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Full text release has been delayed at the author's request until August 04, 2026

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Investigations into the Treatment of Organophosphorus Exposed Acetylcholinesterase Variants by Quinone Methide Precursors.

Buck, Anne Kilborn
http://orcid.org/0009-0003-2107-3946
http://rave.ohiolink.edu/etdc/view?acc_num=osu1721401903518357

Abstract Details

2024, Doctor of Philosophy, Ohio State University, Chemistry.
Organophosphorus (OP) compounds are highly toxic, causing over 200,000 deaths annually. These compounds inhibit acetylcholinesterase (AChE) by covalently modifying its catalytic serine residue, preventing the breakdown of the neurotransmitter acetylcholine (ACh). The resulting ACh buildup can cause severe symptoms and, if untreated, death. Traditional treatments using oximes aim to reactivate OP-inhibited AChE but are limited by their inability to cross the blood-brain barrier and their lack of broad-spectrum efficacy. Moreover, OP-inhibited AChE can undergo a spontaneous O- dealkylation event termed aging, and the OP-aged form is recalcitrant to oximes. Quinone methide precursors (QMP) provide a plausible strategy to realkylate the OP-aged cholinesterase adducts, and then, by subsequent water activation or nucleophilic addition to the phosphorus center, restore native enzyme function. Studies were performed to both determine efficacious compounds and their mechanism of action. Additionally, research was done to explore different enzymatic model systems and develop methodologies to investigate high binding QMPs. Phenolic QMPs can both reactivate OP-inhibited AChE and resurrect OP-aged AChE with both electron-donating and electron-withdrawing groups, primarily off of the 4-position. Electron-donating groups should theoretically promote quinone methide formation, whereas electron-withdrawing groups should hinder it. The fact that OP-aged AChE was resurrected by QMPs with both types of groups indicates that the realkylation or reactivation steps in the resurrection mechanism of the phosphylated oxyanion moiety are likely mediated by the enzyme or water. ii 6-Alkoxypyridin-3-ol QMPs are able to restore activity to both OP-inhibited AChE as well as OP-aged AChE. Resurrection kinetic studies show that efficacy of QMPs with (R)-2-methylpyrrolidine as the leaving group is due to their lower Km values, while QMPs with diethylamine are effective because of their high phosphylated adduct displacement rates. Benzimidazole QMPs produce the highest net resurrection of ethyl paraoxon- aged AChE of any compound tested in the Hadad lab to date. The variations in the efficacies of benzimidazole QMPs are likely due to differences in binding within the active site, especially by protonation state, since alkyl substituents do not significantly affect the QMP’s electronic effects. An AChE variant (AChEEC), expressible in E. coli, was investigated as a potential model enzyme. The AChEEC variant shows significant differences, such as rapid inhibition rates by OP compounds, slower aging rates, and reduced net recovery of OP- inhibited and OP-aged AChE. These trends indicate changes in enzyme dynamics for this bacterially expressed form of AChE. Peripheral anionic site (PAS) binding ligands that are linked to QMPs were shown to have increased the binding efficiency of QMPs to AChE. This created issues with the traditional methodologies for reactivation and resurrection, thus requiring further assay development. An additional step was added to the high-throughput assay, including a 100-fold dilution prior to any addition of colorimetric substrate, to effectively stop any reactivation and allow for the QMPs to diffuse out of the active site. This methodology reduced the inhibition of native enzyme, resulting in a more representative percentage for recovered activity. Additional work was attempted to develop an assay with immobilized AChE such that the QMPs could be washed away prior to the assessment of recovered activity.
Christopher Hadad (Advisor)
Thomas Magliery (Committee Member)
Dehua Pei (Committee Member)
271 p.
Biochemistry; Chemistry

Recommended Citations

Citations

  • Buck, A. K. (2024). Investigations into the Treatment of Organophosphorus Exposed Acetylcholinesterase Variants by Quinone Methide Precursors. [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1721401903518357

    APA Style (7th edition)

  • Buck, Anne. Investigations into the Treatment of Organophosphorus Exposed Acetylcholinesterase Variants by Quinone Methide Precursors. 2024. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1721401903518357.

    MLA Style (8th edition)

  • Buck, Anne. "Investigations into the Treatment of Organophosphorus Exposed Acetylcholinesterase Variants by Quinone Methide Precursors." Doctoral dissertation, Ohio State University, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=osu1721401903518357

    Chicago Manual of Style (17th edition)

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