We propose here a cutting edge biophysical study aimed at identifying structurally imposed limiting factors in oxime reactivation of nerve agent and pesticide organophosphate (OP) inhibited human acetylcholinesterase (hAChE). We will focus on characterizing hAChE protein domain motions in solution critical for facilitating reactivation a well as on precise proton structure in the hAChE active center involved in the nucleophilic substitution of covalently conjugated phosphorus. The study will use cutting edge techniques to characterize reactivation related molecular motions of hAChE in solution based on X-ray and neutron scatter in equilibrium and resolved in time from ps to min intervals. High resolution joint X-ray/neutron diffraction on hAChE crystals at room temperature will be used to resolve precise positions of protons and complete water molecules in the hAChE active center. Both scattering and diffraction studies will rely on comparative analysis of native, OP-conjugated, and aged hAChE alone, and in complex with selected structurally diverse oxime reactivators and will be aimed at identifying structural features of oximes optimal for overcoming limitations imposed by structural dynamics and proton distribution in the hAChE active center. The acquired information coupled with advanced computational analysis and simulation will be used to design prototypic accelerated oxime reactivator of OP-hAChE. The structures of prototypic accelerated reactivators will be made available for synthesis and refinement using our existing drug design pipeline developed in collaboration with researchers of The Scripps Research Institute in La Jolla.
Acetylcholine catalysis;inhibition by organophosphates;and oxime antidote reactivation involving acetylcholinesterase all involve protonation equilibria and conformational dynamics in the enzyme. This collaborative project employs cutting edge neutron and X-ray beam based techniques to understand these related processes since further advances in designing efficient antidotes require atomic level resolution methods using neutron beams to monitor proton inventories and locations in these catalytic turnover and reactivation events. Information emerging from these advanced specialized technologies is required to understand intimate mechanisms and yield the structural and dynamical insights to guide the improved design of future nerve agent antidotes.
|Kovalevsky, Andrey; Blumenthal, Donald K; Cheng, Xiaolin et al. (2016) Limitations in current acetylcholinesterase structure-based design of oxime antidotes for organophosphate poisoning. Ann N Y Acad Sci 1378:41-49|