Vladimir V. Malygin

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.

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.

Biosensor assay of neuropathy target esterase in whole blood as a new approach to OPIDN risk assessment: review of progress.

Organophosphates (OPs) that inhibit neuropathy target esterase (NTE) with subsequent ageing can produce OP-induced delayed neuropathy (OPIDN). NTE inhibition in lymphocytes can be used as a biomarker of exposure to neuropathic OPs. An electrochemical method was developed to assay NTE in whole blood. The high sensitivity of the tyrosinase carbon-paste biosensors for the phenol produced by hydrolysis of the substrate, phenyl valerate, allowed NTE activity to be measured in diluted samples of whole blood, which cannot be done using the standard colorimetric assay. The biosensor was used to establish correlations of NTE inhibitions in blood with that in lymphocytes and brain after dosing hens with a neuropathic OP. The results of further studies demonstrated that whole blood NTE is a reliable biomarker of neuropathic OPs for up to 96 hours after exposure. These validation results suggest that the biosensor NTE assay for whole blood could be developed to measure human exposure to neuropathic OPs as a predictor of OPIDN. The small blood volume required (100 μL), simplicity of sample preparation and rapid analysis times indicate that the biosensor should be useful in biomonitoring and epidemiological studies. The present paper is an overview of our previous and ongoing work in this area. Human & Experimental Toxicology (2007) 26, 273-282

Quantitative structure-activity relationships predict the delayed neurotoxicity potential of a series of O-alkyl-O-methylchloroformimino phenylphosphonates.

Abstract Inhibition of acetylcholinesterase (AChE) versus inhibition and aging of neuropathy target esterase (NTE) by organophosphorus (OP) compounds in vivo can give rise to distinct neurological consequences: acute cholinergic toxicity versus OP compound-induced delayed neurotoxicity (OPIDN). Previous work has shown that the relative potency of an OP compound to react with NTE versus AChE in vitro may predict its capability to produce OPIDN. The present study was conducted to evaluate further the validity of such predictions and to enhance them with quantitative structure-activity relationships (QSAR) using a homologous series of alkyl phenylphos-phonates, (RO)C6H5P(O)ON=CClCH3 (PhP; R=alkyl). Neuropathic potential of PhP was assessed by measuring ki(NTE)/ki(AChE) ratios in vitro and comparing these with ED50 ratios in vivo. Selectivity for NTE increased with rising R-group hydrophobicity. The ki(NTE)/ki(AChE) ratios were 0.42 (methyl), 3.6 (ethyl), 15 (isopropyl), 36 (propyl), 69 (isobutyl), 105 (butyl), and 124 (pentyl). Ratios >1 suggest the potential to produce OPIDN at doses lower than the LD50. Inhibition of NTE and AChE in hen brain in vivo was studied 24 h after im injection of hens with increasing doses of methyl and butyl derivatives. Analysis of dose-response curves yielded ED50(AChE)/ED50(NTE) ratios of 0.86 for methyl PhP and 22.1 for butyl PhP. These results predict that the butyl derivative should be more neuropathic than the methyl analogue. Excellent correspondence between in vivo and in vitro predictions of neuropathic potential indicate that valid predictive QSAR models may be based on the in vitro approach. Adoption of this system would result in reducing experimental animal use, lowering costs, accelerating data production, and enabling standardization of a biochemically based risk assessment of the neuropathic potential of OP compounds.

Fluorinated α-aminophosphonates—a new type of irreversible inhibitors of serine hydrolases

The structural analogs of α -aminoacids, α -aminophosphonic acids and their esters, are widely studied as biologically active substances [1]. At the same time, among the numerous publications of the last twenty years there are only a few communications devoted to biological activity of fluorinated α -aminophosphonates, although it is well known that inclusion of fluorine atoms and fluorine-containing substituents into molecules of organic substances results in profound changes of chemical and physicochemical properties and, consequently, the biological activity of these substances. In particular, it was shown that some fluorinated esters and phosphin-oxides inhibited cholinesterases [2, 3] and thrombin [4], in contrast to their nonfluorinated analogues. In this paper, the results of studies of interaction of fluorinated α -aminophosphonates (FAPs, 3a ‐ 3h ) with four serine hydrolases are presented. Compounds 3a ‐ 3h were synthesized according to the scheme shown below:

A NEW APPROACH FOR DETERMINATION OF NEUROPATHY TARGET ESTERASE ACTIVITY

Neuropathy target esterase (NTE) was shown to be an excellent biochemical marker for screening of organophosphates (OPs) with respect to their ability to result in organophosphate induced delayed neurotoxicity (OPIDN). This paper describes a new biosensor approach to the analysis of NTE and its inhibitors. The method is based on the combination of NTE enzymatic hydrolysis of phenyl valerate (PV) with phenol detection by the Clark-type oxygen electrode modified by immobilized tyrosinase. The validity of this biosensor method is confirmed by the facts that the calibration curves for NTE obtained by colorimetric and flow-through electrochemical methods were nearly identical and the titration of NTE by test inhibitor mipafox was shown to yield the same pI50 values. The developed electrochemical methods can be considered as a promising approach both for serial express NTE analysis and for kinetic characteristics of NTE.

Diethyl [2,2,2-trifluoro-1-phenyl­sulfonyl­amino-1-(trifluoro­meth­yl)eth­yl]phospho­nate

The title compound, C13H16F6NO5PS, is of interest with respect to inhibition of serine hydrolases. Its structure contains a 1.8797 (13) Å P—C bond and two intermolecular N—H⋯O=P hydrogen bonds, resulting in centrosymmetric dimers. An intramolecular N—H⋯O=P hydrogen bond is also present.

SYNTHESIS OF O-ARYL-DIARYLPHOSPHINATES AND STUDY OF THEIR INTERACTION WITH NEUROTOXIC ESTERASE AND ACETYLCHOLINESTERASE

Many organophosphorus compounds (OPCs), includingthose used as drugs for the treatment of Alzheimer’s diseaseand glaucoma [1, 2], exhibit significant acute toxicity relatedto the inhibition of acetylcholinesterase (ACE) activity inneuronal synapses and, in addition, lead to delayed distalneuropathy manifested by a long axon degeneracy in the spi-nal cord and peripheral nerves. The latter phenomenon is re-ferred to as the delayed neurotoxicity of organophosphoruscompounds (DNOPC) [3 – 6]. A target for the axonotoxicOPCs is one of the serine hydrolases of the nerve tissue,which is called neurotoxic esterase (NTE) [3 – 5, 7, 8]. Incontrast to the acute toxicity of OPCs, related to the ACE in-hibition, the mechanism of DNOPC initiation includes twostages: (i) inhibition of the catalytic activity of NTE byphosphorylation of the enzyme followed by (ii) extremelyrapid dealkylation of a phosphoryl group covalently bound toserine. The latter stage is equivalent to “aging” of thephosphorylated enzyme, leading to the appearance of a mod-ified protein that is believed to initiate the axon degenerationprocess [3 – 5, 7, 8].Compounds acylating the enzyme but, for certain rea-sons, excluding the possibility of the aging stage followingdo not induce DNOPC. Moreover, the results of experiments

Synthesis and Antiesterase Activity of Thiophosphorylated Oximes.

The o-phosphorylated oximes (III) are prepared from the dithiophosphites (I) and dichlorofluoronitrosomethane (II).