Comparing oxime-mediated reactivation of nerve agent inhibited acetylcholinesterase from various species Maggie Weese Mentored by Dr. Douglas Cerasoli and Ms. Linn Cadieux Introduction Results Conclusion Organophosphorus (OP) nerve agents are chemical warfare agents that The purpose of this research was to evaluate differences in the rates of inhibit acetylcholinesterase (AChE), an enzyme that hydrolyzes the t1/2 for Reactivation of GB Inhibited AChE oxime-mediated reactivation of HuAChE, mAChE and GPAChE. The neurotransmitter acetylcholine (ACh). This inhibition results in an 30 27.4 comparison of these three enzymes after inhibition by either VX or GB accumulation of ACh in the cholinergic synapse, and causes seizures, 25 21.2 23.9 20.2 and then reactivation by MMB4, 2PAM, or HI-6 revealed that mAChE and convulsions, and eventual death through disruption of cardiac and GPAChE do not accurately represent the reactivation potential of 20 Human respiratory function (Ballantyne & Marrs, 1992). Current treatments for OP 16.1 HuAChE. As shown in Graph 1 and Graph 2, these enzymes varied 15 12.7 Mouse (min) nerve agent poisoning include administration of the cholinesterase Guinea Pig drastically in their t1/2s, indicating differences in their ability to predict the reactivating compound 2-PAM − an oxime that cleaves nerve agent from 10 oxime-mediated reactivation that occurs for HuAChE. The amino acid 5.2 4.8 4.4 the AChE active site – which has only limited efficacy against some OPs. 5 differences between the AChE enzymes from the three animal species, The efficacy of oximes is tested using animal models to ensure that these Half Time of Reactivation 0 especially those that are located near the active site gorge, are likely treatments are both safe and effective before being tested in humans. In 2PAM HI6 MMB4 responsible for this variability. The amino acid 325, highlighted in green on this study, recombinant AChEs from mouse and guinea pig were tested for Graph 1: This graph reveals the time taken for 50% of each enzyme to be Image 1, was a valine amino acid in HuAChE but an isoleucine in both reactivation in the presence of several oximes after inhibition by OPs; the reactivated after being inhibited by sarin (GB). mAChE and GPAChE. This difference could alter the quaternary results were compared to the reactivation rate of recombinant human structures of the enzymes, affecting the ability of a particular oxime to AChE under the same conditions. It was hypothesized that the oxime- enter the active site gorge to reactivate the inhibited enzyme. At amino acid mediated reactivation of inhibited AChE will vary based on species. t1/2 for Reactivation of VX Inhibited AChE 97, highlighted in yellow in Image 1, GPAChE has a isoleucine residue 70 rather than the threonine found in both HuAChE and mAChE. This 57.8 58.7 60 55.2 difference could impact reactivation of GPAChE relative to human and 50 Materials and Methods Human mouse versions of the enzyme due to the differing charges of the amino 40 Mouse acids. Finally, at amino acid 374, mAChE has a valine residue instead of the (min) 27.3 Stock solutions of 20 mM acetylthiocholine (AtCh) and 20 mM 5,5’- 30 22.9 23.8 Guinea Pig alanine found in the other enzymes. This difference may also contribute to dithiobis-(2-nitrobenzoic acid) (DTNB) were made in advance and stored 20 enzyme reactivation variability due to the proximity of this amino acid to 6.3 at -20 C prior to use. One ml of AtCh and 2 ml of DTNB were added to 10 4.3 3.1 the active site gorge. The results suggested that testing oximes for 37 ml of 0.1 M KPO4 buffer (pH 7.0) to create a substrate-reporter Half Time of Reactivation 0 reactivation of nerve agent-inhibited acetylcholinesterase in mice or guinea 2PAM HI6 MMB4 solution. Twenty µL HuAChE, mAChE, and GPAChE (~1mg/mL) were pig models may not provide results that are highly predictive of protection exposed to 5 µL of either VX or GB and incubated for 10 minutes at room Graph 2: This graph reveals the time taken for 50% of each enzyme to be that would be afforded to humans by the same oxime. Future studies could temperature. Five µL of KPO4 buffer were added to 20 µL of HuAChE, reactivated after being inhibited by VX. analyze other nerve agents and other oximes with human, mouse, and mAChE and GPAChE and incubated for 10 minutes at room temperature guinea pig AChE to further explore enzyme reactivation variability. as a positive control. The 20 µL inhibited and uninhibited enzyme samples were then added to G25-150 sepharose size exclusion columns and spun at 3000 RPM for 2 min in a centrifuge, separating excess agent from the References enzyme by gravity flow extraction. Both enzyme samples were then diluted to a concentration which could be used with the substrate-reporter solution Ballantyne, B., & Marrs, T. C. (1992). Overview of the biological and and 150 µL of these dilutions were then added to each well of a serial clinical aspects of organophosphates and carbamates. In: B. Ballantyne dilution reservoir; the activity of this mixture was used as the baseline & T. C. Marrs (Eds.), Clinical and experimental toxicology of organophosphates activity of inhibited and uninhibited AChE respectively. Then, oxime and carbamates (3-14). Oxford: Butterworth-Heinemann. solution (methoxime (MMB4), pralidoxime (2PAM), asoxime chloride (HI- 6 )) or buffer was added to each well, creating a reactivation mixture. Aliquots of this reactivation mixture were removed and tested for Acknowledgements hydrolysis activity against AtCh at several time points. The resulting Image 1: This is an image of the HuAChE molecule. The catalytic triad is highlighted above in teal, the amino acid 325 is highlighted in green, the amino acid I would like to give a special thanks to my mentors Ms. Cadieux and Dr. velocities were compared to the hydrolysis activity of uninhibited enzyme 97 is highlighted in yellow and the amino acid 374 is highlighted in blue. Cerasoli, my faculty advisor Mrs. McDonough, and the MRICD over the same time course and the t1/2 for each enzyme was determined. Bioscavenger lab for all of their help and support. .