Received for publication March 25, 2008.
Revised May 28, 2008.
Accepted for publication June 2, 2008.

Aging-Resistant Organophosphate Bioscavenger Based on PEGylated Phe338Ala Human Acetylcholinesterase

Abstract

The high reactivity of ChEs toward 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 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 AChE-based OP-bioscavenger, the F338A mutation, known to decelerate the rate of aging of AChE-OP conjugates, was incorporated into 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, sarin or VX. Unlike PEGylated WT-AChE, the PEGylated F338A-AChE exhibits significantly reduced aging rates following soman inhibition and can be efficiently reactivated by the HI-6 oxime, both in-vitro and in-vivo. Accordingly, oxime administration to PEG-F338A-AChE pretreated mice enabled them to withstand repeated soman exposure (5.4 and 4 LD₅₀/dose), while same regime treatment of non-PEGylated F338A-AChE or PEGylated WT-AChE pretreated mice failed to protect against the second challenge, due to rapid clearance or irreversible aging of the latter enzymes. Thus, judicious incorporation of selected mutations into the AChE mold in conjunction with its chemical modification, provides means to engineer an optimal ChE based OP-bioscavenger in terms of circulatory longevity, resistance to aging, and reduced doses required for protection even against repeated exposures to nerve agents, such as soman.

Key words: Mutagenesis/Chimeric approaches, Enzymology, Protein targets