Optimization of Cholinesterase-Based Catalytic Bioscavengers Against Organophosphorus Agents

Abstract

Organophosphorus agents (OPs) are irreversible inhibitors of acetylcholinesterase (AChE). OP poisoning causes major cholinergic syndrome. Current medical counter-measures mitigate the acute effects but have limited action against OP-induced brain damage. Bioscavengers are appealing alternative therapeutic approach because they neutralize OPs in bloodstream before they reach physiological targets. First generation bioscavengers are stoichiometric bioscavengers. However, stoichiometric neutralization requires administration of huge doses of enzyme. Second generation bioscavengers are catalytic bioscavengers capable of detoxifying OPs with a turnover. High bimolecular rate constants (k_cat/K_m > 10⁶ M^-1min^-1) are required, so that low enzyme doses can be administered. Cholinesterases (ChE) are attractive candidates because OPs are hemi-substrates. Moderate OP hydrolase (OPase) activity has been observed for certain natural ChEs and for G117H-based human BChE mutants made by site-directed mutagenesis. However, before mutated ChEs can become operational catalytic bioscavengers their dephosphylation rate constant must be increased by several orders of magnitude. New strategies for converting ChEs into fast OPase are based either on combinational approaches or on computer redesign of enzyme. The keystone for rational conversion of ChEs into OPases is to understand the reaction mechanisms with OPs. In the present work we propose that efficient OP hydrolysis can be achieved by re-designing the configuration of enzyme active center residues and by creating specific routes for attack of water molecules and proton transfer. Four directions for nucleophilic attack of water on phosphorus atom were defined. Changes must lead to a novel enzyme, wherein OP hydrolysis wins over competing aging reactions. Kinetic, crystallographic, and computational data have been accumulated that describe mechanisms of reactions involving ChEs. From these studies, it appears that introducing new groups that create a stable H-bonded network susceptible to activate and orient water molecule, stabilize transition states (TS), and intermediates may determine whether dephosphylation is favored over aging. Mutations on key residues (L286, F329, F398) were considered. QM/MM calculations suggest that mutation L286H combined to other mutations favors water attack from apical position. However, the aging reaction is competing. Axial direction of water attack is not favorable to aging. QM/MM calculation shows that F329H+F398H-based multiple mutants display favorable energy barrier for fast reactivation without aging.

Keywords: acetylcholinesterase; bioscavenger; butyrylcholinesterase; computer design; organophosphorus compound; phosphotriesterase.

Scheme 1

Inhibition of ChEs by Ops.

Figure 1

X-ray structure of phosphorylated G117H active site [PDB ID 2XMD (Nachon et al., 2011)]. Carbon atoms of the mutated residue, H117, are shown in cyan, atoms of echothiophate residue conjugated to catalytic serine 198, and crystallographic water closest to the phosphorus atom are shown as balls. Yellow dashes show critical hydrogen bonds in the active site.

Figure 2

Structure of diethylphosphorylated wild-type BChE PDB ID 1XLW (Nachon et al., 2005), positions for introduction of alternative nucleophilic centers near the phosphorylated catalytic serine are shown in pink.

Figure 3

Possible directions for nucleophilic attack of a water molecule on the phosphorus atom of diethylphosphorylated serine with corresponding configurations of the resulting PI. 2D schemes indicate vertices of trigonal bipyramids. Atoms of the activating histidines and water molecules are colored according to the direction of the attack: direction A, magenta; direction B, cyan, direction C, orange.

Figure 4

Energy diagrams for several selected mutants. Energy values are provided in Table 2. The pink, light blue, and orange colors correspond to different directions of nucleophilic attack depicted in Table 2 and Figure 3.

Figure 5

Enzyme-inhibitor conjugate (A) and PI (B) for the L286H/P285A/F329E/F357S mutant. Carbon atoms of the mutated resides are shown in blue. P285A mutation is not shown.

Figure 6

Structures for the proposed F329H/Y332E/D70Q/F398H mutant at various points in the reactivation pathway. (A) Wild-type BChE, residues to be mutated are shown in yellow; (B) conjugate between echothiopate (Echo) and catalytic serine of F329H/Y332E/D70Q/F398H mutant, mutated residues are colored in cyan; (C) first transition state, i.e., nucleophilic attack of the water molecule on the phosphorus atom and transfer of a proton from the water molecule to H329 (violet dashes); (D) pentacoordinate intermediate stabilized by hydrogen bonds with H398 and Q70-E332-H329 system; (E) second transition state, i.e., transfer of a proton from the catalytic H438 to oxygen of the catalytic S198 (violet dashes) accompanied by transfer of a proton from the hydroxyl group of the PI to H398. (F) Products of the reactivation reaction, i.e., diethylphosphate and free mutated BChE with the regenerated operative catalytic triad.