Accession Number : ADA465580


Title :   Generation of Recombinant Human AChE OP-Scavengers with Extended Circulatory Longevity


Descriptive Note : Final rept. 3 Mar 2003-2 Oct 2006


Corporate Author : ISRAEL INST FOR BIOLOGICAL RESEARCH NESS ZIONA


Personal Author(s) : Shafferman, Avigdor


Full Text : https://apps.dtic.mil/dtic/tr/fulltext/u2/a465580.pdf


Report Date : Nov 2006


Pagination or Media Count : 144


Abstract : We demonstrate that chemical conjugation of polyethylene glycol (PEG) moieties to recombinant human acetylcholinesterase (rHuAChE) gives rise to OP bioscavenger species which reside for very long periods of time in the circulation of mice, regardless of their post-translation-modification state, and that circulatory elimination of AChE via specific amino acid-related epitopes can also be efficiently overcome by enzyme PEGylation. Taken together, these findings indicate that the circulatory residence is dictated primarily by the PEG appendage. In line with these findings, we examined the possibility to express human AChE in microorganism-based production systems which do not support animal-cell-related enzyme processing, utilizing a specialized designed synthetic human AChE gene. Although the gene product was rapidly degraded in Bacillus brevis cells, the Pichia pastoris yeast cell system was shown to support production and secretion of bioactive rHuAChE. In a series of studies we demonstrate that selective removal of human AChE lysine residues, which serve as target sites for PEG-conjugation, can generate an enzyme, which upon PEGylation, displays a high degree of homogeneity, extended circulatory longevity and reduced immunogenicity. In structure-function studies of AChE, we compared the reactivities of enantiomers of VX and their noncharged isosters, as well as ecothiophate, in conjunction with a battery of AChE mutants. These studies allowed us to define two subsites, located in the active site and peripheral anionic subsites, which confer enzyme stereoselectivity to CW agents such as VX.


Descriptors :   *PHARMACOKINETICS , *ACETYLCHOLINESTERASE , ISRAEL , MUTATIONS , GENETIC ENGINEERING , VX AGENT , RHESUS MONKEYS , CARDIOVASCULAR SYSTEM , MICE , GENES


Subject Categories : Biochemistry
      Genetic Engineering and Molecular Biology
      Weapons Effects(biological)
      Pharmacology


Distribution Statement : APPROVED FOR PUBLIC RELEASE