Investigating the interaction between Organic Anion Transporter 1 and Ochratoxin A: an in silico structural study to depict early molecular events of substrate recruitment and the impact of single point mutations

Organic anion transporters (OATs) belong to a subgroup of the solute carrier 22 transporter family. OATs have a central role in xenobiotic disposition affecting the toxicokinetics of its substrates and inter-individual differences in their expression, activity and function impact both toxicokinetics and toxicodynamics. Amongst OATs, OAT1 (solute carrier family 22 member 6) is involved in the urinary excretion of many xenobiotics bringing substrates into renal proximal tubular cells which can then be secreted across the apical membrane into the tubule lumen. The mycotoxin ochratoxin A has been shown to have a high affinity for OAT1, which is an important renal transporter involved in its urinary excretion. Nowadays, molecular modeling techniques are widely applied to assess protein-ligand interactions and may provide a tool to depict the mechanistic of xenobiotic action be it toxicokinetics or toxicodynamics. This work provides a structured pipeline consisting of docking and molecular dynamic simulations to study OAT1-ligand interactions and the impact of OAT1 polymorphisms on such interactions. Such a computational structure-based analytical framework allowed to: i) model OAT1-substrate complex formation and depict the features correlating its sequence, structure and its capability to recruit substrates; and ii) investigate the impact of OAT1 missense mutations on substrate recruitment. Perspectives on applying such a structured pipeline to xenobiotic-metabolising enzymes are discussed

Assessing the hydrolytic fate of the masked mycotoxin zearalenone-14-glucoside–A warning light for the need to look at the “maskedome”

Masked mycotoxins are plant metabolites of mycotoxins that contaminate food and feed. They pose health concern as the shortage of toxicological data forces the lack of regulation worldwide. The present work investigated the toxicological relevance of the masked mycotoxin zearalenone-14-glucoside. In vitro, it shows a lower toxicity in respect to the parent compound. However, the major risks related to the consumption of masked mycotoxins depend on the possibility to undergo hydrolysis. Therefore, the hydrolysis and further transformation of zearalenone-14-glucoside in bovine blood and blood components (i.e. plasma, serum and serum albumin) were monitored using LC/MS-MS analysis to gain insights on the possible systemic fate. Hydrolysis was observed in all matrices, and both cell-dependent and eindependent contributions were pointed out. Moreover, further metabolism was observed in the whole blood as zearalenol isomers were found. Serum albumin was identified among the active components, and the protein-ligand interaction was investigated via computational analysis. The blood has been pointed out as possible district of reversion and further activation of zearalenone-14-glucoside, and a similar fate cannot be excluded for other masked mycotoxins. Therefore, the systemic hydrolysis should be evaluated beside the absorption, bioavailability and bioaccessibility to deeply understand the toxicity of masked mycotoxins

Preventing the Interaction between Coronaviruses Spike Protein and Angiotensin I Converting Enzyme 2: An In Silico Mechanistic Case Study on Emodin as a Potential Model Compound

Emodin, a widespread natural anthraquinone, has many biological activities including health-protective and adverse effects. Amongst beneficial effects, potential antiviral activity against coronavirus responsible for the severe acute respiratory syndrome outbreak in 2002–2003 has been described associated with the inhibition of the host cells target receptors recognition by the viral Spike protein. However, the inhibition mechanisms have not been fully characterized, hindering the rational use of emodin as a model compound to develop more effective analogues. This work investigates emodin interaction with the Spike protein to provide a mechanistic explanation of such inhibition. A 3D molecular modeling approach consisting of docking simulations, pharmacophoric analysis and molecular dynamics was used. The plausible mechanism is described as an interaction of emodin at the protein–protein interface which destabilizes the viral protein-target receptor complex. This analysis has been extended to the Spike protein of the coronavirus responsible for the current pandemic hypothesizing emodin’s functional conservation. This solid knowledge-based foothold provides a possible mechanistic rationale of the antiviral activity of emodin as a future basis for the potential development of efficient antiviral cognate compounds. Data gaps and future work on emodin-related adverse effects in parallel to its antiviral pharmacology are explored

In Silico approaches applied to the study of peptide analogs of ile-pro-ile in relation to their dipeptidyl peptidase IV inhibitory properties

