Acetylcholinesterase: converting a vulnerable target to a template for antidotes and detection of inhibitor exposure

Abstract

Applications of recombinant DNA technology, chemical synthesis on biological templates and fluorescence detection of organophosphorylation provide unexplored avenues for development of antidotes and approaches for remote detection of organophosphate nerve agents and pesticides. We discuss here how acetylcholinesterase (AChE), through appropriate mutations, becomes more susceptible to oxime reactivation. Since the reaction between organophosphate and the mutated enzyme remains rapid, regeneration of active enzyme by oxime becomes the rate-limiting step in the process to complete a catalytic cycle for generation of active enzyme. Accordingly, "Oxime-assisted Catalysis" by AChE provides a potential means for catalyzing the hydrolysis of organophosphates in plasma prior to their reaching the cellular target site. In turn, AChE, when conjugated with organophosphate, is employed as a template for 'click-chemistry, freeze-frame' synthesis of new nucleophilic reactivating agents that could potentially prove useful in AChE reactivation at the target site as well as in catalytic scavenging of organophosphates in plasma. Finally, substituted AChE molecules can be conjugated to fluorophores giving rise to shifts in emission spectra for detection of dispersed organophosphates. Since external reagents do not have to be added to detect the fluorescence change, the modified enzyme would serve as a remote sensor.

Figure 1

Reaction of Alkyl Methylphosphonates with the Active Center Serine (203) of Acetylcholinesterase to Form a Conjugate with Three Subsequent Fates: Hydrolysis, Oxime Reactivation and Aging.

Figure 2

Reaction Cycle of Alkyl Methylphosphonfluoridate Hydrolysis Mediated by Acetylcholinesterase (E-OH) and an Oxime (OX). The Resulting Phospho-oxime Hydrolyzes to the Phosphonate Acid and Oxime.

Figure 3

Proposed Reaction of a Peripheral Site Propidium Analogue with an Exocyclic Chain of Variable Chain Length and an Azido Alkyl Pyridine Aldoxime, also with variable Methylene Chain Length, (CH₂)_n and (CH₂)_m. Shown above is the proposed reaction in the mouse AChE active center gorge (Bourne et al., 1995) for the six membered methylene chain connected to propidium and three membered chain connected to the oxime.

Figure 4

A) Emission Spectra of Conjugated Acrylodan for a 81 Glu (E) to Cys (C) mutation prior to and after reaction with S_P-and R_P -3,3-dimethylbutyl methylphosphono thiocholine (DMBMP-TCh). B) The actual color of cuvettes containing the conjugated AChE with fluorophore and organophosphate DDVP (O,O-dimethyl O-(2,2-dichlorovinyl)phosphate) or S_P-and R_P - enantiomers of DMBMP-TCh.