G. F. Makhaeva

Neuropathy target esterase (NTE): overview and future

Neuropathy target esterase (NTE) was discovered by M.K. Johnson in his quest for the entity responsible for the striking and mysterious paralysis brought about by certain organophosphorus (OP) esters. His pioneering work on OP neuropathy led to the view that the biochemical lesion consisted of NTE that had undergone OP inhibition and aging. Indeed, nonaging NTE inhibitors failed to produce disease but protected against neuropathy from subsequently administered aging inhibitors. Thus, inhibition of NTE activity was not the culprit; rather, formation of an abnormal protein was the agent of the disorder. More recently, however, Paul Glynn and colleagues showed that whereas conventional knockout of the NTE gene was embryonic lethal, conditional knockout of central nervous system NTE produced neurodegeneration, suggesting to these authors that the absence of NTE rather than its presence in some altered form caused disease. We now know that NTE is the 6th member of a 9-protein family called patatin-like phospholipase domain-containing proteins, PNPLA1-9. Mutations in the catalytic domain of NTE (PNPLA6) are associated with a slowly developing disease akin to OP neuropathy and hereditary spastic paraplegia called NTE-related motor neuron disorder (NTE-MND). Furthermore, the NTE protein from affected individuals has altered enzymological characteristics. Moreover, closely related PNPLA7 is regulated by insulin and glucose. These seemingly disparate findings are not necessarily mutually exclusive, but we need to reconcile recent genetic findings with the historical body of toxicological data indicating that inhibition and aging of NTE are both necessary in order to produce neuropathy from exposure to certain OP compounds. Solving this mystery will be satisfying in itself, but it is also an enterprise likely to pay dividends by enhancing our understanding of the physiological and pathogenic roles of the PNPLA family of proteins in neurological health and disease, including a potential role for NTE in diabetic neuropathy.

Comparative Analysis of Esterase Activities of Human, Mouse, and Rat Blood

Acetylcholinesterase, butyrylcholinesterase, carboxylesterase, and paraoxonase activities in human, mouse, and rat blood were measured. The proportions of these enzymes activities differed significantly. In humans, the most significant were cholinesterase activities, while in rats and mice the contribution of carboxylesterase activity was the greatest. High arylesterase activity of paraoxonase was observed in all cases. Species-specific differences should be taken into consideration when carrying out preclinical trials on rodents for optimization of the pharmacokinetic characteristics of drugs containing complex ester groups.

Synthesis of organophosphates with fluorine-containing leaving groups as serine esterase inhibitors with potential for Alzheimer disease therapeutics.

Acetylcholinesterase and butyrylcholinesterase inhibitors are potential cognition enhancers in Alzheimer disease. O,O-Dialkylphosphate inhibitors with 1-substituted 2,2,2-trifluoroethoxy leaving groups were synthesized by phosphonate-phosphate rearrangement. Substituents in the 1-position of the leaving group along with the O-alkyl groups modulated potency and selectivity against acetylcholinesterase, butyrylcholinesterase, and carboxylesterase.

Esterase profile and analysis of structure-inhibitor selectivity relationships for homologous phosphorylated 1-hydroperfluoroisopropanols

352 Organophosphorous compounds (OPCs) are widely used not only in agrochemical practice but are also cited in pharmacopoeias of many countries as drugs for treating schistosomiasis, glaucoma, and Alzheimer’s disease [1]. The physiological effect of such compounds is based on competitive interaction with serine esterases including the primary biological targets, such as acetylcholinesterase (AChE) [2], the target enzyme for the acute toxic effect of antiacetylcholinesterase compounds, and the neuropathy target esterase (NTE) [3], the target enzyme for organophosphate-induced delayed neurotoxicity, as well as the secondary biological targets, such as butyrylcholinesterase (BChE) and carboxylesterase (CaE). BChE and CaE are stoichiometric scavengers, interaction with which reduces the content of active phosphoryl compound at the toxicokinetic stage of development of biological response [4, 5]. The preferential binding of anticholinesterase compounds with one or another target esterase largely determines the resultant therapeutic or toxic effect of a given compound as well as the character and degree of expression of this effect.

Biosensor detection of neuropathy target esterase in whole blood as a biomarker of exposure to neuropathic organophosphorus compounds.

