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

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 soman-inhibited 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.

Structural and kinetic evidence of aging after organophosphate inhibition of human Cathepsin A.

Human Cathepsin A (CatA) is a lysosomal serine carboxypeptidase of the renin-angiotensin system (RAS) and is structurally similar to acetylcholinesterase (AChE). CatA can remove the C-terminal amino acids of endothelin I, angiotensin I, Substance P, oxytocin, and bradykinin, and can deamidate neurokinin A. Proteomic studies identified CatA and its homologue, SCPEP1, as potential targets of organophosphates (OP). CatA could be stably inhibited by low µM to high nM concentrations of racemic sarin (GB), soman (GD), cyclosarin (GF), VX, and VR within minutes to hours at pH 7. Cyclosarin was the most potent with a kinetically measured dissociation constant (KI) of 2 µM followed by VR (KI = 2.8 µM). Bimolecular rate constants for inhibition by cyclosarin and VR were 1.3 × 103 M-1sec-1 and 1.2 × 103 M-1sec-1, respectively, and were approximately 3-orders of magnitude lower than those of human AChE indicating slower reactivity. Notably, both AChE and CatA bound diisopropylfluorophosphate (DFP) comparably and had KIDFP = 13 µM and 11 µM, respectively. At low pH, greater than 85% of the enzyme spontaneously reactivated after OP inhibition, conditions under which OP-adducts of cholinesterases irreversibly age. At pH 6.5 CatA remained stably inhibited by GB and GF and <10% of the enzyme spontaneously reactivated after 200 h. A crystal structure of DFP-inhibited CatA was determined and contained an aged adduct. Similar to AChE, CatA appears to have a "backdoor" for product release. CatA has not been shown previously to age. These results may have implications for: OP-associated inflammation; cardiovascular effects; and the dysregulation of RAS enzymes by OP.

Structural Insights of Stereospecific Inhibition of Human Acetylcholinesterase by VX and Subsequent Reactivation by HI-6.

Over 50 years ago, the toxicity of irreversible organophosphate inhibitors targeting human acetylcholinesterase (hAChE) was observed to be stereospecific. The therapeutic reversal of hAChE inhibition by reactivators has also been shown to depend on the stereochemistry of the inhibitor. To gain clarity on the mechanism of stereospecific inhibition, the X-ray crystallographic structures of hAChE inhibited by a racemic mixture of VX (P R/S) and its enantiomers were obtained. Beyond identifying hAChE structural features that lend themselves to stereospecific inhibition, structures of the reactivator HI-6 bound to hAChE inhibited by VX enantiomers of varying toxicity, or in its uninhibited state, were obtained. Comparison of hAChE in these pre-reactivation and post-reactivation states along with enzymatic data reveals the potential influence of unproductive reactivator poses on the efficacy of these types of therapeutics. The recognition of structural features related to hAChE's stereospecificity toward VX shed light on the molecular influences of toxicity and their effect on reactivators. In addition to providing a better understanding of the innate issues with current reactivators, an avenue for improvement of reactivators is envisioned.

Nanosizing a Metal-Organic Framework Enzyme Carrier for Accelerating Nerve Agent Hydrolysis.

We report the synthesis and characterization of a water-stable zirconium metal-organic framework (MOF), NU-1003, featuring the largest mesoporous aperture known for a zirconium MOF. This material has been used to immobilize the nerve agent hydrolyzing enzyme, organophosphorus acid anhydrolase (OPAA). The catalytic efficiency of immobilized OPAA in nanosized NU-1003 is significantly increased compared to that of OPAA immobilized in microsized NU-1003 and even exceeds that of the free OPAA enzyme. This paper highlights a method for rapid and highly efficient hydrolysis of nerve agents using nanosized enzyme carriers.

Encapsulation of a Nerve Agent Detoxifying Enzyme by a Mesoporous Zirconium Metal-Organic Framework Engenders Thermal and Long-Term Stability.

Immobilized enzymes typically have greater thermal and operational stability than their soluble form. Here we report that for the first time, a nerve agent detoxifying enzyme, organophosphorus acid anhydrolase (OPAA), has been successfully encapsulated into a water-stable zirconium metal-organic framework (MOF). This MOF features a hierarchical mesoporous channel structure and exhibits a 12 wt % loading capacity of OPAA. The thermal and long-term stabilities of OPAA are both significantly enhanced after immobilization.