A series of previously synthesized conjugates of tacrine and salicylamide was extended by varying the structure of the salicylamide fragment and using salicylic aldehyde to synthesize salicylimine derivatives. The hybrids exhibited broad-spectrum biological activity. All new conjugates were potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with selectivity toward BChE. The structure of the salicylamide moiety exerted little effect on anticholinesterase activity, but AChE inhibition increased with spacer elongation. The most active conjugates were salicylimine derivatives: IC(50) values of the lead compound 10c were 0.0826 microM (AChE) and 0.0156 microM (BChE), with weak inhibition of the off-target carboxylesterase. The hybrids were mixed-type reversible inhibitors of both cholinesterases and displayed dual binding to the catalytic and peripheral anionic sites of AChE in molecular docking, which, along with experimental results on propidium iodide displacement, suggested their potential to block AChE-induced beta-amyloid aggregation. All conjugates inhibited Abeta(42) self-aggregation in the thioflavin test, and inhibition increased with spacer elongation. Salicylimine 10c and salicylamide 5c with (CH(2))(8) spacers were the lead compounds for inhibiting Abeta(42) self-aggregation, which was corroborated by molecular docking to Abeta(42). ABTS(+)-scavenging activity was highest for salicylamides 5a-c, intermediate for salicylimines 10a-c, low for F-containing salicylamides 7, and non-existent for methoxybenzoylamides 6 and difluoromethoxybenzoylamides 8. In the FRAP antioxidant (AO) assay, the test compounds displayed little or no activity. Quantum chemical analysis and molecular dynamics (MD) simulations with QM/MM potentials explained the AO structure-activity relationships. All conjugates were effective chelators of Cu(2+), Fe(2+), and Zn(2+), with molar compound/metal (Cu(2+)) ratios of 2:1 (5b) and ~1:1 (10b). Conjugates exerted comparable or lower cytotoxicity than tacrine on mouse hepatocytes and had favorable predicted intestinal absorption and blood-brain barrier permeability. The overall results indicate that the synthesized conjugates are promising new multifunctional agents for the potential treatment of AD.
We investigated the inhibitory activities of novel 9-phosphoryl-9,10-dihydroacridines and 9-phosphorylacridines against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CES). We also studied the abilities of the new compounds to interfere with the self-aggregation of beta-amyloid (Abeta(42)) in the thioflavin test as well as their antioxidant activities in the ABTS and FRAP assays. We used molecular docking, molecular dynamics simulations, and quantum-chemical calculations to explain experimental results. All new compounds weakly inhibited AChE and off-target CES. Dihydroacridines with aryl substituents in the phosphoryl moiety inhibited BChE; the most active were the dibenzyloxy derivative 1d and its diphenethyl bioisostere 1e (IC(50) = 2.90 +/- 0.23 microM and 3.22 +/- 0.25 microM, respectively). Only one acridine, 2d, an analog of dihydroacridine, 1d, was an effective BChE inhibitor (IC(50) = 6.90 +/- 0.55 microM), consistent with docking results. Dihydroacridines inhibited Abeta(42) self-aggregation; 1d and 1e were the most active (58.9% +/- 4.7% and 46.9% +/- 4.2%, respectively). All dihydroacridines 1 demonstrated high ABTS(+)-scavenging and iron-reducing activities comparable to Trolox, but acridines 2 were almost inactive. Observed features were well explained by quantum-chemical calculations. ADMET parameters calculated for all compounds predicted favorable intestinal absorption, good blood-brain barrier permeability, and low cardiac toxicity. Overall, the best results were obtained for two dihydroacridine derivatives 1d and 1e with dibenzyloxy and diphenethyl substituents in the phosphoryl moiety. These compounds displayed high inhibition of BChE activity and Abeta(42) self-aggregation, high antioxidant activity, and favorable predicted ADMET profiles. Therefore, we consider 1d and 1e as lead compounds for further in-depth studies as potential anti-AD preparations.
The development of multi-target-directed ligands (MTDLs) would provide effective therapy of neurodegenerative diseases (ND) with complex and nonclear pathogenesis. A promising method to create such potential drugs is combining neuroactive pharmacophoric groups acting on different biotargets involved in the pathogenesis of ND. We developed a synthetic algorithm for the conjugation of indole derivatives and methylene blue (MB), which are pharmacophoric ligands that act on the key stages of pathogenesis. We synthesized hybrid structures and performed a comprehensive screening for a specific set of biotargets participating in the pathogenesis of ND (i.e., cholinesterases, NMDA receptor, mitochondria, and microtubules assembly). The results of the screening study enabled us to find two lead compounds (4h and 4i) which effectively inhibited cholinesterases and bound to the AChE PAS, possessed antioxidant activity, and stimulated the assembly of microtubules. One of them (4i) exhibited activity as a ligand for the ifenprodil-specific site of the NMDA receptor. In addition, this lead compound was able to bypass the inhibition of complex I and prevent calcium-induced mitochondrial depolarization, suggesting a neuroprotective property that was confirmed using a cellular calcium overload model of neurodegeneration. Thus, these new MB-cycloalkaneindole conjugates constitute a promising class of compounds for the development of multitarget neuroprotective drugs which simultaneously act on several targets, thereby providing cognitive stimulating, neuroprotective, and disease-modifying effects.
