Inhibitor Report for: Methylparaoxon

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.

An amperometric biosensor array has been developed to resolve pesticide mixtures of dichlorvos and methylparaoxon. The biosensor array has been used in a Flow Injection system, in order to operate automatically the inhibition procedure. The sensors used were three screen-printed amperometric biosensors that incorporated three different acetylcholinesterase enzymes: the wild type from Electric eel and two different genetically modified enzymes, B1 and B394 mutants, from Drosophila melanogaster. The inhibition response triplet was modelled using an Artificial Neural Network which was trained with mixture solutions that contain dichlorvos from 10(-4) to 0.1 microM and methylparaoxon from 0.001 to 2.5 microM. This system can be considered an inhibition electronic tongue.

Amperometric acetylcholinesterase biosensors have been developed for quantification of the pesticides carbofuran, carbaryl, methylparaoxon, and dichlorvos in phosphate buffer containing 5% acetonitrile. Three different biosensors were built using three different acetylcholinesterase (AChE) enzymes-AChE from electric eel, and genetically engineered (B394) and wild-type (B1) AChE from Drosophila melanogaster. Enzymes were immobilized on cobalt(II) phthalocyanine-modified electrodes by entrapment in a photocrosslinkable polymer (PVA-AWP). Each biosensor was tested against the four pesticides. Good operational stability, immobilisation reproducibility, and storage stability were obtained for each biosensor. The best detection limits were obtained with the B394 enzyme for dichlorvos and methylparaoxon (9.6 x 10(-11) and 2.7 x 10(-9) mol L(-1), respectively), the B1 enzyme for carbofuran (4.5 x 10(-9) mol L(-1)), and both the B1 enzyme and the AChE from electric eel for carbaryl (1.6 x 10(-7) mol L(-1)). Finally, the biosensors were used for the direct detection of the pesticides in spiked apple samples.

Human Cathepsin A (CatA) is a lysosomal serine carboxypeptidase of the renin-angiotensin system (RAS) and is structurally similar to acetylcholinesterase (AChE). CatA can remove the C-terminal amino acids of endothelin I, angiotensin I, Substance P, oxytocin, and bradykinin, and can deamidate neurokinin A. Proteomic studies identified CatA and its homologue SCPEP1 as potential targets of organophosphates (OP). CatA could be stably inhibited by low microM to high nM concentrations of racemic sarin (GB), soman (GD), cyclosarin (GF), VX, and VR within minutes to hours at pH 7. Cyclosarin was the most potent with a kinetically measured dissociation constant (KI) of 2 microM followed by VR (KI = 2.8 microM). Bimolecular rate constants for inhibition by cyclosarin and VR were 1.3 x 10(3) M(-1)sec(-1) and 1.2 x 10(3) M(-1)sec(-1), respectively, and were approximately 3-orders of magnitude lower than those of human AChE indicating slower reactivity. Notably, both AChE and CatA bound diisopropylfluorophosphate (DFP) comparably and had KI(DFP) = 13 microM and 11 microM, respectively. At low pH, greater than 85% of the enzyme spontaneously reactivated after OP inhibition, conditions under which OP-adducts of cholinesterases irreversibly age. At pH 6.5 CatA remained stably inhibited by GB and GF and <10% of the enzyme spontaneously reactivated after 200 h. A crystal structure of DFP-inhibited CatA was determined and contained an aged adduct. Similar to AChE, CatA appears to have a "backdoor" for product release. CatA has not been shown previously to age. These results may have implications for: OP-associated inflammation; cardiovascular effects; and the dysregulation of RAS enzymes by OP.

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.

An amperometric biosensor array has been developed to resolve pesticide mixtures of dichlorvos and methylparaoxon. The biosensor array has been used in a Flow Injection system, in order to operate automatically the inhibition procedure. The sensors used were three screen-printed amperometric biosensors that incorporated three different acetylcholinesterase enzymes: the wild type from Electric eel and two different genetically modified enzymes, B1 and B394 mutants, from Drosophila melanogaster. The inhibition response triplet was modelled using an Artificial Neural Network which was trained with mixture solutions that contain dichlorvos from 10(-4) to 0.1 microM and methylparaoxon from 0.001 to 2.5 microM. This system can be considered an inhibition electronic tongue.

