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

Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) 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 S(p)-cyclosarin by ∼10(5)-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 k(cat)/K(M) ∼ 10(7) 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.

[1]  Dan S. Tawfik,et al.  Enhanced stereoselective hydrolysis of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase , 2006, The FEBS journal.

[2]  F. Raushel,et al.  Stereoselective detoxification of chiral sarin and soman analogues by phosphotriesterase. , 2001, Bioorganic & medicinal chemistry.

[3]  Dan S. Tawfik,et al.  Incorporating Synthetic Oligonucleotides via Gene Reassembly (ISOR): a versatile tool for generating targeted libraries. , 2007, Protein engineering, design & selection : PEDS.

[4]  F. Raushel,et al.  Stereospecificity in the enzymatic hydrolysis of cyclosarin (GF) , 2005 .

[5]  Andrew D Griffiths,et al.  Directed evolution by in vitro compartmentalization , 2006, Nature Methods.

[6]  Wilfred Chen,et al.  Functional analysis of organophosphorus hydrolase variants with high degradation activity towards organophosphate pesticides. , 2006, Protein engineering, design & selection : PEDS.

[7]  P B Laub,et al.  NCOMP--a windows-based computer program for noncompartmental analysis of pharmacokinetic data. , 1996, Journal of pharmaceutical sciences.

[8]  F. Raushel,et al.  Stereoselective hydrolysis of organophosphate nerve agents by the bacterial phosphotriesterase. , 2010, Biochemistry.

[9]  J. Chen,et al.  Reversed enantioselectivity of diisopropyl fluorophosphatase against organophosphorus nerve agents by rational design. , 2009, Journal of the American Chemical Society.

[10]  J. Kassa,et al.  The influence of combinations of oximes on the reactivating and therapeutic efficacy of antidotal treatment of soman poisoning in rats and mice , 2009, Toxicology mechanisms and methods.

[11]  Dan S. Tawfik,et al.  High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments. , 2005, Chemistry & biology.

[12]  K. Courtney,et al.  A new and rapid colorimetric determination of acetylcholinesterase activity. , 1961, Biochemical pharmacology.

[13]  L. Lumley,et al.  Stoichiometric and catalytic scavengers as protection against nerve agent toxicity: a mini review. , 2007, Toxicology.

[14]  A. Saxena,et al.  Mechanism for potent reactivation ability of H oximes analyzed by reactivation kinetic studies with cholinesterases from different species. , 2010, Chemico-biological interactions.

[15]  Dan S. Tawfik,et al.  Directed enzyme evolution via small and effective neutral drift libraries , 2008, Nature Methods.

[16]  Dan S. Tawfik,et al.  In vivo administration of BL-3050: highly stable engineered PON1-HDL complexes , 2009, BMC clinical pharmacology.

[17]  J L Sussman,et al.  Purification and crystallization of a dimeric form of acetylcholinesterase from Torpedo californica subsequent to solubilization with phosphatidylinositol-specific phospholipase C. , 1988, Journal of molecular biology.

[18]  Mikhail G. Shapiro,et al.  Directed evolution of a magnetic resonance imaging contrast agent for noninvasive imaging of dopamine , 2010, Nature Biotechnology.

[19]  Manfred T Reetz,et al.  Addressing the Numbers Problem in Directed Evolution , 2008, Chembiochem : a European journal of chemical biology.

[20]  A. Griffiths,et al.  High-throughput screening of enzyme libraries: in vitro evolution of a beta-galactosidase by fluorescence-activated sorting of double emulsions. , 2005, Chemistry & biology.

[21]  B. P. Doctor,et al.  Bioscavengers for the protection of humans against organophosphate toxicity. , 2005, Chemico-biological interactions.

[22]  Y. Ashani,et al.  Prophylaxis against organophosphate poisoning by an enzyme hydrolysing organophosphorus compounds in mice. , 1991, Life sciences.

[23]  G. Amitai,et al.  Asymmetric fluorogenic organophosphates for the development of active organophosphate hydrolases with reversed stereoselectivity. , 2007, Toxicology.

[24]  J L Sussman,et al.  Stereo-specific synthesis of analogs of nerve agents and their utilization for selection and characterization of paraoxonase (PON1) catalytic scavengers. , 2010, Chemico-biological interactions.

[25]  D. Rochu,et al.  [Engineering of catalytic bioscavengers of organophosphorus compounds]. , 2007, Bulletin de l'Academie nationale de medecine.

[26]  L. Costa,et al.  Paraoxonase protects against chlorpyrifos toxicity in mice. , 1995, Toxicology letters.

[27]  C. Broomfield.  A purified recombinant organophosphorus acid anhydrase protects mice against soman. , 1993, Chemico-biological interactions.

[28]  Dan S. Tawfik,et al.  Latent evolutionary potentials under the neutral mutational drift of an enzyme. , 2007, HFSP journal.

[29]  John C Whitman,et al.  Improving catalytic function by ProSAR-driven enzyme evolution , 2007, Nature Biotechnology.

[30]  B. Lukey,et al.  Chemical warfare agents: chemistry, pharmacology, toxicology, and therapeutics. , 2007 .

[31]  Dan S. Tawfik,et al.  Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Y. Ashani,et al.  Estimation of the upper limit of human butyrylcholinesterase dose required for protection against organophosphates toxicity: a mathematically based toxicokinetic model. , 2004, Toxicological sciences : an official journal of the Society of Toxicology.

[33]  F. Raushel,et al.  Detoxification of organophosphate nerve agents by bacterial phosphotriesterase. , 2005, Toxicology and applied pharmacology.

[34]  Tatyana Belinskaya,et al.  In search of a catalytic bioscavenger for the prophylaxis of nerve agent toxicity. , 2010, Chemico-biological interactions.