Alzheimer's disease (AD) is considered a modern epidemic because of its increasing prevalence worldwide and serious medico-social consequences, including the economic burden of treatment and patient care. The development of new effective therapeutic agents for AD is one of the most urgent and challenging tasks. To address this need, we used an aminoalkylene linker to combine the well-known anticholinesterase drug tacrine with antioxidant 2-tolylhydrazinylidene-1,3-diketones to create 3 groups of hybrid compounds as new multifunctional agents with the potential for AD treatment. Lead compounds of the new conjugates effectively inhibited acetylcholinesterase (AChE, IC(50) 0.24-0.34 M) and butyrylcholinesterase (BChE, IC(50) 0.036-0.0745 M), with weak inhibition of off-target carboxylesterase. Anti-AChE activity increased with elongation of the alkylene spacer, in agreement with molecular docking, which showed compounds binding to both the catalytic active site and peripheral anionic site (PAS) of AChE, consistent with mixed type reversible inhibition. PAS binding along with effective propidium displacement suggest the potential of the hybrids to block AChE-induced beta-amyloid aggregation, a disease-modifying effect. All of the conjugates demonstrated metal chelating ability for Cu(2+), Fe(2+), and Zn(2+), as well as high antiradical activity in the ABTS test. Non-fluorinated hybrid compounds 6 and 7 also showed Fe(3+) reducing activity in the FRAP test. Predicted ADMET and physicochemical properties of conjugates indicated good CNS bioavailability and safety parameters acceptable for potential lead compounds at the early stages of anti-AD drug development.
New conjugates of tacrine and salicylamide with alkylene spacers were synthesized and evaluated as potential multifunctional agents for Alzheimer's disease (AD). The compounds exhibited high acetylcholinesterase (AChE, IC(50) to 0.224microM) and butyrylcholinesterase (BChE, IC(50) to 0.0104microM) inhibitory activities. They were also rather poor inhibitors of carboxylesterase, suggesting a low tendency to exert potential unwanted drug-drug interactions in clinical use. The conjugates were mixed-type reversible inhibitors of both cholinesterases and demonstrated dual binding to the catalytic and peripheral anionic sites of AChE in molecular docking that, along with experimental results on propidium iodide displacement, suggest their potential to block AChE-induced beta-amyloid aggregation. The new conjugates exhibited high ABTS(.+) -scavenging activity. N-(6-(1,2,3,4-Tetrahydroacridin-9-ylamino)hexyl)salicylamide is a lead compound that also demonstrates metal chelating ability toward Cu(2+) , Fe(2+) and Zn(2+) . Thus, the new conjugates have displayed the potential to be multifunctional anti-AD agents for further development.
Using two ways of functionalizing amiridine-acylation with chloroacetic acid chloride and reaction with thiophosgene-we have synthesized new homobivalent bis-amiridines joined by two different spacers-bis-N-acyl-alkylene (3) and bis-N-thiourea-alkylene (5) -as potential multifunctional agents for the treatment of Alzheimer's disease (AD). All compounds exhibited high inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with selectivity for BChE. These new agents displayed negligible carboxylesterase inhibition, suggesting a probable lack of untoward drug-drug interactions arising from hydrolytic biotransformation. Compounds 3 with bis-N-acyl-alkylene spacers were more potent inhibitors of both cholinesterases compared to compounds 5 and the parent amiridine. The lead compounds 3a-c exhibited an IC(50)(AChE) = 2.9-1.4 microM, IC(50)(BChE) = 0.13-0.067 microM, and 14-18% propidium displacement at 20 microM. Kinetic studies of compounds 3a and 5d indicated mixed-type reversible inhibition. Molecular docking revealed favorable poses in both catalytic and peripheral AChE sites. Propidium displacement from the peripheral site by the hybrids suggests their potential to hinder AChE-assisted Abeta(42) aggregation. Conjugates 3 had no effect on Abeta(42) self-aggregation, whereas compounds 5c-e (m = 4, 5, 6) showed mild (13-17%) inhibition. The greatest difference between conjugates 3 and 5 was their antioxidant activity. Bis-amiridines 3 with N-acylalkylene spacers were nearly inactive in ABTS and FRAP tests, whereas compounds 5 with thiourea in the spacers demonstrated high antioxidant activity, especially in the ABTS test (TEAC = 1.2-2.1), in agreement with their significantly lower HOMO-LUMO gap values. Calculated ADMET parameters for all conjugates predicted favorable blood-brain barrier permeability and intestinal absorption, as well as a low propensity for cardiac toxicity. Thus, it was possible to obtain amiridine derivatives whose potencies against AChE and BChE equaled (5) or exceeded (3) that of the parent compound, amiridine. Overall, based on their expanded and balanced pharmacological profiles, conjugates 5c-e appear promising for future optimization and development as multitarget anti-AD agents.
