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

Abstract

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.

The structural and biochemical impacts of monomerizing human acetylcholinesterase

“…To explore whether the flexibility of the active site was overly impacted by the monomerization of hAChE by the mutations L380R/F535K, the kinetic constants of (P R/S ) – VX inhibition and subsequent rescue by HI‐6 were obtained. The determined dissociation constant, K d , and the unimolecular bonding rate constant, k 2 , of hAChE L380R/F535K are slightly different from the values previously recorded for dimeric hAChE; however, the yielded bimolecular rate constant, k i , of 7.9 × 10 7 M −1 min −1 for (P R/S ) – VX‐inhibited hAChE L380R/F535K is consistent with the k i value of dimeric hAChE from previous studies (Tables and ) . As the potency of (P R/S ) – VX appeared consistent for both enzymes, a modified Ellman's assay was utilized to assess the ability of HI‐6 to reactivate (P R/S ) – VX inhibited monomeric hAChE L380R/F535K.…”

“…Recently, the plasticity within the active site of hAChE has been implicated in the ability of the enzyme to be inhibited by nerve agents and rescued by reactivating agents . To explore whether the flexibility of the active site was overly impacted by the monomerization of hAChE by the mutations L380R/F535K, the kinetic constants of (P R/S ) – VX inhibition and subsequent rescue by HI‐6 were obtained.…”

“…Final crystal conditions were obtained by screening pH and sodium citrate concentrations. Using crystals generated under these conditions, a 2.09 Å data set was obtained in the space group P6 5 22, which differs from previous hAChE space groups . Using molecular replacement with one subunit of hAChE (PDB http://firstglance.jmol.org/fg.htm?mol=4EY4), a solution was readily found (Table ).…”

“…Outside the dimer interface, the presence of a loop at residues 491–499 is the most noticeable structural difference between the structures of the dimers and monomers. This loop in structures of the dimer is rarely complete and usually lacks electron density, implicating structural flexibility . As a result, modeling of this region has been sparse.…”

“…Reactivators seek to reverse the covalent modification of hAChE's catalytic serine by OPs, which must occur prior to aging of the nerve agent–AChE complex . The efficacy of reactivators was shown to vary based on the OP, the stereochemistry of the OP, and the species of AChE . The flexibility of the active site, in particular the acyl loop, has recently been shown to play a role in the ability of nerve agents to inhibit hAChE and subsequent reactivation .…”