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Neuropathy target esterase (NTE) is an integral membrane protein whose covalent modification by organophosphorus compounds (OPs) results in a delayed axonopathy (called OP-induced delayed neuropathy (OPIDN)) whose mechanisms of initiation and progression are unknown. The current model for OPIDN states that inhibition of NTE by a neuropathic OP followed by a post-inhibitory aging reaction results in NTE gaining a new function that is detrimental to the survival of the axon. Thus, we hypothesized that if NTE gains a new function, it would also need to experience a change in its structure. To this end, we employed molecular modeling along with x-ray crystallography of NTE’s plant homologue patatin-17 (pat17) to investigate conformational changes in tertiary structure associated with inhibition and aging of a neuropathic OP-pat17 conjugate. Analysis of NTE’s catalytic (patatin-homology) domain (PNTE) revealed it to have 30% sequence homology and 18% identity to pat17, allowing us to build a homology model for PNTE using the crystal structure of pat17 as a template. This model revealed that contrary to previously published data, NTE has a Ser966-Asp1086 catalytic dyad with the Asp1086 acting as the general base during catalysis. Simulated inhibition (SI) and simulated aging (SA) work undertaken with this model using the neuropathic OP diisopropylphosphorofluoridate (DFP) indicated the absence of significant conformational changes following inhibition (Cα root mean square deviation (RMSD): 1.4Å) and aging (RMSD: 1.6Å), suggesting that the current model for OPIDN’s pathogenesis may be flawed. Furthermore, biochemical analysis of pat17 revealed that it could be inhibited and aged by OPs like DFP (IC50: 181μM; t1/2 aging: 11.4 min). We also showed that pat17 could be inhibited by methyl arachidonyl fluorophosphonate (MAFP) (IC50: 116nM). The crystal structures of pat17 aged with DFP along with that of pat17 inhibited with MAFP were solved via molecular replacement in space-group P43212. Analysis of these crystal structures confirmed our SI and SA results, indicating no significant conformational change associated with inhibition by MAFP (RMSD: 1.07Å) and aging by DFP (0.98Å). In conclusion, a new model for the initiation and progression of OPIDN, based on disruption of membrane lipids is presented.

Organophosphorus (OP) compounds, used in insecticides, pharmaceuticals, and weapons of biochemical warfare inhibit serine hydrolases. Exposure to OP compounds has shown that a phosphylation of certain serine esterases results in two distinct types of toxicities: an acute cholinergic toxicity associated with inhibition of acetylcholinesterase (AChE), and a more chronic toxicity associated with the inhibition and aging of neuropathy target esterase (NTE). OP induced delayed neurotoxicity (OPIDN) occurs when a threshold of NTE is inhibited and aged, and is characterized by axonopathies in the peripheral and central nervous systems 1-4 weeks after exposure. An accurate in vivo model of OPIDN is difficult to develop, due to interspecies variations of inhibitor sensitivity and metabolism. Understanding the mechanism of inhibition and aging of serine esterases by OP compounds and correlating this with pathological axonopathies are important for research on OPIDN. Fluorinated aminophosphonates (FAP) are a group of OP compounds that were hypothesized to inhibit serine esterases through a scission in a chemically stable carbon-phosphorus bond. Through the use of surface enhanced laser desorption/absorption time of flight mass spectrometry, the FAP compounds were shown to covalently phosphorylate the active site serine of butyrylcholinesterase and subsequently age through dealkylation. To begin modeling OPIDN, correlations were found in the bimolecular rate constants of inhibition of AChE and NTE using hen brain, mouse brain, and human recombinant enzymes. Furthermore, correlations in relative inhibitory potentials were found that could predict the neuropathic potential of OP compounds. Finally, two point mutations in NTE were found in patients with a hereditary spastic paraplegia that had clinical presentations similar to OPIDN. Through site-directed mutagenesis, these mutations were created in the catalytic domain of NTE and found to have altered enzymological properties, including reduced kinetic rates of substrate hydrolysis, inhibition, and aging. This research reveals that the mechanism of inhibition by OP compounds can be elucidated using mass spectrometry. Additionally, associations of kinetic values between rodents, hens, and humans may lead to further modeling of OPIDN. In conclusion, alterations in the enzymological properties of NTE may be associated with pathology presented in patients with and associated motor neuron disease.