A new series of conjugates of aminoadamantane and gamma-carboline, which are basic scaffolds of the known neuroactive agents, memantine and dimebon (Latrepirdine) was synthesized and characterized. Conjugates act simultaneously on several biological structures and processes involved in the pathogenesis of Alzheimer's disease and some other neurodegenerative disorders. In particular, these compounds inhibit enzymes of the cholinesterase family, exhibiting higher inhibitory activity against butyrylcholinesterase (BChE), but having almost no effect on the activity of carboxylesterase (anti-target). The compounds serve as NMDA-subtype glutamate receptor ligands, show mitoprotective properties by preventing opening of the mitochondrial permeability transition (MPT) pore, and act as microtubule stabilizers, stimulating the polymerization of tubulin and microtubule-associated proteins. Structure-activity relationships were studied, with particular attention to the effect of the spacer on biological activity. The synthesized conjugates showed new properties compared to their prototypes (memantine and dimebon), including the ability to bind to the ifenprodil-binding site of the NMDA receptor and to occupy the peripheral anionic site of acetylcholinesterase (AChE), which indicates that these compounds can act as blockers of AChE-induced beta-amyloid aggregation. These new attributes of the conjugates represent improvements to the pharmacological profiles of the separate components by conferring the potential to act as neuroprotectants and cognition enhancers with a multifunctional mode of action.
An expanded series of alkyl 2-arylhydrazinylidene-3-oxo-3-polyfluoroalkylpropionates (HOPs) 3 was obtained via Cu(OAc)(2)-catalyzed azo coupling. All were nanomolar inhibitors of carboxylesterase (CES), while moderate or weak inhibitors of acetylcholinesterase and butyrylcholinesterase. Steady-state kinetics studies showed that HOPs 3 are mixed type inhibitors of the three esterases. Molecular docking studies demonstrated that two functional groups in the structure of HOPs, trifluoromethyl ketone (TFK) and ester groups, bind to the CES active site suggesting subsequent reactions: formation of a tetrahedral adduct, and a slow hydrolysis reaction. The results of molecular modeling allowed us to explain some structure-activity relationships of CES inhibition by HOPs 3: their selectivity toward CES in comparison with cholinesterases and the high selectivity of pentafluoroethyl-substituted HOP 3p to hCES1 compared to hCES2. All compounds were predicted to have good intestinal absorption and blood-brain barrier permeability, low cardiac toxicity, good lipophilicity and aqueous solubility, and reasonable overall drug-likeness. HOPs with a TFK group and electron-donor substituents in the arylhydrazone moiety were potent antioxidants. All compounds possessed low cytotoxicity and low acute toxicity. Overall, a new promising type of bifunctional CES inhibitors has been found that are able to interact with the active site of the enzyme with the participation of two functional groups. The results indicate that HOPs have the potential to be good candidates as human CES inhibitors for biomedicinal applications.
We synthesized eleven new amiridine-piperazine hybrids 5a-j and 7 as potential multifunctional agents for Alzheimer's disease (AD) treatment by reacting N-chloroacetylamiridine with piperazines. The compounds displayed mixed-type reversible inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Conjugates were moderate inhibitors of equine and human BChE with negligible fluctuation in anti-BChE activity, whereas anti-AChE activity was substantially dependent on N4-substitution of the piperazine ring. Compounds with para-substituted aromatic moieties (5g, 5h, and bis-amiridine 7) had the highest anti-AChE activity in the low micromolar range. Top-ranked compound 5h, N-(2,3,5,6,7,8-hexahydro-1H-cyclopenta[b]quinolin-9-yl)-2-[4-(4-nitro-phenyl)-piperazin-1-yl]-acetamide, had an IC(50) for AChE = 1.83 +/- 0.03 microM (K(i) = 1.50 +/- 0.12 and alphaK(i) = 2.58 +/- 0.23 microM). The conjugates possessed low activity against carboxylesterase, indicating a likely absence of unwanted drug-drug interactions in clinical use. In agreement with analysis of inhibition kinetics and molecular modeling studies, the lead compounds were found to bind effectively to the peripheral anionic site of AChE and displace propidium, indicating their potential to block AChE-induced beta-amyloid aggregation. Similar propidium displacement activity was first shown for amiridine. Two compounds, 5c (R = cyclohexyl) and 5e (R = 2-MeO-Ph), exhibited appreciable antioxidant capability with Trolox equivalent antioxidant capacity values of 0.47 +/- 0.03 and 0.39 +/- 0.02, respectively. Molecular docking and molecular dynamics simulations provided insights into the structure-activity relationships for AChE and BChE inhibition, including the observation that inhibitory potencies and computed pK(a) values of hybrids were generally lower than those of the parent molecules. Predicted ADMET and physicochemical properties of conjugates indicated good CNS bioavailability and safety parameters comparable to those of amiridine and therefore acceptable for potential lead compounds at the early stages of anti-AD drug development.
