Surface screening, molecular modeling and in vitro studies on the interactions of aflatoxin M1 and human enzymes acetyl- and butyrylcholinesterase
Introduction
Aflatoxin M1 (AFM1) (Fig. 1) is a secondary metabolite of fungi Aspergillus flavus and A. parasiticus [1]. It can be found mainly in contaminated milk and dairy products, being a great threat to humans, especially to children due to the high consumption of milk and its derivatives. AFM1 (Fig. 1) is also produced as the major metabolite of aflatoxin B1 (AFB1) by hydroxylation, and like AFB1, it is highly toxic and carcinogenic to human and other mammal species [2]. It affects preferentially the liver, but can also attack lungs, kidney and gastro enteric tissues [3]. Every condition that favors the growing of these fungi species also favors the contamination by AFB1 [4] and, in consequence, by AFM1. Therefore, it is important to eliminate or reduce the sources of contamination, mainly during storage, such as high temperature and humidity. When cows ingest some feed contaminated by AFB1, it is metabolized in their livers by cytochrome P450 into the monohydroxy analogue AFM1 [5]. In consequence their milk and dairy products will carry the contamination.
In recent studies with infant feed products conducted in India [6], 100% of the baby feed analyzed presented amounts of AFM1 higher than the level allowed by the European Union, that is 25 ng kg−1 or 50 ng L−1 in milk [7]. Other studies performed in Portugal in 2018 pointed to the importance of measuring the occurrence of AFM1 in breast milk [8]. It means that not only commercial children feed but also maternal milk can also transmit AFM1 to children. It was observed that 32.8% of the tested samples were contaminated by AFM1 in levels exceeding the allowed limits. The contamination was especially associated to the consumption of rice and chocolate by the mothers [8].
AFB1 is a strong inhibitor of the enzyme acetylcholinesterase (AChE) [9]. Previous studies [10,11] have shown that its metabolites are also potential AChE inhibitors and, consequently, potential neurotoxic agents. These studies suggested that the peripheral anionic site (PAS) (residues Tyr72, Trp286 and Arg296) and the catalytic anionic site (CAS) (residues Asp74, Trp86, Tyr337, and Tyr341) are the preferential binding sites for its metabolites, including AFM1, having the affinity for the CAS higher than for the PAS. Literature reports that AFB1 is not able to inhibit the enzyme butyrylcholinesterase (BChE) [12], but no studies were performed about its metabolites yet. In this work, a surface screening (SS) study of AFM1 on human AChE (HssAChE) was performed to corroborate previous results [10,11] and also an inedited SS study of AFM1 on human BChE (HssBChE) was done to verify if AFM1 is able to inhibit HssBChE. In addition, inedited in vitro inhibition assays of AFM1, with HssAChE and HssBChE, were performed to corroborate the theoretical results.
Section snippets
Surface screening and binding energy calculations
The crystallographic structures used in this study were 3LII, for HssAChE, and 6ESJ, for HssBChE, selected among others [13,14] available in the Protein Data Bank [15]. HssAChE crystal was defective and thus a model was constructed to complete missing residues, using Swiss-Model Server [16]. It was validated using the server PDBsum [17], from where Ramachandran plot and other validation parameters were obtained and analyzed.
The tridimensional structure of AFM1 was built using the software PC
HssAChE/AFM1
Fig. 2 shows the clusters of docking solutions (poses) obtained from the SS studies of AFM1 over HssAChE. Most of the poses were located in the gorge of the enzyme, close to the PAS and CAS. The remaining poses spread around the enzyme, in sites not related to the catalytic function of HssAChE. These results corroborate our previous studies [10,11] pointing to the PAS and CAS as the preferential binding sites for AFB1 metabolites, suggesting that AFM1 comes closer to the active site, presenting
Conclusion
Previous studies [10,11] showed that AFB1 metabolites were potential inhibitors of HssAChE, such as AFB1, being the PAS and CAS the preferential binding sites, with preference for the CAS over the PAS. In this work, AFM1 was submitted to SS on HssAChE to corroborate previous data. An inedited SS study of AFM1 in HssBChE was also performed to verify if AFM1 present the same behavior of AFB1, which is not able to inhibit HssBChE. In addition, inedited in vitro inhibition assays for AFM1, HssAChE
Conflicts of interest
All the authors declare that there are no conflicts of interest related to the publishing of this manuscript on CBI as stated in the signed copies of the Chemico-Biological interactions.
Conflict of Interest policy attached to this submission.
Acknowledgments
The authors wish to thank the Military Institute of Engineering, University of Hradec Kralove and University of Defense for the infrastructure; the Brazilian financial agencies CNPq (no 308225/2018-0), and FAPERJ (no E-02/202.961/2017) the Czech Science Foundation (no 18-01734S) the University of Hradec Kralove (Faculty of Science, VT2019-2021) and the OPCW Research Project Support Programme (L/ICA/ICB/201062/15), for financial support. This work was also supported by the Ministry of Defence of
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