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Directed evolution of hydrolases for prevention of G-type nerve agent intoxication

Nature Chemical Biology volume 7, pages 120125 (2011) | Download Citation


Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >107 (M−1 min−1). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic Sp isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of Sp-cyclosarin by 105-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with kcat/KM 107 M−1 min−1. We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.

  • Compound C7H14FO2P


  • Compound C4H10FO2P


  • Compound C7H16FO2P


  • Compound C18H20NO5P

    3-Cyano-4-methyl-2-oxo-2H-coumarin-7-ylcyclohexyl methylphosphonate

  • Compound C15H16NO5P

    3-Cyano-4-methyl-2-oxo-2H-coumarin-7-ylisopropyl methylphosphonate

  • Compound C18H22NO5P

    3-Cyano-4-methyl-2-oxo-2H-coumarin-7-ylpinacolyl methylphosphonate

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Financial support by US National Institutes of Health (W81XWH-07-2-0020) and the Defense Threat Reduction Agency (HDTRA 1-07-C-0024) is gratefully acknowledged. We thank D. Tal and the Israel Structural Proteomics Centre for assistance with protein production and T. Magliery, D. Lenz and D. Cerasoli for a fruitful collaboration.

Author information

Author notes

    • Rinkoo D Gupta
    •  & Moshe Goldsmith

    These authors contributed equally to this work.


  1. Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.

    • Rinkoo D Gupta
    • , Moshe Goldsmith
    • , Yair Simo
    • , Gavriel Mullokandov
    • , Hagit Bar
    •  & Dan S Tawfik
  2. Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.

    • Yacov Ashani
    • , Moshe Ben-David
    •  & Joel L Sussman
  3. Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel.

    • Yacov Ashani
    •  & Israel Silman
  4. Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot, Israel.

    • Haim Leader
  5. Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

    • Raanan Margalit


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R.D.G. constructed and screened variant libraries by FACS and plate screening with coumarin substrates, characterized evolved variants, analyzed data and wrote the paper. M.G. constructed and screened variant libraries using coumarin and in situ–generated substrates, characterized selected variants, performed in vivo experiments, analyzed data and wrote the paper. Y.A. designed and synthesized coumarin and fluoride substrates, established the AChE assay, characterized selected variants, designed and performed in vivo experiments and contributed to writing and editing the paper. Y.S. screened variant libraries. G.M. constructed and screened the first-generation variant libraries. H.B. generated recombinant AChE. M.B.-D. assisted the design of targeted libraries. H.L. synthesized the coumarin and fluoride substrates. R.M. performed in vivo experiments. I.S. contributed to writing and editing the paper. J.L.S. performed structural analysis and contributed to writing the paper. D.S.T. designed the experiments, analyzed data and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Dan S Tawfik.

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