New hybrid compounds of 4-amino-2,3-polymethylene-quinoline containing different sizes of the aliphatic ring and linked to p-tolylsulfonamide with alkylene spacers of increasing length were synthesized as potential drugs for treatment of Alzheimer's disease (AD). All compounds were potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with selectivity toward BChE. The lead compound 4-methyl-N-(5-(1,2,3,4-tetrahydro-acridin-9-ylamino)-pentyl)-benzenesulfonamide (7h) exhibited an IC(50) (AChE) = 0.131 +/- 0.01 muM (five times more potent than tacrine), IC(50)(BChE) = 0.0680 +/- 0.0014 muM, and 17.5 +/- 1.5% propidium displacement at 20 muM. The compounds possessed low activity against carboxylesterase, indicating a likely absence of unwanted drug-drug interactions in clinical use. Kinetics studies were consistent with mixed-type reversible inhibition of both cholinesterases. Molecular docking demonstrated dual binding sites of the conjugates in AChE and clarified the differences in the structure-activity relationships for AChE and BChE inhibition. The conjugates could bind to the AChE peripheral anionic site and displace propidium, indicating their potential to block AChE-induced beta-amyloid aggregation, thereby exerting a disease-modifying effect. All compounds demonstrated low antioxidant activity. Computational ADMET profiles predicted that all compounds would have good intestinal absorption, medium blood-brain barrier permeability, and medium cardiac toxicity risk. Overall, the results indicate that the novel conjugates show promise for further development and optimization as multitarget anti-AD agents.
New hybrids of 4-amino-2,3-polymethylenequinoline with different sizes of the aliphatic ring linked to butylated hydroxytoluene (BHT) by enaminoalkyl (7) or aminoalkyl (8) spacers were synthesized as potential multifunctional agents for Alzheimer's disease (AD) treatment. All compounds were potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with selectivity toward BChE. Lead compound 8c, 2,6-di-tert-butyl-4-{[2-(7,8,9,10- tetrahydro-6H-cyclohepta[b]quinolin-11-ylamino)-ethylimino]-methyl}-phenol exhibited an IC(50)(AChE) = 1.90 +/- 0.16 microM, IC(50)(BChE) = 0.084 +/- 0.008 microM, and 13.6 +/- 1.2% propidium displacement at 20 M. Compounds possessed low activity against carboxylesterase, indicating likely absence of clinically unwanted drug-drug interactions. Kinetics were consistent with mixed-type reversible inhibition of both cholinesterases. Docking indicated binding to catalytic and peripheral AChE sites; peripheral site binding along with propidium displacement suggest the potential of the hybrids to block AChE-induced beta-amyloid aggregation, a disease-modifying effect. Compounds demonstrated high antioxidant activity in ABTS and FRAP assays as well as inhibition of luminol chemiluminescence and lipid peroxidation in mouse brain homogenates. Conjugates 8 with amine-containing spacers were better antioxidants than those with enamine spacers 7. Computational ADMET profiles for all compounds predicted good blood-brain barrier distribution (permeability), good intestinal absorption, and medium cardiac toxicity risk. Overall, based on their favorable pharmacological and ADMET profiles, conjugates 8 appear promising as candidates for AD therapeutics.
Systemic inhibition of neuropathy target esterase (NTE) with certain organophosphorus (OP) compounds produces OP compound-induced delayed neurotoxicity (OPIDN), a distal degeneration of axons in the central nervous system (CNS) and peripheral nervous system (PNS), thereby providing a powerful model for studying a spectrum of neurodegenerative diseases. Axonopathies are important medical entities in their own right, but in addition, illnesses once considered primary neuronopathies are now thought to begin with axonal degeneration. These disorders include Alzheimer's disease, Parkinson's disease, and motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Moreover, conditional knockout of NTE in the mouse CNS produces vacuolation and other degenerative changes in large neurons in the hippocampus, thalamus, and cerebellum, along with degeneration and swelling of axons in ascending and descending spinal cord tracts. In humans, NTE mutations cause a variety of neurodegenerative conditions resulting in a range of deficits including spastic paraplegia and blindness. Mutations in the Drosophila NTE orthologue SwissCheese (SWS) produce neurodegeneration characterized by vacuolization that can be partially rescued by expression of wild-type human NTE, suggesting a potential therapeutic approach for certain human neurological disorders. This chapter defines NTE and OPIDN, presents an overview of OP compounds, provides a rationale for NTE research, and traces the history of discovery of NTE and its relationship to OPIDN. It then briefly describes subsequent studies of NTE, including practical applications of the assay; aspects of its domain structure, subcellular localization, and tissue expression; abnormalities associated with NTE mutations, knockdown, and conventional or conditional knockout; and hypothetical models to help guide future research on elucidating the role of NTE in OPIDN.
