Organophosphorus nerve agents (OPs) are an extremely toxic class of compounds that have found application as pesticides and, for the more toxic varieties, chemical warfare agents. The OPs toxicity is brought about by their ability to inhibit activity of the serine hydrolase acetylcholinesterase (AChE), leading to a buildup of the neurotransmitter acetylcholine and causing uncontrollable muscle contractions and, in cases of severe exposure, death by respiratory failure. Acetylcholinesterase has been the ongoing focus of academic and industrial interest due not only to its inhibition by OPs, but also because of its rapid catalysis rates in spite of its buried active site; moreover, inhibition of acetylcholine catalysis has been exploited as a means to treat the symptoms of Alzheimer’s disease.
In this thesis, the reaction mechanisms for hydrolysis of organophosphorus compounds are modeled using computational methods at a variety of levels, ranging from aqueous phosphate ester hydrolysis via ab initio calculations on small model systems, to enzymatic phosphate hydrolysis mechanisms in the active site of acetylcholinesterase using quantum mechanical/molecular mechanical (QM/MM) methods. Using QM/MM simulations, the inhibition mechanism of AChE by OPs was determined, and important residues leading to stereospecificity were discerned. As the interactions between a ligand and its receptor are dynamic by nature, a combination of molecular dynamics and molecular docking simulations were employed in order to study conformational dynamics in the enzyme as well as binding distributions of the OPs in the AChE gorge. In addition to the chemical nerve agents, the interactions of OP analogues, used for experimental testing due to their decreased toxicity when compared to their parent nerve agents, were evaluated. The role of substituents as well as the chirality at phosphorus were explored for various OPs.
Having presented the enzyme inhibition mechanism and the interactions controlling stereospecificity in the AChE catalytic site, we have applied this knowledge to the design of more effective therapeutics to nerve agent exposure. In this thesis, two different classes of therapeutics are described: pyridinium oximes for the reactivation of inhibited AChE, and alkylating agents for the reversal of enzymatic aging following inhibition by OPs.
Pyridinium oximes present a unique challenge for therapeutic design, as they exhibit varied potency with regard to structurally similar OPs. Experimental studies have illustrated that small structural perturbations can have a drastic impact on pyridinium oxime potency, even though the oxime functional group remains constant throughout the series of therapeutics. Even more daunting is the observation that crystallographic structures of the oxime-bound inhibited AChE suggest that the different oximes bind in a nearly identical manner, in spite of their activity differences. Molecular dynamics and docking simulations suggest that the distribution of oximes bound to the residue Y341, and their dynamics at that site, can be correlated to observed oxime potency. These results suggest that oxime dynamics in the gorge, when coupled with conformational dynamics of the O-alkyl side chain of the OP bound in AChE are the likely origin of reactivity differences.
The design of an effective alkylating agent for aged acetylcholinesterase was attempted several decades ago but was unsuccessful, in part due to the lack of structural information on the acetylcholinesterase enzyme. We have investigated the interactions of a previous set of potential alkylating agents with the aged enzyme, and propose a new class of compounds for the alkylation of aged AChE known as quinone methide precursors (QMPs). Computational modeling was carried out using ab initio methods to evaluate the alkylation mechanism of the QMPs. These modeling efforts suggest that the reactivity of QMPs can be modified significantly by varying substituents on the precursor compound. We have also investigated the binding and dynamics of QMPs in the gorge of AChE. The QMPs also exhibit favorable and stable binding in the catalytic site of aged AChE, maintaining a reactive orientation between the alkylating group and the aged OP, suggesting that they would serve as a suitable template for improved alkylating agent design.