title: 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.affiliation: Blum-Scientific Services, Hamburg, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Melzer
authors.forename: Anne
authors.initials: A
authors.lastname: Heidenreich
authors.forename: Frederic
authors.initials: F
authors.lastname: Dorandeu
authors.forename: Jürgen
authors.initials: J
authors.lastname: Gäb
authors.forename: Kai
authors.initials: K
authors.lastname: Kehe
authors.forename: Horst
authors.initials: H
authors.lastname: Thiermann
authors.forename: Thomas
authors.initials: T
authors.lastname: Letzel
authors.forename: Marc-Michael
authors.initials: MM
authors.lastname: Blum
mesh_terms: D000818:Animals; D000931:Antidotes / Q000737:chemistry / Q000627:therapeutic use*; D002619:Chemical Warfare Agents / Q000506:poisoning*; D002800:Cholinesterase Inhibitors / Q000506:poisoning*; D049834:Loligo / Q000201:enzymology* / Q000235:genetics; D008297:Male; D013058:Mass Spectrometry; D009154:Mutation; D044345:Phosphoric Triester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000627:therapeutic use*; D011092:Polyethylene Glycols / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D012999:Soman / Q000506:poisoning*
chemical_list: D000931:Antidotes; D002619:Chemical Warfare Agents; D002800:Cholinesterase Inhibitors; D011092:Polyethylene Glycols; D012999:Soman; D044345:Phosphoric Triester Hydrolases; C020061:diisopropyl-fluorophosphatase