Human acetylcholinesterase (AChE) is a significant target for therapeutic drugs. Here we present high resolution crystal structures of human AChE, alone and in complexes with drug ligands; donepezil, an Alzheimer's disease drug, binds differently to human AChE than it does to Torpedo AChE. These crystals of human AChE provide a more accurate platform for further drug development than previously available.

Over 50 years ago, the toxicity of irreversible organophosphate inhibitors targeting human acetylcholinesterase (hAChE) was observed to be stereospecific. The therapeutic reversal of hAChE inhibition by reactivators has also been shown to depend on the stereochemistry of the inhibitor. To gain clarity on the mechanism of stereospecific inhibition, the X-ray crystallographic structures of hAChE inhibited by a racemic mixture of VX (P R/S ) and its enantiomers were obtained. Beyond identifying hAChE structural features that lend themselves to stereospecific inhibition, structures of the reactivator HI-6 bound to hAChE inhibited by VX enantiomers of varying toxicity, or in its uninhibited state, were obtained. Comparison of hAChE in these pre-reactivation and post-reactivation states along with enzymatic data reveals the potential influence of unproductive reactivator poses on the efficacy of these types of therapeutics. The recognition of structural features related to hAChE’s stereospecificity toward VX shed light on the molecular influences of toxicity and their effect on reactivators. In addition to providing a better understanding of the innate issues with current reactivators, an avenue for improvement of reactivators is envisioned.

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The recent use of organophosphate nerve agents in Syria, Malaysia, Russia, and the United Kingdom has reinforced the potential threat of their intentional release. These agents act through their ability to inhibit human acetylcholinesterase (hAChE; E.C. 3.1.1.7), an enzyme vital for survival. The toxicity of hAChE inhibition via G-series nerve agents has been demonstrated to vary widely depending on the G-agent used. To gain insight into this issue, the structures of hAChE inhibited by tabun, sarin, cyclosarin, soman, and GP were obtained along with the inhibition kinetics for these agents. Through this information, the role of hAChE active site plasticity in agent selectivity is revealed. With reports indicating that the efficacy of reactivators can vary based on the nerve agent inhibiting hAChE, human recombinatorially expressed hAChE was utilized to define these variations for HI-6 among various G-agents. To identify the structural underpinnings of this phenomenon, the structures of tabun, sarin, and somaninhibited hAChE in complex with HI-6 were determined. This revealed how the presence of G-agent adducts impacts reactivator access and placement within the active site. These insights will contribute toward a path of next-generation reactivators and an improved understanding of the innate issues with the current reactivators.

Serving a critical role in neurotransmission, human acetylcholinesterase (hAChE) is the target of organophosphate nerve agents. Hence, there is an active interest in studying the mechanism of inhibition and recovery of enzymatic activity, which could lead to better countermeasures against nerve agents. As hAChE is found in different oligomeric assemblies, certain approaches to studying it have been problematic. Herein, we examine the biochemical and structural impact of monomerizing hAChE by using two mutations: L380R/F535K. The activities of monomeric hAChE L380R/F535K and dimeric hAChE were determined to be comparable utilizing a modified Ellman's assay. To investigate the influence of subunit–subunit interactions on the structure of hAChE, a 2.1 Å X‐ray crystallographic structure was determined. Apart from minor shifts along the dimer interface, the overall structure of the hAChE L380R/F535K mutant is similar to that of dimeric hAChE. To probe whether the plasticity of the active site was overtly impacted by monomerizing hAChE, the kinetic constants of (PR/S) − VX (ethyl({2‐[bis(propan‐2‐yl)amino]ethyl}sulfanyl)(methyl)phosphinate) inhibition and subsequent rescue of hAChE L380R/F535K activity with HI‐6 (1‐(2′‐hydroxyiminomethyl‐1′‐pyridinium)‐3‐(4′‐carbamoyl‐1‐pyridinium)) were determined and found to be comparable to those of dimeric hAChE. Thus, hAChE L380R/F535K could be used as a substitute for dimeric hAChE when experimentally probing the ability of the hAChE active site to accommodate future nerve agent threats or judge the ability of new therapeutics to access the active site.

The threat of a deliberate release of chemical nerve agents has underscored the need to continually improve field effective treatments for these types of poisonings. The oxime containing HLö-7 is a potential second-generation therapeutic reactivator. A synthetic process for HLö-7 is detailed with improvements to the DIBAL reduction and ion exchange steps. HLö-7 was visualized for the first time within the active site of human acetylcholinesterase and its relative ex vivo potency confirmed against various nerve agents using a phrenic nerve hemidiaphragm assay.

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