peer-reviewedInhibition of dipeptidyl peptidase IV (DPP-IV) may be exploited to maintain the incretin effect during the postprandial phase. As a result, glycemic regulation and energy homeostasis may be improved. Food protein-derived peptides have been identified as natural agents capable of inhibiting DPP-IV. Ile-Pro-Ile is the most potent DPP-IV inhibitory peptide identified to date. A minimum analog peptide set approach was used to study peptide analogs of Ile-Pro-Ile. The DPP-IV half maximal inhibitory concentration (IC50) values of the 25 peptides evaluated ranged from 3.9 ± 1.0 μM (Ile-Pro-Ile) to 247.0 ± 32.7 μM (Phe-Pro-Phe). The presence of Pro at position 2 of tripeptides was required to achieve high DPP-IV inhibition. Most peptides behaved as competitive inhibitors of DPP-IV with the exception of peptides with a N-terminal Trp, which were mixed-type inhibitors. While possessing the structure of preferred DPP-IV substrates, most peptides studied were particularly stable during 30 min incubation with DPP-IV. Molecular docking revealed that Ile-Pro-Ile and its peptide analogs interacted in a very similar manner with the active site of DPP-IV. In addition, no correlation was found between the Hydropathic INTeraction score and the DPP-IV IC50 values of the peptides studied. This outcome suggests that free energy may not be directly responsible for enzyme inhibition by the peptides. Finally, novel DPP-IV inhibitory peptides were identified using the strategy employed herein. These results may be relevant for the development of food protein-derived peptides with serum glucose lowering and food intake regulatory properties in humans

A Computational Understanding of Inter-Individual Variability in CYP2D6 Activity to Investigate the Impact of Missense Mutations on Ochratoxin A Metabolism

Cytochrome P-450 (CYP) enzymes have a key role in the metabolism of xenobiotics of food origin, and their highly polymorphic nature concurs with the diverse inter-individual variability in the toxicokinetics (TK) and toxicodynamics (TD) of food chemicals. Ochratoxin A is a well-known mycotoxin which contaminates a large variety of food and is associated with food safety concerns. It is a minor substrate of CYP2D6, although the effects of CYP2D6 polymorphisms on its metabolism may be overlooked. Insights on this aspect would provide a useful mechanistic basis for a more science-based hazard assessment, particularly to integrate inter-individual differences in CYP2D6 metabolism. This work presents a molecular modelling approach for the analysis of mechanistic features with regard to the metabolic capacity of CYP2D6 variants to oxidise a number of substrates. The outcomes highlighted that a low-frequency CYP2D6 variant (CYP2D6*110) is likely to enhance ochratoxin A oxidation with possible consequences on TK and TD. It is therefore recommended to further analyse such TK and TD consequences. Generally speaking, we propose the identification of mechanistic features and parameters that could provide a semi-quantitative means to discriminate ligands based on the likelihood to undergo transformation by CYP2D6 variants. This would support the development of a fit-for-purpose pipeline which can be extended to a tool allowing for the bulk analysis of a large number of compounds. Such a tool would ultimately include inter-phenotypic differences of polymorphic xenobiotic-metabolising enzymes in the hazard assessment and risk characterisation of food chemicals

Alternaria toxins as casein kinase 2 inhibitors and possible consequences for estrogenicity: a hybrid in silico/in vitro study

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Persistence of the antagonistic effects of a natural mixture of Alternaria mycotoxins on the estrogen-like activity of human feces after anaerobic incubation

Several Alternaria mycotoxins are believed to act as endocrine disruptive chemicals (EDCs), since they are reported to bind estrogen receptors in several experimental models. After ingestion of contaminated food commodities, the mycotoxins reach the intestine, where they come into direct contact with food constituents as well as the gut microbiota. Thus, the aim of the present work was to evaluate the modulatory potential of a complex extract of cultured Alternaria fungi (CE; containing eleven chemically characterized compounds) on the estrogenic signaling cascade of mammalian cells before and after anaerobic incubation with fecal slurries, in order to simulate an in vivo-like condition in the gut. Assessing alkaline phosphatase expression in Ishikawa cells as a measure for estrogenicity, we found the CE to partially quench the intrinsic estrogenic properties of fecal slurries and fecal waters, even after 3 h of fecal incubation. Investigation of the mechanisms underlying the effects observed carried out through an in vitro/in silico approach revealed the ability of the extract to decrease the ERα/ERβ nuclear ratio, while a possible action of the mycotoxins as ER-antagonists was excluded. Our results suggest that Alternaria mycotoxins might act as EDCs in vivo, and warrant further investigation in animal models

Response of intestinal HT-29 cells to the trichothecene mycotoxin deoxynivalenol and its sulfated conjugates

Abstract The sulfated forms of the Fusarium toxin deoxynivalenol (DON), deoxynivalenol-3-sulfate (DON-3-Sulf) and deoxynivalenol-15-sulfate (DON-15-Sulf) were recently described, however little is known about their mechanism of action in mammalian cells. DON-3-Sulf and DON-15-Sulf were taken up by HT-29 colon carcinoma cells, although to a lesser extent compared to DON. All three compounds were found to enhance the intracellular ROS level in the dichlorofluorescein assay (≥ 1μM), even though substantial differences were observed in their cytotoxic potential. In silico modelling highlighted that DON-sulfates do not share the classical mechanism of action of DON, being unable to fit into the ribosomal pocket and trigger the classical ribotoxic stress response. However, DON-3-Sulf and DON-15-Sulf sustained a distinctive proliferative stimulus in HT-29 and activated autophagy. The mechanisms of action of DON-3-Sulf and DON-15-Sulf suggest a potential interplay between the onset of ribosomal inhibition and autophagy activation as an alternative and/or complementary mode of action for DON and its sulfated analogues