Avaliação de novos compostos sobre a atividade de colinesterases em modelos in silico e in vitro

Abstract

The enzyme acetylcholinesterase (EC 3.1.1.7, AChE) is responsible to terminate acetylcholine activity in the terminal nervous junctions with its effector organs or post-synaptic sites. The activity of this enzyme could be inhibited by organophosphorus (OP) compounds, and this inactivation leads to an accumulation of acetylcholine in the cholinergic receptors, leading to a cholinergic crisis that may result in death. In this way, the OP compound methamidophos has been related to its broad use in various agriculture cultures in Brazil, with high intoxication rate. Actually, the only compounds able to revert the AChE inhibition by OP, are the oxime, such compounds may reactive the enzyme activity due its high nuclephilic power, attacking the phosphoryl group of the inhibited enzyme and displacing it. However, such compounds show toxic effects, and its use is limited by the high specificity, with each oxime acting only in the reactivation of AChE induced by specific OP coumponds. These limitations raise the need of development of new compounds with AChE reactivator potency, with minor side effects. In this way, have been utilized a series of computational tools (in silico models), with the aim of understand the interaction occurring at a molecular level and, so, rationalize the new compounds development. Thus, the aim of this thesis is to evaluate the activity of three new compounds in reactivate the AChE inhibited by methamidophos, in comparison with two others oximes used in clinical (obidoxime and pralidoxime), both in in silico and in vitro models. Our work demonstrate that the newly synthesized compounds are able to reactivate human erythrocyte AChE, however less efficiently than pralidoxime and obidoxime, and reactivate human plasma butyrylcholinesterase (BChE), where the classical oximes failed. We also show that pralidoxime, which obtained the best reactivation constant among all tested compounds, attack the phosphorus-oxygen moiety (formed between the methamidophos and the AChE catalytic triad residue Ser203) via a region known as oxyanion-hole , composed by the residues Gly120, Gly121 and Ala204. Such found may help in the development of new compounds with better reactivatory activity on AChE inhibited by OP compounds. Furthermore, we show for the first time the individual contribution of each amino acid of AChE, in a radii of 14 Å from the ligand, to the oxime bonding to its active site, using quantum chemistry methods. Here, we demonstrate the important of a quaternary nitrogen to the stabilization of the oximes into the active site; as well as, we obtained evidences that the active form of oximes should be the unprotonated one, instead the protonated, which has been target of debate in the scientific society. Particularly important, we show the critical contribution of amino acids that lies distant from the ligand to the adopted conformation and stabilization of the compounds into the active site of AChE, which has been neglected until far. Finally, our study also evaluates the toxic effects of the compound isatin-3-N4-benzilthiosemicarbazone (IBTC) in mice, which presented low toxicity, with median lethal dose superior at 500 mg/kg. Concluding, this study contributes significantly to the development of new drugs able to restore the AChE activity with minor toxic effects.