The Reactivation Of Organophosphorus Inhibited Acetylcholinesterase

Cholinesterases (ChEs) are the most important physiological targets in human organophosphorus (OP) poisoning, especially for acetylcholinesterase (AChE). AChE could be irreversibly inhibited by OP compounds, resulting in disorder in nervous system and even fatality in serious situation. The oxime is the only approved antidote for OP poisoning in clinics. However, the detoxification efficiency is low and it cannot be used as a general antidote for various OPs. The development of more efficient oxime antidote is therefore of high importance, and the revealing of fundamental detoxification reaction mechanism is necessary, in which the accurate understanding of binding mode of oxime in AChE is crucial. In this study, we focus on the attaining and analyzing of the binding modes in two aspects:1). the protonation state determination of oxime in AChE; 2). improving the reliability of Autodock in binding mode prediction. In addition, the OP-AChE adduct might undergo an aging process in which the enzyme can no longer be reactivated by oxime, resulting in permanent inactivity of the enzyme. However, if the aging reaction could be reversed or inhibited, the phosphorylated enzyme still has the chance to be reactivated by oximes, which will significantly increase the detoxification efficiency. Therefore, we have also explored the possibilities of the reversing of aged OP-AChE adducts.In Chapter One, we have established a method for protonation state determination of ligand in complex with protein, aiming at determining the protonation state of HI6 binding with AChE. This method requires accurate pKa value of ligand in aqueous solution, which may be achieved by the calculations employing solvation models. To evaluate the performance of various solvation models, we have calculated the pKa values of 13 rigid compounds in aqueous solution by using three different solvation models, i.e., IEFPCM, SMD, and SMVLE. The results from SMD and SMVLE are in general better than those from IEFPCM, and SMVLE performed slightly better than SMD, suggesting that the short-range interactions between solute and solvent is very important for accurate evaluation of solvation effect. There is a controversy on solvation calculation, in which a highly cited paper claimed that the correction to Gibbs free energy should be obtained from gas phase calculation, instead of from aqueous phase calculation. We disagree with this and believe that the correction to Gibbs free energy obtained from aqueous phase calculation is theoretically correct way for solvation calculations. In addition, we found the geometry optimization in aqueous solution is important even for small rigid molecules. Afterwards, a new method that reliably determine the protonation state of ligand in complex with protein through a thermodynamic cycle was developed. The method was validated by a case study on guanine binding with purine nucleoside phosphorylase (PNP). Finally, we have used the new method to determine the protonation state of HI6 in complex with AChE. To further verify our protonation state determination, we have incorporated the computationally more expensive thermodynamic integration (TI) method and have achieved consistent results on protonation state determination.In Chapter Two, we have developed a reliable method for predicting the binding mode of oxime in complex with AChE. Water molecules having strong affinity to protein play important role in the docking process, and therefore, in prior to the prediction of the binding modes, we have developed a method to determine the key waters which having high affinity to the protein pocket. The molecular docking was used to sample possible locations of key water molecules in the pocket, and the key waters were selected by using criteria composing of two energy terms. Our results showed that more than 50% of key water molecules are overlapped with those resolved in crystal structures. The remaining key waters which do not overlap with any resolved crystal waters are still having good interaction with protein. On the basis of such key-water prediction method, a new scoring function employing MM/PBSA was developed to include 1). the effects of oxime's conformational difference upon binding; 2). the replacement of key waters by oxime upon binding. In comparison to those by default scoring function implemented in Autodock, our new scoring function significantly improved the accuracy of binding mode prediction of HI6, obidoxime and HLo7 in complex with AChE.In Chapter Three, we have explored the possibilities of reversing the aging process of OP-AChE. The methyl methane-phosphonate monoanion was reported in literature to be methylated by N-methyl-2-methoxy pyridinium species, which mimics the reverse of the aging process of sarin-AChE. Our calculated results support the SN2 reaction mechanism for these methylation reactions, and the calculated free energy barriers are in good agreement with the experimental data. Afterwards, we have performed Quantum Mechanical/Molecular Mechanical (QM/MM) calculations to reveal the fundamental reaction mechanism for the methylation of the aged sarin-AChE adduct by N-methyl-2-methoxy pyridinium compound (compound 2). Our calculations showed that the reverse of the aged sarin-AChE employs the SN2 reaction mechanism with an extremely high averaged free energy barrier of 30.4±3.5 (or 26.6) kcal/mol, implying that this reaction in enzyme is hardly to occur. By carefully investigating the details of both the enzymatic and nonenzymatic reactions, it turns out that the stable ?-? stacking interaction between compound 2 and the aromatic ring of W86 residue is most likely responsible for the hindrance of the reaction in enzyme, which cause compound 2 to be unable to approach sarin close enough forming a more energy-favorable transition state. Based on these findings, several possible strategies have been suggested for designing methylating agents with higher activity against the aged sarin-AChE adduct.