Evolved Stereoselective Hydrolases for Broad-Spectrum G-Type Nerve Agent Detoxification

SummaryA preferred strategy for preventing nerve agents intoxication is catalytic scavenging by enzymes that hydrolyze them before they reach their targets. Using directed evolution, we simultaneously enhanced the activity of a previously described serum paraoxonase 1 (PON1) variant for hydrolysis of the toxic SP isomers of the most threatening G-type nerve agents. The evolved variants show ≤340-fold increased rates and catalytic efficiencies of 0.2-5 × 107 M−1 min−1. Our selection for prevention of acetylcholinesterase inhibition also resulted in the complete reversion of PON1's stereospecificity, from an enantiomeric ratio (E) < 6.3 × 10−4 in favor of the RP isomer of a cyclosarin analog in wild-type PON1, to E > 2,500 for the SP isomer in an evolved variant. Given their ability to hydrolyze G-agents, these evolved variants may serve as broad-range G-agent prophylactics

Molecular Toxicology Efficacy of the rePON1 mutant IIG1 to prevent cyclosarin toxicity

in vivo and to detoxify structurally different nerve agents in vitr

Engineering V‑Type Nerve Agents Detoxifying Enzymes Using Computationally Focused Libraries

VX and its Russian (RVX) and Chinese (CVX) analogues rapidly inactivate acetylcholinesterase and are the most toxic stockpile nerve agents. These organophosphates have a thiol leaving group with a choline-like moiety and are hydrolyzed very slowly by natural enzymes. We used an integrated computational and experimental approach to increase <i>Brevundimonas diminuta</i> phosphotriesterase’s (PTE) detoxification rate of V-agents by 5000-fold. Computational models were built of the complex between PTE and V-agents. On the basis of these models, the active site was redesigned to be complementary in shape to VX and RVX and to include favorable electrostatic interactions with their choline-like leaving group. Small libraries based on designed sequences were constructed. The libraries were screened by a direct assay for V-agent detoxification, as our initial studies showed that colorimetric surrogates fail to report the detoxification rates of the actual agents. The experimental results were fed back to improve the computational models. Overall, five rounds of iterating between experiment and model refinement led to variants that hydrolyze the toxic <i>S</i>_P isomers of all three V-agents with <i>k</i>_cat/<i>K</i>_M values of up to 5 × 10⁶ M^–1 min^–1 and also efficiently detoxify G-agents. These new catalysts provide the basis for broad spectrum nerve agent detoxification

Engineering V‑Type Nerve Agents Detoxifying Enzymes Using Computationally Focused Libraries

VX and its Russian (RVX) and Chinese (CVX) analogues rapidly inactivate acetylcholinesterase and are the most toxic stockpile nerve agents. These organophosphates have a thiol leaving group with a choline-like moiety and are hydrolyzed very slowly by natural enzymes. We used an integrated computational and experimental approach to increase <i>Brevundimonas diminuta</i> phosphotriesterase’s (PTE) detoxification rate of V-agents by 5000-fold. Computational models were built of the complex between PTE and V-agents. On the basis of these models, the active site was redesigned to be complementary in shape to VX and RVX and to include favorable electrostatic interactions with their choline-like leaving group. Small libraries based on designed sequences were constructed. The libraries were screened by a direct assay for V-agent detoxification, as our initial studies showed that colorimetric surrogates fail to report the detoxification rates of the actual agents. The experimental results were fed back to improve the computational models. Overall, five rounds of iterating between experiment and model refinement led to variants that hydrolyze the toxic <i>S</i>_P isomers of all three V-agents with <i>k</i>_cat/<i>K</i>_M values of up to 5 × 10⁶ M^–1 min^–1 and also efficiently detoxify G-agents. These new catalysts provide the basis for broad spectrum nerve agent detoxification