A Novel, Modified Human Butyrylcholinesterase Catalytically Degrades the Chemical Warfare Nerve Agent, Sarin.

Chemical warfare nerve agents (CWNAs) present a global threat to both military and civilian populations. The acute toxicity of CWNAs stems from their ability to effectively inhibit acetylcholinesterase (AChE). This inhibition can lead to uncontrolled cholinergic cellular signaling, resulting in cholinergic crisis and, ultimately, death. Although the current FDA-approved standard of care is moderately effective when administered early, development of novel treatment strategies is necessary. Butyrylcholinesterase (BChE) is an enzyme which displays a high degree of structural homology to AChE. Unlike AChE, the roles of BChE are uncertain and possibilities are still being explored. However, BChE appears to primarily serve as a bioscavenger of toxic esters due to its ability to accommodate a wide variety of substrates within its active site. Like AChE, BChE is also readily inhibited by CWNAs. Due to its high affinity for binding CWNAs, and that null-BChE yields no apparent health effects, exogenous BChE has been explored as a candidate therapeutic for CWNA intoxication. Despite years of research, minimal strides have been made to develop a catalytic bioscavenger. Furthermore, BChE is only in early clinical trials as a stoichiometric bioscavenger of CWNAs, and large quantities must be administered to treat CWNA toxicity. Here, we describe previously unidentified mutations to residues within and adjacent to the acyl binding pocket (positions 282-285 were mutagenized from YGTP to NHML) of BChE that confer catalytic degradation of the CWNA, sarin. These mutations, along with corresponding future efforts, may finally lead to a novel therapeutic to combat CWNA intoxication.

Characterization of Cholinesterases From Multiple Large Animal Species for Medical Countermeasure Development Against Chemical Warfare Nerve Agents.

Organophosphorus (OP) compounds, which include insecticides and chemical warfare nerve agents (CWNAs) such as sarin (GB) and VX, continue to be a global threat to both civilian and military populations. It is widely accepted that cholinesterase inhibition is the primary mechanism for acute OP toxicity. Disruption of cholinergic function through the inhibition of acetylcholinesterase (AChE) leads to the accumulation of the neurotransmitter acetylcholine. Excess acetylcholine at the synapse results in an overstimulation of cholinergic neurons which manifests in the common signs and symptoms of OP intoxication (miosis, increased secretions, seizures, convulsions, and respiratory failure). The primary therapeutic strategy employed in the United States to treat OP intoxication includes reactivation of inhibited AChE with the oxime pralidoxime (2-PAM) along with the muscarinic acetylcholine receptor antagonist atropine and the benzodiazepine, diazepam. CWNAs are also known to inhibit butyrylcholinesterase (BChE) without any apparent toxic effects. Therefore, BChE may be viewed as a "bioscavenger" that stoichiometrically binds CWNAs and removes them from circulation. The degree of inhibition of AChE and BChE and the effectiveness of 2-PAM are known to vary among species. Animal models are imperative for evaluating the efficacy of CWNA medical countermeasures, and a thorough characterization of available animal models is important for translating results to humans. Thus, the objective of this study was to compare the circulating levels of each of the cholinesterases as well as multiple kinetic properties (inhibition, reactivation, and aging rates) of both AChE and BChE derived from humans to AChE and BChE derived from commonly used large animal models.

Kinetic analysis of oxime-assisted reactivation of human, Guinea pig, and rat acetylcholinesterase inhibited by the organophosphorus pesticide metabolite phorate oxon (PHO).

Phorate is a highly toxic agricultural pesticide currently in use throughout the world. Like many other organophosphorus (OP) pesticides, the primary mechanism of the acute toxicity of phorate is acetylcholinesterase (AChE) inhibition mediated by its bioactivated oxon metabolite. AChE reactivation is a critical aspect in the treatment of acute OP intoxication. Unfortunately, very little is currently known about the capacity of various oximes to rescue phorate oxon (PHO)-inhibited AChE. To help fill this knowledge gap, we evaluated the kinetics of inhibition, reactivation, and aging of PHO using recombinant AChE derived from three species (rat, guinea pig and human) commonly utilized to study the toxicity of OP compounds and five oximes that are currently fielded (or have been deemed extremely promising) as anti-OP therapies by various nations around the globe: 2-PAM Cl, HI-6 DMS, obidoxime Cl2, MMB4-DMS, and HLö7 DMS. The inhibition rate constants (ki) for PHO were calculated for AChE derived from each species and found to be low (i.e., 4.8×103 to 1.4×104M-1min-1) compared to many other OPs. Obidoxime Cl2 was the most effective reactivator tested. The aging rate of PHO-inhibited AChE was very slow (limited aging was observed out to 48h) for all three species. CONCLUSIONS: (1) Obidoxime Cl2 was the most effective reactivator tested. (2) 2-PAM Cl, showed limited effectiveness in reactivating PHO-inhibited AChE, suggesting that it may have limited usefulness in the clinical management of acute PHO intoxication. (3) The therapeutic window for oxime administration following exposure to phorate (or PHO) is not limited by aging.