Title: Inhibition of Acetylcholinesterases by Stereoisomeric Organophosphorus Compounds Containing Both Thioester and p-Nitrophenyl Leaving Groups Talley TT, Chao CK, Berkman CE, Richardson RJ, Thompson CM Ref: Chemical Research in Toxicology, 33:2455, 2020 : PubMed
Studies with acetylcholinesterase (AChE) inhibited by organophosphorus (OP) compounds with two chiral centers can serve as models or surrogates for understanding the rate, orientation, and postinhibitory mechanisms by the nerve agent soman that possesses dual phosphorus and carbon chiral centers. In the current approach, stereoisomers of O-methyl, [S-(succinic acid, diethyl ester), O-(4-nitrophenyl) phosphorothiolate (MSNPs) were synthesized, and the inhibition, reactivation, and aging mechanisms were studied with electric eel AChE (eeAChE) and recombinant mouse brain AChE (rmAChE). The MSNP R(P)R(C) isomer was the strongest inhibitor of both eeAChE and rmAChE at 8- and 24-fold greater potency, respectively, than the weakest S(P)S(C) isomer. eeAChE inhibited by the R(P)R(C)- or R(P)S(C)-MSNP isomer underwent spontaneous reactivation -10- to 20-fold faster than the enzyme inhibited by S(P)R(C)- and S(P)S(C)-MSNP, and only 4% spontaneous reactivation was observed from the S(P)R(C)-eeAChE adduct. Using 2-pyridine aldoxime methiodide (2-PAM) or trimedoxime (TMB-4), eeAChE inhibited by R(P)R(C)- or S(P)R(C)-MSNP reactivated up to 90% and 3- to 4-fold faster than eeAChE inhibited by the R(P)S(C)- or S(P)S(C)-MSNP isomer. Spontaneous reactivation rates for rmAChE were 1.5- to 10-fold higher following inhibition by R(P)S(C)- and S(P)S(C)-MSNPs than inhibition by either R(C) isomer, a trend opposite to that found for eeAChE. Oxime reactivation of rmAChE following inhibition by R(P)R(C)- and S(P)R(C)-MSNPs was 2.5- to 5-fold faster than inhibition by R(P)S(C)- or S(P)S(C)-MSNPs. Due to structural similarities, MSNPs that phosphylate AChE with the loss of the p-nitrophenoxy (PNP) group form identical, nonreactivatable adducts to those formed from S(P)-isomalathion; however, all the MSNP isomers inhibited AChE to form adducts that reactivated. Thus, MSNPs inactivate AChE via the ejection of either PNP or thiosuccinyl groups to form a combination of reactivatable and nonreactivatable adducts, and this differs from the mechanism of AChE inhibition by isomalathion.
We studied the inhibitory activity of methylene blue (MB) gamma-carbolines (gC) conjugates (MB-gCs) against human erythrocyte acetylcholinesterase (AChE), equine serum butyrylcholinesterase (BChE), and a structurally related enzyme, porcine liver carboxylesterase (CaE). In addition, we determined the ability of MB-gCs to bind to the peripheral anionic site (PAS) of Electrophorus electricus AChE (EeAChE) and competitively displace propidium iodide from this site. Moreover, we examined the ability of MB-gCs to scavenge free radicals as well as their influence on mitochondrial potential and iron-induced lipid peroxidation. We found that MB-gCs effectively inhibited AChE and BChE with IC50 values in the range 1.73-10.5 muM and exhibited low potencies against CaE (9.8-26% inhibition at 20 muM). Kinetic studies showed that MB-gCs were mixed-type reversible inhibitors of both cholinesterases. Molecular docking results showed that the MB-gCs could bind both to the catalytic active site and to the PAS of human AChE and BChE. Accordingly, MB-gCs effectively displaced propidium from the peripheral anionic site of EeAChE. In addition, MB-gCs were extremely active in both radical scavenging tests. Quantum mechanical DFT calculations suggested that free radical scavenging was likely mediated by the sulfur atom in the MB fragment. Furthermore, the MB-gCs, in like manner to MB, can restore mitochondrial membrane potential after depolarization with rotenone. Moreover, MB-gCs possess strong antioxidant properties, preventing iron-induced lipid peroxidation in mitochondria. Overall, the results indicate that MB-gCs are promising candidates for further optimization as multitarget therapeutic agents for neurodegenerative diseases.
A series of 2-arylhydrazinylidene-3-oxo-4,4,4-trifluorobutanoic acids was synthesized via dealkylation of ethyl 2-arylhydrazinylidene-3-oxo-4,4,4-trifluorobutanoates under the action of a Lewis acid. Under the same conditions, ethyl 2-arylhydrazinylidene-3-oxobutanoates were also found to undergo dealkylation rather than the previously described cyclization into cinnolones. Study of the esterase profile of these compounds showed that trifluoromethyl-containing acids, in contrast to non-fluorinated analogs, were effective and selective inhibitors of carboxylesterase (CES), without substantially inhibiting structurally related cholinesterases (acetylcholinesterase and butyrylcholinesterase). Moreover, both 3-oxo-4,4,4-trifluorobutanoic and 3-oxobutanoic acids having methyl or methoxy substituent in the arylhydrazinylidene fragment showed high antioxidant activity in the ABTS test. Thus, 2-arylhydrazinylidene-3-oxo-4,4,4-trifluorobutanoic acids were found to constitute a new class of effective and selective CES inhibitors that also possess high radical-scavenging activity.
