stood out, emulating the pharmacophoric fingerprint and interaction of the enzyme's natural substrate. Serendipitously, in vitro trials revealed ED as an activity enhancer. Considering the growth of C. jejuni in protein‐rich matrices, this outlined a possibly relevant matrix‐dependent effect worthy of dedicated investigations. The underpinning mechanisms are computationally investigated, describing possible ED‐dependent effects on substrate/product turnover and enzyme structural stability. This study deepened the understanding of chorismate synthase and opened new directions in designing food‐grade peptide‐based modulators. This may provide ground to improve controlling bacterial growth in diverse contexts, including food safety and environmental/agricultural hygiene.