Efficacy of Recommended Prehospital Human Equivalent Doses of Atropine and Pralidoxime Against the Toxic Effects of Carbamate Poisoning in the Hartley Guinea Pig.

PURPOSE: Aldicarb and methomyl are carbamate pesticides commonly implicated in human poisonings. The primary toxic mechanism of action for carbamate poisoning is cholinesterase (ChE) inhibition. As such, it is logical to assume that the currently accepted therapies for organophosphate poisoning (muscarinic antagonist atropine and the oxime acetylcholinesterase reactivator pralidoxime chloride [2-PAM Cl]) could afford therapeutic protection. However, oximes have been shown to be contraindicated for poisoning by some carbamates. METHODS: A protective ratio study was conducted in guinea pigs to evaluate the efficacy of atropine and 2-PAM Cl. The ChE activity was determined in both the blood and the cerebral cortex. RESULTS: Coadministration of atropine free base (0.4 mg/kg) and 2-PAM Cl (25.7 mg/kg) demonstrated protective ratios of 2 and 3 against aldicarb and methomyl, respectively, relative to saline. The data reported here show that this protection was primarily mediated by the action of atropine. The reactivator 2-PAM Cl had neither positive nor negative effects on survival. Both blood acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were significantly reduced at 15 minutes postchallenge but gradually returned to normal within 24 hours. Analysis of cerebral cortex showed that BChE, but not AChE, activity was reduced in animals that succumbed prior to 24 hours after challenge. CONCLUSION: The results suggest that coadministration of atropine and 2-PAM Cl at the currently recommended human equivalent doses for use in the prehospital setting to treat organophosphorus nerve agent and pesticide poisoning would likely also be effective against aldicarb or methomyl poisoning.

Acute toxicity of phorate oxon by oral gavage in the Sprague-Dawley rat.

The oral toxicity of phorate oxon (PHO), with emphasis on gender- and age-related effects, was characterized in the Sprague-Dawley rat. The oral LD50 (95% fiducial limits) for PHO in corn oil was 0.88 (0.79, 1.04) mg/kg in males and 0.55 (0.46, 0.63) mg/kg in females with a probit slope of 15. Females had higher baseline blood cholinesterase titers, but males were significantly more tolerant. Younger rats generally had lower absolute cholinesterase blood titers. However as PHO challenges increased, baseline-normalized cholinesterase inhibition was independent of age and gender. Butyrylcholinesterase (BChE) and especially acetylcholinesterase (AChE) in brains of younger females were affected more than that in either males or older females. In summary, while female rats, especially older females, had higher titers relative to males, female rats were more susceptible in terms of absolute cholinesterase inhibition and 24-hr lethality data, but the differences were not observed when titers were normalized to baseline levels.

Evaluation of HemogloBind™ treatment for preparation of samples for cholinesterase analysis.

Acetylcholine is an essential neurotransmitter found throughout the nervous system. Its action on postsynaptic receptors is regulated through hydrolysis by various carboxylesterases, especially cholinesterases (ChEs). The acute toxicity of organophosphate (OP) compounds is directly linked to their action as inhibitors of ChE. One widely used assay for evaluating ChE activity is a spectrophotometric method developed by Ellman et al. When the enzyme source is from tissues or, in particular, blood, hemoglobin displays a spectrophotometric peak at the same wavelength used to analyze cholinergic activity. This creates a substantial background that interferes with the Ellman's assay and must be overcome in order to accurately monitor cholinesterase activity. Herein, we directly compare blood processing methods: classical method (1.67 ± 0.30 U/mL) and HemogloBind™ treatment (1.51 ± 0.17 U/mL), and clearly demonstrate that pretreatment of blood samples with Hemoglobind™ is both a sufficient and rapid sample preparation method for the assessment of ChE activity using the Ellman's method.

Destabilization of the P site codon-anticodon helix results from movement of tRNA into the P/E hybrid state within the ribosome.

Retention of the reading frame in ribosomal complexes after single-round translocation depends on the acylation state of the tRNA. When tRNA lacking a peptidyl group is translocated to the P site, the mRNA slips to allow re-pairing of the tRNA with a nearby out-of-frame codon. Here, we show that this ribosomal activity results from movement of tRNA into the P/E hybrid state. Slippage of mRNA is suppressed by 3' truncation of the translocated tRNA, increased MgCl2 concentration, and mutation C2394A of the 50S E site, and each of these conditions inhibits P/E-state formation. Mutation G2252U of the 50S P site stimulates mRNA slippage, suggesting that decreased affinity of tRNA for the P/P state also destabilizes mRNA in the complex. The effects of G2252U are suppressed by C2394A, further implicating the P/E state in mRNA destabilization. This work uncovers a functional attribute of the P/E state crucial for understanding translation.