To search for effective and selective inhibitors of carboxylesterase (CES), a series of 3-oxo-2-tolylhydrazinylidene-4,4,4-trifluorobutanoates bearing higher or natural alcohol moieties was synthesized via pre-transesterification of ethyl trifluoroacetylacetate with alcohols to isolate transesterificated oxoesters as lithium salts, which were then subjected to azo coupling with tolyldiazonium chloride. Inhibitory activity against porcine liver CES, along with two structurally related serine hydrolases, acetylcholinesterase and butyrylcholinesterase, were investigated using enzyme kinetics and molecular docking. Kinetics studies demonstrated that the tested keto-esters are reversible and selective mixed-type CES inhibitors. Analysis of X-ray crystallographic data together with our IR and NMR spectra and QM calculations indicated that the Z-isomers were the most stable. The kinetic data were well explained by the molecular docking results of the Z-isomers, which showed specific binding of the compounds in the CES catalytic active site with carbonyl oxygen atoms in the oxyanion hole and non-specific binding outside it. Some compounds were studied as inhibitors of the main human isozymes involved in biotransformation of ester-containing drugs, hCES1 and hCES2. Esters of geraniol (3d) and adamantol (3e) proved to be highly active and selective inhibitors of hCES2, inhibiting the enzyme in the nanomolar range, whereas esters of borneol (3f) and isoborneol (3g) were more active and selective against hCES1. Computational ADMET studies revealed that all test compounds had excellent intestinal absorption, medium blood-brain barrier permeability, and low hERG liability risks. Moreover, all test compounds possessed radical-scavenging properties and low acute toxicity. Overall, the results indicate that members of this novel series of esters have the potential to be good candidates as hCES1 or hCES2 inhibitors for biomedicinal applications.
Alzheimer's disease (AD) is a multifactorial neurodegenerative process whose effective treatment will require drugs that can act simultaneously on multiple pathogenic targets. Here, we present an overview of our previous multitarget studies of five groups of novel hybrid structures that combine, through spacers, five pharmacophores that have been found promising for AD treatment: gamma-carbolines, carbazoles, tetrahydrocarbazoles, phenothiazines, and aminoadamantanes. Biological activity of the compounds was assessed by a battery of assays. These included inhibitory potency against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) as indicators of potential for cognition enhancement and against carboxylesterase (CaE) to exclude unwanted inhibition of this biotransformation pathway. Displacement of propidium from the peripheral anionic site of AChE was determined as a predictor of anti-aggregation activity. Binding to the two sites of the NMDA subtype of the glutamate receptor was conducted as an additional indicator of potential cognition enhancement and neuroprotection. Propensity to protect against mitochondrial triggers of cell death was evaluated by tests of mitochondrial potential and calcium-induced swelling as indicators of mitochondrial permeability transition. Antioxidant potential was measured to evaluate the tendency to prevent oxidative stress. Potential for disease modification was gauged by the ability to stimulate microtubule assembly. Finally, binding modes of conjugates to AChE and BChE were studied using quantum mechanical-assisted molecular docking. We found selective BChE inhibitors (conjugates of gamma-carbolines and phenothiazine I, gamma-carbolines and carbazoles II, and aminoadamantanes and carbazoles III) as well as inhibitors of both cholinesterases (conjugates of gamma-carbolines and methylene blue IV and bis-gamma-carbolines with ditriazole-containing spacers V). These compounds combined potentials for cognition enhancement, neuroprotection, and disease modification. None of the conjugates exhibited high potency against CaE, thereby precluding potential drug-drug interactions from CaE inhibition. Thus, the studied compounds exhibited positive characteristics of multitarget drugs, indicating their potential for the next generation of AD therapeutics.
We synthesized conjugates of tacrine with 1,2,4-thiadiazole derivatives linked by two different spacers, pentylaminopropene (compounds 4) and pentylaminopropane (compounds 5), as potential drugs for the treatment of Alzheimer's disease (AD). The conjugates effectively inhibited cholinesterases with a predominant effect on butyrylcholinesterase (BChE). They were also effective at displacing propidium from the peripheral anionic site (PAS) of acetylcholinesterase (AChE), suggesting that they could block AChE-induced beta-amyloid aggregation. In addition, the compounds exhibited high radical-scavenging capacity. Conjugates 5 had higher anti-BChE activity and greater anti-aggregant potential as well relatively lower potency against carboxylesterase than compounds 4. Quantum-mechanical (QM) characterization agreed with NMR data to identify the most stable forms of conjugates for docking studies, which showed that the compounds bind to both CAS and PAS of AChE consistent with mixed reversible inhibition. Conjugates 4 were more potent radical scavengers, in agreement with HOMO localization in the enamine-thiadiazole system. Computational studies showed that all of the conjugates were expected to have good intestinal absorption, whereas conjugates 4 and 5 were predicted to have medium and high blood-brain barrier permeability, respectively. All conjugates were predicted to have medium cardiac toxicity risks. Overall, the results indicated that the conjugates are promising candidates for further development and optimization as multifunctional therapeutic agents for the treatment of AD.
