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Research ArticleToxicology

Identification of Human Butyrylcholinesterase Organophosphate-Resistant Variants through a Novel Mammalian Enzyme Functional Screen

Jun Zhang, Sigeng Chen, Erik C. Ralph, Mary Dwyer and John R. Cashman
Journal of Pharmacology and Experimental Therapeutics December 2012, 343 (3) 673-682; DOI: https://doi.org/10.1124/jpet.112.198499
Jun Zhang
Human BioMolecular Research Institute, San Diego, California
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Sigeng Chen
Human BioMolecular Research Institute, San Diego, California
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Erik C. Ralph
Human BioMolecular Research Institute, San Diego, California
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Mary Dwyer
Human BioMolecular Research Institute, San Diego, California
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John R. Cashman
Human BioMolecular Research Institute, San Diego, California
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Abstract

Human butyrylcholinesterase (hBChE) is currently being developed as a detoxication enzyme for the catalytic hydrolysis or stoichiometric binding of organophosphates (OPs). Previously, rationally designed hBChE mutants (G117H and E197Q) were reported in the literature and showed the feasibility of engineering OP hydrolytic functional activity into hBChE. However, the OP hydrolysis rate for G117H is too low for clinical utility. Additional OP-resistant hBChE variants with greater hydrolysis rates are needed as OP nerve-agent countermeasures for therapeutic utility. As described herein, a directed molecular evolution process was used to identify amino acid residues that contribute to OP-resistant functional activity of hBChE variants. In this article, we describe the development and validation of a novel method to identify hBChE variants with OP-resistant functional activity (decreased rate of OP inhibition). The method reported herein used an adenoviral protein expression system combined with a functional screening protocol of OP nerve-agent model compounds that have been shown to have functional properties similar to authentic OP nerve-agent compounds. The hBChE screening method was robust for transfection efficiency, library diversity, and reproducibility of positive signals. The screening approach not only identified the previously reported hBChE G117H variant, but also identified a series of additional hBChE variants, including hBChE G117N, G117R, E197C, and L125V, that exhibited OP-resistant functional activities not reported previously. The mammalian functional screening approach can serve as a cornerstone for further optimization and screening for OP-resistant hBChEs for potential therapeutic applications.

Footnotes

  • This work was funded by the National Institutes of Health CounterACT Program through the National Institutes of Health National Institute of Neurological Disorders and Stroke awards [Grants NS058183, NS058038].

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

    http://dx.doi.org/10.1124/jpet.112.198499.

  • ↵Embedded Image The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

  • ABBREVIATIONS:

    OP
    organophosphate
    AChE
    acetylcholinesterase
    BChE
    butyrylcholinesterase
    hBChE
    human BChE
    ChE
    cholinesterase
    ETP
    echothiophate
    GB
    sarin
    GD
    soman
    GF
    cyclosarin
    GA
    tabun
    ATC
    acetylthiocholine
    BTC
    butyrylthiocholine iodide
    DTNB
    5,5′-dithiobis(2-nitrobenzoic acid)
    SpGF-Met
    Sp-O-cyclohexyl S-methyl methylphosphonothioate hydrochloride
    RpGF-Met
    Rp-O-cyclohexyl S-methyl methylphosphonothioate hydrochloride
    AD
    adenovirus
    pAD
    AD plasmid vector
    PCR
    polymerase chain reaction
    CHO
    Chinese hamster ovary
    WT
    wild type
    CMV
    cytomegalovirus
    DMEM
    Dulbecco's modified Eagle's medium
    FBS
    fetal bovine serum
    PBS
    phosphate-buffered saline
    HRP
    horseradish peroxidase
    HEK
    human embryonic kidney
    NNK
    nitrosamine ketone
    AU
    arbitrary units.

  • Received July 24, 2012.
  • Accepted September 5, 2012.
  • Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 343 (3)
Journal of Pharmacology and Experimental Therapeutics
Vol. 343, Issue 3
1 Dec 2012
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Research ArticleToxicology

Molecular Evolution of Human BChE OP-Resistant Variants

Jun Zhang, Sigeng Chen, Erik C. Ralph, Mary Dwyer and John R. Cashman
Journal of Pharmacology and Experimental Therapeutics December 1, 2012, 343 (3) 673-682; DOI: https://doi.org/10.1124/jpet.112.198499

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Research ArticleToxicology

Molecular Evolution of Human BChE OP-Resistant Variants

Jun Zhang, Sigeng Chen, Erik C. Ralph, Mary Dwyer and John R. Cashman
Journal of Pharmacology and Experimental Therapeutics December 1, 2012, 343 (3) 673-682; DOI: https://doi.org/10.1124/jpet.112.198499
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