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 >10⁷ (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 -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 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.