We investigated the biological activity of a series of substituted chromeno[3,2-c]pyridines, including compounds previously synthesized by our group and novel compounds whose syntheses are reported here. Tandem transformation of their tetrahydropyridine ring under the action of activated alkynes yielding 2-vinylsubstituted chromones was used to prepare nitrogen-containing derivatives of a biologically active chromone system. The inhibitory activity of these chromone derivatives against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE) was investigated using the methods of enzyme kinetics and molecular docking. Antioxidant (antiradical) activity of the compounds was assessed in the ABTS assay. The results demonstrated that a subset of the studied chromone derivatives selectively inhibit BChE but do not exhibit antiradical activity. In addition, the results of molecular docking effectively explained the observed features in the efficacy, selectivity, and mechanism of BChE inhibition by the chromone derivatives.
We investigated the inhibitory activity of 4 groups of novel acridine derivatives against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE) using the methods of enzyme kinetics and molecular docking. Antioxidant activity of the compounds was determined using the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(+)) radical decolorization assay as their ability to scavenge free radicals. Analysis of the esterase profiles and antiradical activities of the acridine derivatives showed that 9-aryl(heteroaryl)-N-methyl-9,10-dihydroacridines have a high radical-scavenging activity but low potency as AChE and BChE inhibitors, whereas 9-aryl(heteroaryl)-N-methyl-acridinium tetrafluoroborates effectively inhibit cholinesterases but do not exhibit antiradical activity. In contrast, a group of derivatives of 9-heterocyclic amino-N-methyl-9,10-dihydroacridine has been found that combine effective inhibition of AChE and BChE with rather high radical-scavenging activity. The results of molecular docking well explain the observed features in the efficacy, selectivity, and mechanism of cholinesterase inhibition by the acridine derivatives. Thus, in a series of acridine derivatives we have found compounds possessing dual properties of effective and selective cholinesterase inhibition together with free radical scavenging, which makes promising the use of the acridine scaffold to create multifunctional drugs for the therapy of neurodegenerative diseases.
To search for effective and selective inhibitors of carboxylesterase (CaE), a series of 7-hydroxy-7-polyfluoroalkyl-4,7-dihydroazolo[5,1-c][1,2,4]triazines has been synthesized. Their inhibitory activity against acetylcholinesterase, butyrylcholinesterase, and CaE were investigated using the methods of enzyme kinetics and molecular docking. It was shown that the tested compounds are reversible selective CaE inhibitors of mixed type. Elongation of the polyfluoroalkyl substituent and the presence of an ester, preferably the ethoxycarbonyl group, enhance inhibitory activity toward CaE. Furthermore, the compounds with a tetrazole ring are more active against CaE than their triazole analogues. The obtained kinetic data are well explained by the results of molecular docking, according to which there is a similar orientation of triazolo- and tetrazolotriazines in the active site of CaE and the opposite one for pyrazolotriazines. In the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay, all of the studied tetrazolotriazines and some pyrazolotriazines demonstrated good antiradical activity comparable with a standard antioxidant, Trolox. The leading compounds were nonafluorobutyl substituted tetrazolo- and 7-phenylpyrazolotriazines, which possess effective and selective CaE inhibitory activity as well as additional useful radical-scavenging properties.
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 0.22-2.3muM. 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 (<30muM), 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.
We studied 4 serine esterases (EOHs) that are associated with the following consequences from their inhibition by organophosphorus compounds (OPCs): acetylcholinesterase (AChE: acute neurotoxicity; cognition enhancement), butyrylcholinesterase (BChE: inhibition of drug metabolism and/or stoichiometric scavenging of EOH inhibitors; cognition enhancement), carboxylesterase (CaE; inhibition of drug metabolism and/or stoichiometric scavenging of EOH inhibitors), and neuropathy target esterase (NTE: delayed neurotoxicity, OPIDN). The relative degree of inhibition of these EOHs constitutes the "esterase profile" of an OPC, which we hypothesize can serve as a predictor of its overall physiological effects. To test this hypothesis, we selected 3 OPCs known from previous work on reference enzymes to span a wide range of esterase profiles, neuropathic potential, and acute cholinergic toxicity. For each compound, we determined in vitro IC50 and in vivo ED50 values for inhibition of AChE, BChE, CaE, and NTE in mouse brain and blood. The results showed good correlations between in vitro and in vivo measures of potency and selectivity except for brain CaE, a tissue-specific isoform of the enzyme that was less sensitive to the test compounds than expected. Thus, this synthesis of new and previously published results indicates that the concept of the esterase profile of OPCs is useful for the prediction of therapeutic and toxic effects in vivo.
