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Computational And Experimental Studies Towards The Development Of Novel Therapeutics Against Organophosphorus Nerve Agents: Butyrylcholinesterase And Paraoxonase

Vyas, Shubham

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2011, Doctor of Philosophy, Ohio State University, Chemistry.

Organophosphorus (OP) compounds are chemical nerve agents that interact with cholinesterases (ChEs). Since there is no effective remedy available for nerve agent poisoning, OP compounds pose a severe threat to civilian and military personnel. Research efforts described in this dissertation are focused on developments of therapeutics against OP nerve agent exposure.

Butyrylcholinesterase (BuChE) is a stoichiometric bioscavenger of OP compounds. To design catalytically active BuChE variants, extensive molecular dynamics (MD) simulations were carried out on BuChE as well as several known and novel mutants. Two transient binding modes of the catalytic serine of BuChE were identified, and the MD simulations revealed a backdoor of the active site. Mutants at the G116 position led to severe alterations around the active site. Simulations on both G117H and G117N variants showed a conserved water molecule that is in close proximity to S198. Modeling of the E197Q mutant suggested that Q197 is placed in a different position and away from the active site relative to the wild-type E197 residue. The double mutant, G117H/E197Q, was found to have structural characteristics of both G117H and E197Q.

Pyridinium oximes (PyOX) can work with BuChE to detoxify the nerve agents catalytically. Interaction of OP compounds and pyridinium oximes (PyOXs) with BuChE was studied using molecular docking simulations. The computational protocol reproduced the binding mode of acetylcholine with cholinesterases (ChEs). The oxyanion hole was the key element for OP binding, and the computed results provided molecular level explanations for available experimental results. PyOXs were found to interact with BuChE at the choline binding residues, oxyanion hole as well as D70 and Y332 residues. Binding of PyOXs become significantly poor with BuChE when an OP molecule was bound in the active site, contrary to acetylcholinesterase, and due to the lack of aromatic residues around the active site of BuChE.

Reactivation of OP bound ChEs by PyOXs generates phosphyl oximes (POXs) that can re-inhibit ChEs. For reactivation purposes, 2-PAM is predicted to be more efficient than 3- and 4-PAM, and 2-POXs were found to be more inclined towards the decomposition process using ab initio calculations.

Human paraoxonase-1 (PON1) has an inherent capability of hydrolyzing OP compounds. In order to increase the catalytic efficiency of PON1, mutants at position H115 were designed and studied using MD simulations. The H115K variant was found to have severe distortion at the putative active site and thus predicted to have a reduced catalytic efficiency. Atomic level fluctuations in all other H115 mutants (A, V, I, L, S, T, F, Y) suggested a mild displacement of the catalytic calcium (0.5 – 0.7 Å) and the presence of conserved bridging water molecular between E53 and D269. We propose a new operative mechanism of PON1 against OP nerve agents that suggests enhanced catalytic efficiency of H115 mutants, in accord with experimental findings.

The photochemistry of diphenylphosphoryl azide (DPP–N3), a potential photoaffinity label of PON1, was studied by femtosecond transient absorption spectroscopy, chemical analysis of light-induced reaction products, and computational methods. This study provided the first direct experimental detection of a singlet phosphorylnitrene. Theoretical methods yielded two possible mechanisms for singlet diphenylphosphorylnitrene (¹DPP–N) formation: (i) Energy transfer from the (π,π*) singlet phenyl excited state to the azide moiety, which subsequently loses molecular nitrogen to form ¹DPP–N, (ii) direct irradiation of the azide moiety to form an excited singlet state of the azide, which in turn loses molecular nitrogen to form ¹DPP–N. Two transient species centered at 430 nm (τ ∼ 28 ps) and at 525 nm (τ ∼ 480 ps) were observed upon 260 nm excitation. Ultrafast time-resolved studies performed on DPP–N3 with the singlet nitrene quencher, confirmed the spectroscopic assignment of ¹DPP–N to the 525 nm absorption band.

In summary, we investigated several ways to treat chemical nerve agent exposure in this dissertation that provided molecular level explanations for previous experimental findings and suggested future work in this area.

Christopher M. Hadad (Advisor)
Matthew S. Platz (Committee Member)
James V. Coe (Committee Member)
Jovica D. Badjic (Committee Member)

Recommended Citations

Citations

  • Vyas, S. (2011). Computational And Experimental Studies Towards The Development Of Novel Therapeutics Against Organophosphorus Nerve Agents: Butyrylcholinesterase And Paraoxonase [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1309974326

    APA Style (7th edition)

  • Vyas, Shubham. Computational And Experimental Studies Towards The Development Of Novel Therapeutics Against Organophosphorus Nerve Agents: Butyrylcholinesterase And Paraoxonase. 2011. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1309974326.

    MLA Style (8th edition)

  • Vyas, Shubham. "Computational And Experimental Studies Towards The Development Of Novel Therapeutics Against Organophosphorus Nerve Agents: Butyrylcholinesterase And Paraoxonase." Doctoral dissertation, Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1309974326

    Chicago Manual of Style (17th edition)