Amperometric acetylcholinesterase biosensors have been developed for quantification of the pesticides carbofuran, carbaryl, methylparaoxon, and dichlorvos in phosphate buffer containing 5% acetonitrile. Three different biosensors were built using three different acetylcholinesterase (AChE) enzymes-AChE from electric eel, and genetically engineered (B394) and wild-type (B1) AChE from Drosophila melanogaster. Enzymes were immobilized on cobalt(II) phthalocyanine-modified electrodes by entrapment in a photocrosslinkable polymer (PVA-AWP). Each biosensor was tested against the four pesticides. Good operational stability, immobilisation reproducibility, and storage stability were obtained for each biosensor. The best detection limits were obtained with the B394 enzyme for dichlorvos and methylparaoxon (9.6 x 10(-11) and 2.7 x 10(-9) mol L(-1), respectively), the B1 enzyme for carbofuran (4.5 x 10(-9) mol L(-1)), and both the B1 enzyme and the AChE from electric eel for carbaryl (1.6 x 10(-7) mol L(-1)). Finally, the biosensors were used for the direct detection of the pesticides in spiked apple samples.

There is a clear need for broad-spectrum cholinesterase reactivators (active against a multitude of organophosphorus ester enzyme inhibitors) with a higher efficacy than pralidoxime. The purpose of the study was to quantify in vivo the extent of oxime-conferred protection, using methyl-paraoxon [dimethyl p-nitrophenyl phosphate; (methyl-POX)] as a cholinesterase inhibitor. There were seven groups of six rats in each cycle of the experiment. Group 1 (G1) received 2 micromol methyl-POX ( approximately LD(50)), the other groups (G2-7) received 2 micromol methyl-POX + one of the six reactivators. The animals were monitored for 48 h and the time of mortality was recorded. The procedure was repeated six times. All substances were applied i.p. The experiments were repeated using 3 and 5 micromol methyl-POX. Mortality data were compared and hazards ratios (relative risks) ranked using the Cox proportional hazards model with methyl-POX dose and group (reactivator) as time-independent covariables. The relative risk of death estimated by Cox analysis (95% CI) in oxime-treated animals when compared with untreated animals, adjusted for methyl-POX dose (high/low) was K-27, 0.58 (0.42-0.80); K-48, 0.60 (0.43-0.83); trimedoxime, 0.76 (0.55-1.04); pralidoxime, 0.88 (0.65-1.20); obidoxime, 0.93 (0.68-1.26); HI-6, 0.96 (0.71-1.31). Only K-27 and K-48 provided statistically significant protection in rats exposed to methyl-POX. Despite the lower inhibitory potency (higher IC(50)) of methyl-POX compared with POX (ratio 4:1), the ability of oxime reactivators to protect from methyl-POX induced mortality was reduced compared with protection from POX (ethyl-analog).

The mechanism of acute toxicity of the organophosphorus insecticides has been known for many years to be inhibition of the critical enzyme acetylcholinesterase (EC 3.1.1.7), with the resulting excess acetylcholine accumulation leading to symptoms of cholinergic excess. The bimolecular inhibition rate constant k(i) has been used for decades to describe the inhibitory capacity of organophosphates toward acetylcholinesterase. In the current study, a new approach based on continuous systems modeling was used to determine the appk(i)s of paraoxon and methyl paraoxon towards mouse brain acetylcholinesterase over a wide range of oxon concentrations. These studies revealed that the bimolecular inhibition rate constants for paraoxon and methyl paraoxon appeared to change as a function of oxon concentrations. For example, the appk(i) found with a paraoxon concentration of 1000 nM was 0.16 nM-1h-1, whereas that for 0.1 nM paraoxon was 1.60 nM-1h-1, indicating that the efficiency of phosphorylation appeared to decrease as the paraoxon concentration increased. These data suggested that the current understanding of how these organophosphates interact with acetylcholinesterase is incomplete. Modeling studies using several different kinetic schemes, as well as studies using recombinant monomeric mouse brain acetylcholinesterase, suggested the existence of a second binding site in addition to the active site of the enzyme, to which paraoxon and methyl paraoxon bound, probably in a reversibly manner. Occupation of this site likely rendered more difficult the subsequent phosphorylation of the active site by other oxon molecules, probably by steric hindrance or allosteric modification of the active site. It cannot be ascertained from the current study whether the putative second binding site is identical to or shares common elements with the well-characterized propidium-specific peripheral binding site of acetylcholinesterase.