Analysis of the binding of mycotoxins to proteins involved in ASD with a combined computational/experimental approach

Autism spectrum disorder (ASD) is a group of neurodevelopmental disabilities characterized by persistent deficits that manifestwith impaired social communication and social interaction, restricted and repetitive patterns of behavior, interests or activities [1]. The etiology of ASDis unknown, but it is believed that it involves genetic and environmental components. The purpose of this work is to assess the possible involvement of food contaminants, such as mycotoxins, in the etiology of ASD. The hypothesis is that the mycotoxins ingested with the diet could bind to proteins and expose the entire organism,including CNS, to the negative effects of xenobiotics, in genetically predisposed patients. In this study some possible protein targets for the mycotoxinswere identified to evaluate if the bond between any protein target and the mycotoxin in exam could play a role in ASD. Twelve mycotoxins were selected (ochratoxin A, gliotoxin, aflatoxin B1, aflatoxin B2, aflatoxin M1, aflatoxin M2, aflatoxicol, a-zearalanol, b-zeralanol, zearalenone, deoxynivalenol, patulin),which are contaminants of milk and cereals. For each of these molecules,possible protein targets were searched by a reverse docking approach using the idTargetserver[2].From the results given by idTarget, human protein targets expressed in the brain or involved in brain diseaseswere selected. Subsequently, a direct docking was made using Auto- Dock 4.2 [3], in orderto verify the strength of the interaction between selected proteins and each mycotoxin, and to identify the mycotoxins' binding site on each of the selected protein. Finally, the bond of some mycotoxins to selected protein targets has been experimentally tested. For each mycotoxin, idTarget returned thousands of possible protein targets,and only those with the best binding energy were selected and evaluated. Among them, human protein targets that are expressed in the brain or that are involved in cerebral diseases,have been selected; moreover the protein targets that were not human but that idTargetselected for five or more mycotoxins, were replaced with their human counterparts. At the end of the procedure, nineteen protein targets have been identified for the following direct docking approach. From the docking results, eight proteins have been selected for experimental tests, having a predicted binding energy lower than 27 kcal/mol. Finally, the interactions between Acetylcholinesterase (AChE), b-secretase (BACE1) and Neuroligin-4, X-linked (NLG4X) with Aflatoxin B1, Aflatoxin B2, Gliotoxin, Ochratoxin A and Deoxynivalenol, were evaluatedusing fluorescence spectroscopy and microscale thermophoresis. These experiments confirmed the presence of an interaction between BACE1 and Aflatoxin B1; NLG4X and Aflatoxin B1,Gliotoxin and Ochratoxin A; and Deoxynivalenol,AChE and Aflatoxin B1. These results suggest that the interaction between mycotoxins and proteins involved in neuronal plasticity is possible also in vivo, supporting the hypothesis of a putative role of mycotoxins in the etiology of ASD.