Abstract Neuropathy target esterase (NTE) is the target protein for neuropathic organophosphorus (OP) compounds that produce OP compound-induced delayed neurotoxicity (OPIDN). Inhibition/aging of brain NTE within hours of exposure predicts the potential for development of OPIDN in susceptible animal models. Lymphocyte NTE has also found limited use as a biomarker of human exposure to neuropathic OP compounds. Recently, a highly sensitive biosensor was developed for NTE activity using a tyrosinase carbon-paste electrode for amperometric detection of phenol produced by hydrolysis of the substrate, phenyl valerate. The I50 (20 min at 37°C) for N,N′-di-2-propylphosphorodiamid ofluoridate (mipafox) against hen lymphocyte NTE was 6.94 ± 0.28 μM amperometrically and 6.02 ± 0.71 μM colorimetrically. For O,O-di-1-propyl O-2,2-dichlorvinyl phosphate (PrDChVP), the I50 against hen brain NTE was 39 ±8 nM amperometrically and 42 ±2 nM colorimetrically. The biosensor enables NTE to be assayed in whole blood, whereas this cannot be done with the usual colorimetric method. Amperometrically, I50 values for PrDChVP against hen and human blood NTE were 66 ±3 and 70 ± 14nM, respectively. To study the possibility of using blood NTE inhibition as a biochemical marker of neuropathic OP compound exposure, NTE activities in brain and lymphocytes as well in brain and blood were measured 24 h after dosing hens with PrDChVP. Brain, lymphocyte, and blood NTE were inhibited in a dose-responsive manner, and NTE inhibition was highy correlated between brain and lymphocyte (r=.994) and between brain and blood (r=.997). The results suggest that the biosensor NTE assay for whole blood could serve as a biomarker of exposure to neuropathic OP compounds as well as a predictor of OPIDN and an adjunct to its early diagnosis.

Combined QSAR studies of inhibitor properties of O-phosphorylated oximes toward serine esterases involved in neurotoxicity, drug metabolism and Alzheimer's disease

Oxime reactivation of serine esterases (EOHs) inhibited by organophosphorus (OP) compounds can produce O-phosphorylated oximes (POXs). Such oxime derivatives are of interest, because some of them can have greater anti-EOH potencies than the OP inhibitors from which they were derived. Accordingly, inhibitor properties of 58 POXs against four EOHs, along with pair-wise selectivities between them, have been analysed using different QSAR approaches. EOHs (with their abbreviations and consequences of inhibition in parentheses) comprised acetylcholinesterase (AChE: acute neurotoxicity; cognition enhancement), butyrylcholinesterase (BChE: inhibition of drug metabolism or stoichiometric scavenging of EOH inhibitors; cognition enhancement), carboxylesterase (CaE: inhibition of drug metabolism or stoichiometric scavenging of EOH inhibitors), and neuropathy target esterase (NTE: delayed neurotoxicity). QSAR techniques encompassed linear regression and backpropagation neural networks in conjunction with fragmental descriptors containing labelled atoms, Molecular Field Topology Analysis (MFTA), Comparative Molecular Similarity Index Analysis (CoMSIA), and molecular modelling. All methods provided mostly consistent and complementary information, and they revealed structural features controlling the ‘esterase profiles’, i.e. patterns of anti-EOH activities and selectivities of the compounds of interest. In addition, MFTA models were used to design a library of compounds having a cognition-enhancement esterase profile suitable for potential application to the treatment of Alzheimers disease.

Conjugates of γ-Carbolines and Phenothiazine as new selective inhibitors of butyrylcholinesterase and blockers of NMDA receptors for Alzheimer Disease

Alzheimer disease is a multifactorial pathology and the development of new multitarget neuroprotective drugs is promising and attractive. We synthesized a group of original compounds, which combine in one molecule γ-carboline fragment of dimebon and phenothiazine core of methylene blue (MB) linked by 1-oxo- and 2-hydroxypropylene spacers. Inhibitory activity of the conjugates toward acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and structurally close to them carboxylesterase (CaE), as well their binding to NMDA-receptors were evaluated in vitro and in silico. These newly synthesized compounds showed significantly higher inhibitory activity toward BChE with IC50 values in submicromolar and micromolar range and exhibited selective inhibitory action against BChE over AChE and CaE. Kinetic studies for the 9 most active compounds indicated that majority of them were mixed-type BChE inhibitors. The main specific protein-ligand interaction is π-π stacking of phenothiazine ring with indole group of Trp82. These compounds emerge as promising safe multitarget ligands for the further development of a therapeutic approach against aging-related neurodegenerative disorders such as Alzheimer and/or other pathological conditions.

Kinetics and mechanism of inhibition of serine esterases by fluorinated aminophosphonates.

