Novel Nerve-Agent Antidote Design Based on Crystallographic and Mass Spectrometric Analyses of Tabun-Conjugated Acetylcholinesterase in Complex with Antidotes

Abstract

Organophosphorus compound-based nerve agents inhibit the essential enzyme acetylcholinesterase (AChE) causing acute toxicity and death. Clinical treatment of nerve-agent poisoning is to use oxime-based antidotes to reactivate the inhibited AChE. However, the nerve agent tabun is resistant to oximes. To design improved oximes, crystal structures of a tabun-conjugated AChE in complex with different oximes are needed to guide the structural modifications of known antidotes. However, this type of structure is extremely challenging to obtain because both deamidation of the tabun conjugate and reactivation of AChE occur during crystallographic experiments. Here we report, for the first time, the crystal structures of Ortho-7 and HLö-7 in complex with AChE that is conjugated to an intact tabun. These structures were determined by our new strategy of combining crystallographic and mass spectrometric analyses of AChE crystals. The results explain the relative reactivation potencies of the two oximes and offer insights into improving known medical antidotes.

Crystal structures of oxime-bound fenamiphos-acetylcholinesterases: Reactivation involving flipping of the His447 ring to form a reactive Glu334–His447–oxime triad

“…It is conceivable that the Glu334-His447-Ortho-7 triad increases the nucleophilicity of the oxime just as the Glu334-His447-Water-HI-6 tetrad [9]. In excellent agreement with this finding, the imidazole ring flip that permits the formation of the Glu334-His447-Ortho-7 triad is also present in our previously reported structure of tabun-inhibited mAChE in complex with Ortho-7 [8]. An imidazole ring flip has also been reported to produce a catalytic triad in His108-GAR (glycinamide ribonucleotide) triad deprotonates the GAR molecule [33].…”

“…In the previously reported Ortho-7tabun-mAChE structure (2JF0; [8]), the triad (Glu334-His447-Ortho-7) is also observed. The His447 residue is in a similar position and has a flipped imidazole ring just as described in Ortho-7fep-mAChE.…”

“…The catalytic site (CAS) of AChE is located close to the bottom of a ~20 Å-deep and narrow active-site gorge which accommodates the conjugate through nonbonded interactions at the oxyanion hole (main-chain nitrogen atoms of Gly121, Gly122, and Ala204), the acyl pocket (side chains of the aromatic residues Trp236, Phe295, Phe297 and Phe338) and the choline-binding site (side chains of Trp86 and Tyr337; [1]). At the entrance of the gorge, the peripheral anionic site (PAS; side chains of Tyr72, Asp74, Tyr124, Trp286 and Tyr341) constitutes a binding site for various allosteric modulators and reactivators [2][3][4][5][6][7][8][9].…”

“…Previous crystallographic studies showed that carboxyaminopyridinium-containing oximes and bispyridiniumaldoximes form cation-pi interactions with the side chains of Tyr124 and Tyr72, respectively, although they all interact with Trp286, when complexing with nonphosphylated and phosphylated mAChE [7][8][9]. M a n u s c r i p t…”

Organophosphorus compounds as chemical warfare agents: a review

“…143 Structural models for the mechanism of action and the differences in activity among oximes have been proposed. 122,147,150,155,156 It is known that an adequate orientation of the phosphoryl bond inside the active site is necessary for both the enzyme efficient inhibition and its reactivation; however, the orientation of the oximes in the active site and their angles for attacking the phosphylated serine are different. 150 Many new oximes have been synthesized, and their capacities of AChE reactivation evaluated.…”