Structural and Biochemical Insights into the Inhibition of Human Acetylcholinesterase by G-Series Nerve Agents and Subsequent Reactivation by HI-6

Abstract

The recent use of organophosphate nerve agents in Syria, Malaysia, Russia, and the United Kingdom has reinforced the potential threat of their intentional release. These agents act through their ability to inhibit human acetylcholinesterase (hAChE; E.C. 3.1.1.7), an enzyme vital for survival. The toxicity of hAChE inhibition via G-series nerve agents has been demonstrated to vary widely depending on the G-agent used. To gain insight into this issue, the structures of hAChE inhibited by tabun, sarin, cyclosarin, soman, and GP were obtained along with the inhibition kinetics for these agents. Through this information, the role of hAChE active site plasticity in agent selectivity is revealed. With reports indicating that the efficacy of reactivators can vary based on the nerve agent inhibiting hAChE, human recombinatorially expressed hAChE was utilized to define these variations for HI-6 among various G-agents. To identify the structural underpinnings of this phenomenon, the structures of tabun, sarin, and somaninhibited hAChE in complex with HI-6 were determined. This revealed how the presence of G-agent adducts impacts reactivator access and placement within the active site. These insights will contribute toward a path of next-generation reactivators and an improved understanding of the innate issues with the current reactivators.

Backbone Conformation Shifts in X-ray Structures of Human Acetylcholinesterase upon Covalent Organophosphate Inhibition

“…This stabilization then pulls the whole substituted phosphorus towards W86. A similar shift is observed in the structure the just-slightly-smaller Soman-hAChE conjugate [20].…”

Broad‐Spectrum Antidote Discovery by Untangling the Reactivation Mechanism of Nerve‐Agent‐Inhibited Acetylcholinesterase

“…h AChE and m AChE are very similar in sequence and 3D structure, and studies have shown similar inhibition kinetics [66,67] . Also, binary OPNA‐AChE complex structures determined in both species are similar [12,13,15,16] . The molecular structure confirmed that the aromatic scaffold of 1 a interacts with amino acid residues at the entrance of the gorge, with the amine substituent extending towards the catalytic site (Figures 2B and S3).…”

“…The electron density maps show that binding modes A and B of the aromatic scaffold position the pyridinium oxime in close proximity to the serine phosphoesters of tabun and RVX (mode A) and VX (mode B) whereas in mode C the oxime is far from the catalytic site inhibited by sarin. The promiscuity of the peripheral site of OPNA‐ h AChE in accommodating aromatic scaffolds has also been observed for HI‐6 [12,13] . In this case different arene‐arene interactions lead to different binding poses for the aromatic scaffold of HI‐6, while the linker and pyridinium oxime are directed towards the active site for all HI‐6 ⋅ OPNA‐ h AChE species.…”

“…Three loops at the entrance of the gorge constitute the peripheral site, while the the active site with its catalytic triad (Ser203, His447 and Glu334) is located near the bottom of the gorge (Figure 2A) [11] . OPNAs impair the function of AChE by acting as selective electrophiles that react with the catalytic serine to form a covalent bond (Figure 1) while also forming various non‐covalent interactions with other amino acid residues [12–16] . The rate of hydrolysis (i. e., the rate of spontaneous AChE reactivation) is low for most OPNA‐AChE adducts, so the inhibition of AChE is effectively irreversible.…”

“…[11] OPNAs impair the function of AChE by acting as selective electrophiles that react with the catalytic serine to form a covalent bond (Figure 1) while also forming various non-covalent interactions with other amino acid residues. [12][13][14][15][16] The rate of hydrolysis (i. e., the rate of spontaneous AChE reactivation) is low for most OPNA-AChE adducts, so the inhibition of AChE is effectively irreversible. Reactivators carry a nucleophilic oxime functionality that can cleave the bond between the OPNA and AChE, thereby restoring the enzyme's activity (Figure 1).…”