In vitro spectrophotometric method for determination of various mycotoxins and their in Silico Assessment Based on Сholinesterase Inhibitions through the Molecular Docking Approach

Objective: This article discusses approaches to the in vitro spectrophotometric determination of various mycotoxins and their in silico assessment using the molecular docking method based on cholinesterase inhibition effects. Methods: The following tools were used in this study: the Ellman spectrophotometric method for assessing the formation of thiocholine, a product of enzymatic hydrolysis of acetylthiocholine and butyrylthiocholine in the presence of enzymes, respectively; the ChemBio3D Ultra software package; the AutoDock Vina 1.1.2 docking softvare; and the PyMol visualization tool. Results: The type and degree of enzyme inhibition for mycotoxin zearalenone (ZEN), depending on incubation time and Acetylcholinesterase (AchE) enzyme concentration, was determined, showing the competitive type of inhibition. The inhibition constant is 15±4μmol/l. Km, Vmax/E0 and Vmax/(E0·Km) were established for the enzymatic hydrolysis of acetylthiocholine and butyrylthiocholine by AchE and Butyrylcholinesterase (BchE) enzymes, respectively. For AchE: Km=98±23µmol/l, Vmax/E0=316±26min-1, Vmax/(E0·Km)=3.22±1.02 l/µmol•min. For BchE: Km=319±35µmol/l, Vmax/E0=8.3±0.2 min-1, Vmax/(E0`Km)=0.027±0.003 l/µmol•min. For ZEN, the concentration range is 0.5-50 mg/l, sterigmatocystin (STC) - 0.25-0.8 mg/l, deoxynivalenol (DON) - 1.25-25 mg/l, ochratoxin A (OTA) - 2.5-10 mg/l. Molecular docking analysis of four mycotoxins, specific substrates, and co-crystallized ligand inhibitors was performed, affinities (binding energies, kcal/mol) for those compounds were found. Conclusion:. A method for determining mycotoxins using biosensors based on AchE and BchE was developed. The corresponding calibration graphs and meaningful inhibitory effects of four mycotoxins on AchE and BchE enzyme activity were constructed.