Aging-Resistant Organophosphate Bioscavenger Based on Polyethylene Glycol-Conjugated F338A Human Acetylcholinesterase

Abstract

The high reactivity of cholinesterases (ChEs) toward organophosphorus (OP) compounds has led to propose recombinant ChEs as bioscavengers of nerve agents. The bioscavenging potential of recombinant ChEs can be enhanced by conjugation of polyethylene glycol (PEG) moieties, to extend their circulatory residence. However, the ability of exogenously administered ChEs to confer long-term protection against repeated exposures to nerve agents is still limited due to the aging process, whereby organophosphate-ChE adducts undergo irreversible dealkylation, which precludes oxime-mediated reactivation of the enzyme. To generate an optimal acetylcholinesterase (AChE)-based OP bioscavenger, the F338A mutation, known to decelerate the rate of aging of AChE-OP conjugates, was incorporated into polyethylene glycol-conjugated (PEGylated) human AChE. The PEGylated F338A-AChE displayed unaltered rates of hydrolysis, inhibition, phosphylation, and reactivation and could effectively protect mice against exposure to soman (pinacolylmethyl phosphonofluoridate), sarin (O-isopropyl methylphosphonofluoridate), or O-ethyl-S-

Oxime-assisted Acetylcholinesterase Catalytic Scavengers of Organophosphates That Resist Aging

“…However, the moiety was dependent upon synergy with the F338A mutation, as the single mutants alone yielded only minimal enhancements. Similar to a previous study of the F338A single mutant (15), the Y337A/F338A mutant showed promising reactivation rates when inhibited with soman, an OP well known for causing fast aging. Much to our excitement, the Y337A/F338A double mutant displayed faster reactivation kinetics than the single F338A mutant alone when inhibited with soman.…”

“…In a recent study, a human AChE F338A mutant was characterized as an effective aging-resistant bio-scavenger of OPs (15). However, the reactivation rates for the F338A mutant enzyme are relatively slow when compared with the wild-type enzyme.…”

Preclinical and first-in-human evaluation of PRX-105, a PEGylated, plant-derived, recombinant human acetylcholinesterase-R

“…The small number of subjects only provides us with trends rather than statistically significant values; nevertheless, similar ranges of half-life were reported in other studies involving PEGylated AChE-based bio-scavengers (Cohen et al, 2006;Mazor et al, 2008).…”

Catalytic Bioscavengers Against Toxic Esters, an Alternative Approach for Prophylaxis and Treatments of Poisonings

“…Alternatively, in vitro polysialylation of purified enzymes is possible with a sialyltransferase or by using a chemical method (Gregoriadis et al, 1999). PEGylation was also proved to be an effective chemical modification for increasing the circulatory half-life of administered recombinant ChEs (Cohen et al, 2006, 2007; Huang et al, 2007; Kronman et al 2007; Mazor et al, 2008; Chilukuri et al, 2008). Recently, a 150 kDa recombinant fusion protein human albumin-human BChE showed substantially improved pharmacokinetics when administered to juvenile pigs, t1/2 ≈ 32h against ≈ 3h for recombinant 70%-tetrameric BChE (Huang et al, 2008).…”

“…Thus, ChE mutants associated with oxime reactivators act as pseudo-catalysts in displacing the OP moiety bound to the enzyme. These enzyme-reactivator-coupled systems could lead to a new family of pseudo-catalytic bioscavengers (Taylor et al, 2007; Kovarik et al, 2007, 2008; Mazor et al, 2008). …”