In vitro and in vivo efficacy of PEGylated diisopropyl fluorophosphatase (DFPase).

Abstract

Highly toxic organophosphorus compounds that irreversibly inhibit the enzyme acetycholinesterase (AChE), including nerve agents like tabun, sarin, or soman, still pose a credible threat to civilian populations and military personnel. New therapeutics that can be used as a pretreatment or after poisoning with these compounds, complementing existing treatment schemes such as the use of atropine and AChE reactivating oximes, are currently the subject of intense research. A prominent role among potential candidates is taken by enzymes that can detoxify nerve agents by hydrolysis. Diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris is known to effectively hydrolyze DFP and the range of G-type nerve agents including sarin and soman. In the present work, DFPase was PEGylated to increase biological half-life, and to lower or avoid an immunogenic reaction and proteolytic digest. Addition of linear polyethylene glycol (PEG) chains was achieved using mPEG-NHS esters and conjugates were characterized by electrospray ionization--time of flight--mass specrometry (ESI-ToF-MS). PEGylated wildtype DFPase and a mutant selective for the more toxic stereoisomers of the agents were tested in vivo with rats that were challenged with a subcutaneous 3x LD(50) dose of soman. While wildtype DFPase prevented death only at extremely high doses, the mutant was able keep the animals alive and to minimize or totally avoid symptoms of poisoning. The results serve as a proof of principle that engineered variants of DFPase are potential candidates for in vivo use if substrate affinity can be improved or the turnover rate enhanced to lower the required enzyme dose.

• Authors: Marco Melzer, Anne Heidenreich, Frederic Dorandeu, Jürgen Gäb, Kai Kehe, Horst Thiermann, Thomas Letzel, Marc-Michael Blum
• Journal: Drug testing and analysis (Drug Test Anal) 2012 Mar-Apr Vol. 4 Issue 3-4 Pg. 262-70 ISSN: 1942-7611 [Electronic] England
• PMID: 22174192 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)
• Copyright: Copyright © 2011 John Wiley & Sons, Ltd.

Chemical References

• Antidotes
• Chemical Warfare Agents
• Cholinesterase Inhibitors
• Polyethylene Glycols
• Soman
• Phosphoric Triester Hydrolases
• diisopropyl-fluorophosphatase

Topics

• Animals
• Antidotes (chemistry, therapeutic use)
• Chemical Warfare Agents (poisoning)
• Cholinesterase Inhibitors (poisoning)
• Loligo (enzymology, genetics)
• Male
• Mass Spectrometry
• Mutation
• Phosphoric Triester Hydrolases (chemistry, genetics, therapeutic use)
• Polyethylene Glycols (chemistry)
• Rats
• Rats, Wistar
• Soman (poisoning)