Comparison of inhibition kinetics of several organophosphates, including some nerve agent surrogates, using human erythrocyte and rat and mouse brain acetylcholinesterase

Abstract

Because testing of nerve agents is limited to only authorized facilities, our laboratory developed several surrogates that resemble nerve agents because they phosphylate the acetylcholinesterase (AChE) with the same moiety as the actual nerve agents. The inhibition kinetic parameters were determined for AChE by surrogates of cyclosarin (NCMP), sarin (NIMP, PIMP and TIMP) and VX (NEMP and TEMP) and other organophosphorus compounds derived from insecticides. All compounds were tested with rat brain and a subset was tested with mouse brain and purified human erythrocyte AChE. Within the compounds tested on all AChE sources, chlorpyrifos-oxon had the highest molecular rate constant followed by NCMP and NEMP. This was followed by NIMP then paraoxon and DFP with rat and mouse brain AChE but DFP was a more potent inhibitor than NIMP and paraoxon with human AChE. With the additional compounds tested only in rat brain, TEMP was slightly less potent than NEMP but more potent than PIMP which was more potent than NIMP. Methyl paraoxon was slightly less potent than paraoxon but more potent than TIMP which was more potent than DFP. Overall, this study validates that the pattern of inhibitory potencies of our surrogates is comparable to the pattern of inhibitory potencies of actual nerve agents (i.e., cyclosarin>VX>sarin), and that these are more potent than insecticidal organophosphates.

Positron emission tomography studies of organophosphate chemical threats and oxime countermeasures

“…HuAChE or huBChE were treated with F-VX-surrogate (from 10 to 160 nM) to > 95% inhibition and the recovery of enzyme activity measured following dilution to halt further inhibition or possible re-inhibition. The spontaneous reactivation rate constants ( k r ) for were9.31 × 10 −5 min −1 (t ½ = 124 h; HuAChE) and 1.13 × 10 −4 min −1 (t ½ = 102 h; HuBChE), which data is in general agreement with k r rates found for VX and the p -nitrophenoxy VX surrogates (Albuquerque et al, 2006; Coban et al, 2016; Kuca et al, 2010a; Sidell and Groff, 1974). The data implies that ChE-OP(O)(CH 3 )(OCH 2 CH 2 F) adducts are stable and likely measurable in vivo as the corresponding [ 18 F]containing OP-AChE adduct over the lifetime of the tracer, although the lack of restoration of enzyme activity cannot be attributed solely to the ChE-OP(O)(CH 3 )(OCH 2 CH 2 F) adduct.…”

“…The bimolecular inhibition rate constants ( k i ) were determined for electric eel (EEAChE) k i = 4.2 × 10 6 M −1 min −1 , recombinant human AChE k i = 1.1 × 10 7 M −1 min −1 , recombinant human butyrylcholinesterase (HuBChE) k i = 1.95 × 10 5 M −1 min −1 and rat brain AChE k i = 6.1 × 10 6 M −1 min −1 indicating that the F-VX surrogate is a potent, in vitro inhibitor and 5-fold more potent than paraoxon against rat brain AChE. (Johnson and Wallace, 1987) Work by Chambers (Coban et al, 2016; Meek et al, 2012) also showed that (Me)(EtO)P(O)(OPNP) was a strong inhibitor ( k i = 10 5 M −1 min −1 ) of acetyl- and butyrylcholinesterases matching the mechanism of VX inhibition. The k i values and likelihood of the PNP acting as leaving group strongly suggests that the mechanism of AChE inhibition proceeds via formation of the (2-fluoroethyl) methylphosphonate (Scheme 5) that would by analogy, afford 18 [F]-labeled enzyme from F-VX surrogate.…”

Straightforward, Economical Procedures for Microscale Ellman’s Test for Cholinesterase Inhibition and Reactivation

“…30,31 We have also successfully employed nerve agents surrogates using the described methodology. [32][33][34]…”

An improved radiosynthesis of O-(2-[18 F]fluoroethyl)-O-(p -nitrophenyl)methylphosphonate: A first-in-class cholinesterase PET tracer

“…Given these common structural components, we developed a hybrid OP structure based upon the nerve agent VX and paraoxon (insecticide oxon) modified to incorporate an [ 18 F]fluorine atom to produce an OP‐based PET imaging tracer (Figure ). The resultant fluoro‐VX surrogate, O‐(2‐fluoroethyl)‐O‐( p ‐nitrophenyl) methylphosphonate, uses a p ‐nitrophenoxy leaving group and acts similarly in vitro and in vivo to VX without handling concerns . The fluorine tracer atom is strategically positioned 4 bonds from the reactive phosphorus atom to minimize steric or electronic perturbation .…”

“…The resultant fluoro-VX surrogate, O-(2fluoroethyl)-O-(p-nitrophenyl) methylphosphonate, uses a p-nitrophenoxy leaving group and acts similarly in vitro and in vivo to VX without handling concerns. 6,7 The fluorine tracer atom is strategically positioned 4 bonds from the reactive phosphorus atom to minimize steric or electronic perturbation. 8 Injection of high specific activity O-(2-[ 18 F] fluoroethyl)-O-(p-nitrophenyl) methylphosphonate, [ 18 F]1, in rats shows localization of radioactivity in AChE-rich regions of brain and other tissues 5 where it likely exists as (CH 3 )( 18 FCH 2 CH 2 O)P(O)-AChE and other esterase adducts.…”