Nerve Agent Hydrolysis Activity Designed into a Human Drug Metabolism Enzyme

Organophosphorus (OP) nerve agents are potent suicide inhibitors of the essential neurotransmitter-regulating enzyme acetylcholinesterase. Due to their acute toxicity, there is significant interest in developing effective countermeasures to OP poisoning. Here we impart nerve agent hydrolysis activity into the human drug metabolism enzyme carboxylesterase 1. Using crystal structures of the target enzyme in complex with nerve agent as a guide, a pair of histidine and glutamic acid residues were designed proximal to the enzyme's native catalytic triad. The resultant variant protein demonstrated significantly increased rates of reactivation following exposure to sarin, soman, and cyclosarin. Importantly, the addition of these residues did not alter the high affinity binding of nerve agents to this protein. Thus, using two amino acid substitutions, a novel enzyme was created that efficiently converted a group of hemisubstrates, compounds that can start but not complete a reaction cycle, into bona fide substrates. Such approaches may lead to novel countermeasures for nerve agent poisoning

Human carboxylesterase 1 active site structure.

<p>Active site of human carboxylesterase 1 covalently inhibited via S221 with cyclosarin (magenta) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017441#pone.0017441-Hemmert1" target="_blank">[8]</a>. The other catalytic residues, in addition to S221, are H468 and E354 (yellow), and are surrounded by hydrophobic residues (grey surface) including V146 and L363 (light blue), as well as the oxyanion hole (white).</p

Bimolecular rates of inhibition, Michaelis-Menten constants, and rates of reactivation for wild-type and V146H/L363E hCE1 against racemic cyclosarin and stereoisomers of cyclosarin model compounds.

<p>N = 3, s.d., N.D. is not determined, N.R. is no reactivation, pH 7.4, 25°C.</p>a<p>Racemic <i>bona fide</i> cyclosarin,</p>b<p>stereoisomers of cyclosarin model compounds,</p>c<p>(8).</p

Organophosphate (OP) inhibition of human carboxylesterase 1 (hCE1).

<p><b>A</b>. Three G-type OP nerve agents and OP model compound (R represents respective <i>O</i>-alkoxy groups). Wild-type hCE1 preferentially binds the stereoisomers shown (7). <b>B</b>. Schematic mechanism of OP hydrolysis by hCE1. X represents the leaving group and * denotes a non-reactive state.</p

Catalytic efficiencies (<i>k</i>_cat/<i>K</i>_m) of engineered enzymes towards hemisubstrates.

a<p>(16).</p>b<p>(32).</p>c<p>(32).</p

Reactivation of hCE1 following nerve agent exposure.

<p><b>A</b>. Spontaneous reactivation of V146H/L363E hCE1 following inhibition by racemic sarin (blue), soman (green), or cyclosarin (red). Wild type hCE1 (grey) only reactivates following sarin inhibition (7). n = 6, s.d. <b>B</b>. Rates of dephosphonylation for hCE1 variants in the presence of sarin (blue), soman (green) and cyclosarin (red). n = 3, s.d.</p

Inhibition and Michaelis-Menten constants for wild-type and V146H/L363E hCE1 against stereoisomers of sarin and soman model compounds.

<p>(N = 3, s.d.)</p>a<p>(7).</p

Mechanism of reactivation by V146H/L363E hCE1 after cyclosarin binding.

<p><b>A</b>. Model of V146H/L363E (cyan) hCE1 with P<i>_R</i> cyclosarin (magenta) including a water molecule (red) between E363 and the central phosphorus. <b>B</b>. Proposed mechanism for enhanced reactivation following cyclosarin inhibition. <b>C</b>. pH dependence of V146H/L363E (black) and L363E (grey) hCE1 dephosphonylation following cyclosarin inhibition.</p