This paper reviews previously published data and presents new results to address the hypothesis that fluorinated aminophosphonates (FAPs), (RO)(2)P(O)C(CF(3))(2)NHS(O)(2)C(6)H(5), R=alkyl, inhibit serine esterases by scission of the P-C bond. Kinetics studies demonstrated that FAPs are progressive irreversible inhibitors of acetylcholinesterase (AChE, EC 3.1.1.7.), butyrylcholinesterase (BChE, EC 3.1.1.8.), carboxylesterase (CaE, EC 3.1.1.1.), and neuropathy target esterase (NTE, EC 3.1.1.5.), consistent with P-C bond breakage. Chemical reactivity experiments showed that diMe-FAP and diEt-FAP react with water to yield the corresponding dialkylphosphates and (CF(3))(2)CHNHS(O)(2)C(6)H(5), indicating lability of the P-C bond. X-ray crystallography of diEt-FAP revealed an elongated (and therefore weaker) P-C bond (1.8797 (13)A) compared to P-C bonds in dialkylphosphonates lacking alpha-CF(3) groups (1.805-1.822A). Semi-empirical and non-empirical molecular modeling of diEt-FAP and (EtO)(2)P(O)C(CH(3))(2)NHS(O)(2)C(6)H(5) (diEt-AP), which lacks CF(3) groups, indicated lengthening and destabilization of the P-C bond in diEt-FAP compared to diEt-AP. Active site peptide adducts formed by reacting diEt-FAP with BChE and diBu-FAP with NTE catalytic domain (NEST) were identified using peptide mass mapping with mass spectrometry (MS). Mass shifts (mean+/-SE, average mass) for peaks corresponding to active site peptides with diethylphosphoryl and monoethylphosphoryl adducts on BChE were 136.1+/-0.1 and 108.0+/-0.1Da, respectively. Corresponding mass shifts for dibutylphosphoryl and monobutylphosphoryl adducts on NEST were 191.8+/-0.2 and 135.5+/-0.1Da, respectively. Each of these values was statistically identical to the theoretical mass shift for each dialkylphosphoryl and monoalkylphosphoryl species. The MS results demonstrate that inhibition of BChE and NEST by FAPs yields dialkylphosphoryl and monoalkylphosphoryl adducts, consistent with phosphorylation via P-C bond cleavage and aging by net dealkylation. Taken together, predictions from enzyme kinetics, chemical reactivity, X-ray crystallography, and molecular modeling were confirmed by MS and support the hypothesis that FAPs inhibit serine esterases via scission of the P-C bond.

A stable preparation of hen brain neuropathy target esterase for rapid biochemical assessment of neurotoxic potential of organophosphates.

Neuropathy target esterase (NTE) is a molecular target for organophosphate-induced delayed neurotoxicity (OPIDN). This enzyme has proved to be an excellent tool for the assessment of neuropathic potential of organophosphates (OP), in particular by comparison of an OP inhibitory activity in vitro against NTE and acetylcholinesterase. A large-scale OP screening for delayed neurotoxicity was largely prevented by the lack of an available stable preparation of NTE. To obtain a stable NTE preparation the influence of intensive freezing and subsequent lyophilization of paraoxon-preinhibited (P2 + P3) hen brain membrane fraction on NTE properties has been studied using two neuropathic OP: mipafox and O,O-dipropyldichlorovinyl phosphate (PrDChVP). It was shown that lyophilization preserved a high NTE specific activity and did not alter the inhibitor characteristics of the enzyme. A long-term storage study showed that lyophilized NTE preparation exhibited inhibitory features actually identical to those of the native enzyme during 1 year and retained rather high specific activity; in this case some loss of NTE specific activity has been observed. Comparative studies of inhibition of the native and lyophilized NTE preparations by a model series of phenyl phosphonates RO(C6H5)P(O)ON=CClCH3 (R = alkyl), demonstrated a good correlation between the values pI50 obtained with both enzyme preparations as well as identical structure-activity relationships for the lyophilized and native enzymes. The results allow the conclusion that the obtained NTE preparation can be used as a standard, stable and readily available source of NTE for assessing the anti-NTE activity of OP.

Synthesis, molecular docking and biological evaluation of N,N-disubstituted 2-aminothiazolines as a new class of butyrylcholinesterase and carboxylesterase inhibitors

A series of 31 N,N-disubstituted 2-amino-5-halomethyl-2-thiazolines was designed, synthesized, and evaluated for inhibitory potential against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE). The compounds did not inhibit AChE; the most active compounds inhibited BChE and CaE with IC50 values of 0.22-2.3μM. Pyridine-containing compounds were more selective toward BChE; compounds with the para-OMe substituent in one of the two dibenzyl fragments were more selective toward CaE. Iodinated derivatives were more effective BChE inhibitors than brominated ones, while there was no influence of halogen type on CaE inhibition. Inhibition kinetics for the 9 most active compounds indicated non-competitive inhibition of CaE and varied mechanisms (competitive, non-competitive, or mixed-type) for inhibition of BChE. Docking simulations predicted key binding interactions of compounds with BChE and CaE and revealed that the best docked positions in BChE were at the bottom of the gorge in close proximity to the catalytic residues in the active site. In contrast, the best binding positions for CaE were clustered rather far from the active site at the top of the gorge. Thus, the docking results provided insight into differences in kinetic mechanisms and inhibitor activities of the tested compounds. A cytotoxicity test using the MTT assay showed that within solubility limits (<30μM), none of the tested compounds significantly affected viability of human fetal mesenchymal stem cells. The results indicate that a new series of N,N-disubstituted 2-aminothiazolines could serve as BChE and CaE inhibitors for potential medicinal applications.