Conclusion

References

Results (cont.) Results

Evaluation of atypical butyrylcholinesterase from Oryzias latipes as a scavenger of nerve agents

Nidhi Shah Mentored by Dr. Tamara Otto

Introduction

Materials and Methods

In this evaluation of aBuChE from O. latipes as a scavenger of nerve agents, comparison of cell lysates and conditioned media from cells transfected with aBuChE (Graph 1) confirmed that the protein remained in the cell, but was also secreted into the media. Fetal bovine serum in the media contributed to background activity, which necessitated usage of cell lysates. Similarity of KM values (Table 1) assured that transfected aBuChE matched the enzyme found by Pezzementi, Nachon, and Chatonnet (2011). Incubation of aBuChE lysates with nerve agents abolished enzymatic activity, demonstrating that aBuChE binds to the nerve agents. Surprisingly, VR-inhibited aBuChE spontaneously reactivated in the absence of oximes (Graph 2). VR may not bind covalently to the active site of the enzyme, thus aiding spontaneous reactivation. This may be a result of the positioning of a water molecule at the active site of aBuChE acting as a nucleophile to remove the VR adduct (Figure 1). In the presence of oximes, inhibited aBuChE reactivated at a much faster rate (Table 2). Further analyses, such as inhibition and full reactivation studies, will be completed.

Mumford, H., Docx, C. J., Price, M. E., Green, A. C., Tatterstall, J. E., & Armstrong S. J., (2012). Human plasma-derived BuChE as a stoichiometric bioscavenger for treatment of nerve agent poisoning. Chemico-Biological Interactions, 203(1). doi:10.1016/j.cbi.2012.08.018

Pezzementi, L., Nachon, F., & Chatonnet, A. (2011) Evolution of Acetylcholinesterase and Butyrylcholinesterase in the Vertebrates: An Atypical Butyrylcholinesterase from the Medaka Oryzias latipes. PLoS ONE 6(2): e17396.doi:10.1371/journal.pone.0017396

Expression of aBuChE The cDNA encoding aBuChE as well as an empty vector, pcDNA3.1

(control), were transfected into Human Embryonic Kidney 293T cells using Lipofectamine™ 2000. Cells were incubated at 37 °C in a CO2 incubator for 48 hours. Media was harvested, filtered, and stored at 4

C. Cells were lysed in 100 mM KPO4, 1 M NaCl, 1% Triton X-100, pH 7.4 for 5 min at room temperature. Extracts were centrifuged at 20,000g for 20 min, and the supernatants were kept at -20 °C. Reactivation of aBuChE

Fifty µL enzyme was incubated with 100 µL substrate (2.6 µM – 5.28 mM acetylthiocholine (AtCh); 2.6 µM – 1.32 mM butyrylthiocholine (BtCh) or propionylthiocholine (PtCh)) and reporter (2 mM 5,5’-dithiobis-(2-nitrobenzoic acid)) in 100 mM KPO4, pH 7.4. The rate of hydrolysis of each substrate was monitored at A405 for 5 min in a spectrophotometer. For nerve agent inhibition and reactivation studies, 1 µL nerve agent was added to 50 µL enzyme and incubated for 5 min at room temperature. After incubation, samples were passed over a gel-filtration column to remove unbound nerve agent. Samples were diluted in 100 mM KPO4, pH 7.4 and at specific time intervals were tested for hydrolysis of PtCh in the presence or absence of 1 mM oxime.

Graph 1: Comparison of PtCh hydrolysis by cells transfected with aBuChE and pcDNA in media and cell lysate.

Table 1: Observed KM values were determined using Michaelis-Menten kinetics and compared to KM values provided by Pezzementi, Nachon, and Chatonnet (2011).

020406080

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Spontaneous Reactivation of Inhibited aBuChE GAGBGDGFVXVR

Graph 2: Reactivation of inhibited aBuChE in the absence of oxime. The following nerve agents were used: tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), VX, and VR.

Table 2: t1/2 values (minutes) for inhibited-aBuChE with and without oximes: pralidoxime (2PAM), methoxime (MMB4), trimedoxime (TMB4), and toxogonin.

Acknowledgements I would like to thank Dr. Tamara Otto, Mrs. McDonough, Mr. Rich

Sweeney, the Bioscavenger group at MRICD, and my family.

Organophosphorus (OP) nerve agents, when introduced to the human body, bind covalently to the active site serine of acetylcholinesterase (AChE) and inhibit the enzyme. This inhibition prevents the hydrolysis of the neurotransmitter acetylcholine (ACh) by AChE, which results in multi-organ failure, and ultimately death. Current post-OP exposure therapy includes administration of an ACh receptor antagonist, an anticonvulsant, and an oxime. Oximes are nucleophiles that can remove the nerve agent molecule from the active site of the enzyme. Human butyrylcholinesterase (BuChE) has widely gained success as a stoichiometric bioscavenger due to its ability to mimic the irreversible binding of nerve agent to AChE. Bioscavengers bind and remove OP compounds from the bloodstream before they inhibit AChE (Mumford et al., 2011). Atypical BuChE (aBuChE) from Medaka Oryzias latipes has intermediate properties of both AChE and BuChE (Pezzementi, Nachon, & Chatonnet, 2011). It was hypothesized that if aBuChE binds to nerve agents, then the enzyme may have the potential to act as a bioscavenger. The purpose of this study was to assess the ability of aBuChE to bind to nerve agents and reactivate in the presence or absence of oximes.

Figure 1: Active site of VR inhibited-aBuChE. Dashed line shows a distance of 3.03 angstroms between the phosphorus atom of VR (in gold) and water molecule of aBuChE.

KM Values of aBuChE with Thiocholine Substrates

Substrate Observed KM Referenced KM

AtCh 0.22 + 0.08 0.27 + 0.18

BtCh 0.22 + 0.09 0.072 + 0.60

PtCh 0.12 + 0.003 0.16 + 0.15

t1/2 Values for Nerve Agent-Inhibited aBuChE

GA GB GD GF VX VR No oxime >1440 >1440 >1440 >1440 >1440 372 + 6

2PAM 56 + 4 24 + 2 >1440 27 + 0.1 28 + 2 3 + 1

MMB4 >1440 68 + 8 >1440 110 + 24 867 144 + 2

TMB4 294 20 + 3 >1440 28 + 3 58 + 9 30 + 6

Toxogonin 199 + 6 24 + 3 >1440 28 + 0 96 + 0 28 + 5

1.25

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Lysate pcDNA Lysate aBuChE Media pcDNA Media aBuChE

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pcDNA and aBuChE Activity in Media and Cell Lysate