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

Novel Genetically Modified Mouse Model to Assess Soman-Induced Toxicity and Medical Countermeasure Efficacy: Human Acetylcholinesterase Knock-in Serum Carboxylesterase Knockout Mice

The identification of improved medical countermeasures against exposure to chemical warfare nerve agents (CWNAs), a class of organophosphorus compounds, is dependent on the choice of animal model used in preclinical studies. CWNAs bind to acetylcholinesterase and prevent the catalysis of acetylcholine, causing a plethora of peripheral and central physiologic manifestations, including seizure. Rodents are widely used to elucidate the effects of CWNA-induced seizure, albeit with a caveat: they express carboxylesterase activity in plasma. Carboxylesterase, an enzyme involved in the detoxification of some organophosphorus compounds, plays a scavenging role and decreases CWNA availability, thus exerting a protective effect. Furthermore, species-specific amino acid differences in acetylcholinesterase confound studies that use oximes or other compounds to restore its function after inhibition by CWNA. The creation of a human acetylcholinesterase knock-in/serum carboxylesterase knockout (C57BL/6-Ces1ctm1.1LocAChEtm1.1Loc/J; a.k.a KIKO) mouse may facilitate better modeling of CWNA toxicity in a small rodent species. The current studies characterize the effects of exposure to soman, a highly toxic CWNA, and evaluate the efficacy of anti-seizure drugs in this newly developed KIKO mouse model. Data demonstrate that a combination of midazolam and ketamine reduces seizure duration and severity, eliminates the development of spontaneous recurrent seizures, and protects certain brain regions from neuronal damage in a genetically modified model with human relevance to organophosphorus compound toxicity. This new animal model and the results of this study and future studies using it will enhance medical countermeasures development for both defense and homeland security purposes

Purification, characterization, and N-glycosylation of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures.

Recombinant butyrylcholinesterase produced in a metabolically regulated transgenic rice cell culture (rrBChE) was purified to produce a highly pure (95%), active form of enzyme. The developed downstream process uses common manufacturing friendly operations including tangential flow filtration, anion-exchange chromatography, and affinity chromatography to obtain a process recovery of 42% active rrBChE. The purified rrBChE was then characterized to confirm its comparability to the native human form of the molecule (hBChE). The recombinant and native enzyme demonstrated comparable enzymatic behavior and had an identical amino acid sequence. However, rrBChE differs in that it contains plant-type complex N-glycans, including an α-1,3 linked core fucose, and a β-1,2 xylose, and lacking a terminal sialic acid. Despite this difference, rrBChE is demonstrated to be an effective stoichiometric bioscavenger for five different organophosphorous nerve agents in vitro. Together, the efficient downstream processing scheme and functionality of rrBChE confirm its promise as a cost-effective alternative to hBChE for prophylactic and therapeutic use

Evaluating mice lacking serum carboxylesterase as a behavioral model for nerve agent intoxication

<p>Mice and other rodents are typically utilized for chemical warfare nerve agent research. Rodents have large amounts of carboxylesterase in their blood, while humans do not. Carboxylesterase nonspecifically binds to and detoxifies nerve agent. The presence of this natural bioscavenger makes mice and other rodents poor models for studies identifying therapeutics to treat humans exposed to nerve agents. To obviate this problem, a serum carboxylesterase knockout (Es1 KO) mouse was created. In this study, Es1 KO and wild type (WT) mice were assessed for differences in gene expression, nerve agent (soman; GD) median lethal dose (MLD) values, and behavior prior to and following nerve agent exposure. No expression differences were detected between Es1 KO and WT mice in more than 34 000 mouse genes tested. There was a significant difference between Es1 KO and WT mice in MLD values, as the MLD for GD-exposed WT mice was significantly higher than the MLD for GD-exposed Es1 KO mice. Behavioral assessments of Es1 KO and WT mice included an open field test, a zero maze, a Barnes maze, and a sucrose preference test (SPT). While sex differences were observed in various measures of these tests, overall, Es1 KO mice behaved similarly to WT mice. The two genotypes also showed virtually identical neuropathological changes following GD exposure. Es1 KO mice appear to have an enhanced susceptibility to GD toxicity while retaining all other behavioral and physiological responses to this nerve agent, making the Es1 KO mouse a more human-like model for nerve agent research.</p

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

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

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

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