The adult hen is the standard animal model for testing organophosphorus (OP) compounds for organophosphorus compound-induced delayed neurotoxicity (OPIDN). Recently, we developed a mouse model for biochemical assessment of the neuropathic potential of OP compounds based on brain neuropathy target esterase (NTE) and acetylcholinesterase (AChE) inhibition. We carried out the present work to further develop the mouse model by testing the hypothesis that whole blood NTE inhibition could be used as a biochemical marker for exposure to neuropathic OP compounds. Because brain NTE and AChE inhibition are biomarkers of OPIDN and acute cholinergic toxicity, respectively, we compared NTE and AChE 20-min IC50 values as well as ED50 values 1 h after single intraperitoneal (i.p.) injections of increasing doses of two neuropathic OP compounds that differed in acute toxicity potency. We found good agreement between the brain and blood for in vitro sensitivity of each enzyme as well for the ratios IC50 (AChE)/IC50 (NTE). Both OP compounds inhibited AChE and NTE in the mouse brain and blood dose-dependently, and brain and blood inhibitions in vivo were well correlated for each enzyme. For both OP compounds, the ratio ED50 (AChE)/ED50 (NTE) in blood corresponded to that in the brain despite the somewhat higher sensitivity of blood enzymes. Thus, our results indicate that mouse blood NTE could serve as a biomarker of exposure to neuropathic OP compounds. Moreover, the data suggest that relative inhibition of blood NTE and AChE provide a way to assess the likelihood that OP compound exposure in a susceptible species would produce cholinergic and/or delayed neuropathic effects. Copyright (c) 2016 John Wiley & Sons, Ltd.
BACKGROUND: Oliver-McFarlane syndrome is characterised by trichomegaly, congenital hypopituitarism and retinal degeneration with choroidal atrophy. Laurence-Moon syndrome presents similarly, though with progressive spinocerebellar ataxia and spastic paraplegia and without trichomegaly. Both recessively inherited disorders have no known genetic cause. METHODS: Whole-exome sequencing was performed to identify the genetic causes of these disorders. Mutations were functionally validated in zebrafish pnpla6 morphants. Embryonic expression was evaluated via in situ hybridisation in human embryonic sections. Human neurohistopathology was performed to characterise cerebellar degeneration. Enzymatic activities were measured in patient-derived fibroblast cell lines. RESULTS: Eight mutations in six families with Oliver-McFarlane or Laurence-Moon syndrome were identified in the PNPLA6 gene, which encodes neuropathy target esterase (NTE). PNPLA6 expression was found in the developing human eye, pituitary and brain. In zebrafish, the pnpla6 curly-tailed morphant phenotype was fully rescued by wild-type human PNPLA6 mRNA and not by mutation-harbouring mRNAs. NTE enzymatic activity was significantly reduced in fibroblast cells derived from individuals with Oliver-McFarlane syndrome. Intriguingly, adult brain histology from a patient with highly overlapping features of Oliver-McFarlane and Laurence-Moon syndromes revealed extensive cerebellar degeneration and atrophy. CONCLUSIONS: Previously, PNPLA6 mutations have been associated with spastic paraplegia type 39, Gordon-Holmes syndrome and Boucher-Neuhauser syndromes. Discovery of these additional PNPLA6-opathies further elucidates a spectrum of neurodevelopmental and neurodegenerative disorders associated with NTE impairment and suggests a unifying mechanism with diagnostic and prognostic importance.
Title: A review of epidemiologic studies of low-level exposures to organophosphorus insecticides in non-occupational populations Reiss R, Chang ET, Richardson RJ, Goodman M Ref: Crit Rev Toxicol, 45:531, 2015 : PubMed
This paper systematically reviews epidemiologic studies related to low-level non-occupational exposures to organophosphorus (OP) insecticides. Many of the studies evaluate levels of maternal OP metabolites and subsequent health outcomes in offspring. The studies focused primarily on birth outcomes (e.g., infant body weight or head circumference) and neurodevelopmental (e.g., mental and psychomotor) testing results. The evidence from these studies was reviewed under the Bradford Hill guidelines. Most of the studies assessing exposure based on urinary levels of OP insecticide metabolites used only one or two measurements during pregnancy. The potential for exposure misclassification with this method is largely due to (1) preformed metabolites that are ingested with food, (2) the short elimination half-life of OP insecticides, and (3) lack of specificity to particular OP insecticides for many of the metabolites. For birth outcomes, the majority of reported results are not statistically significant, and the associations are inconsistent within and across studies. There is more within-study consistency for some of the neurodevelopmental testing results, although few associations were examined across several studies. These associations are generally weak, have been replicated only to a limited extent, and require further confirmation before they can be considered established. The OP insecticide levels measured in the epidemiologic studies are too low to cause biologically meaningful acetylcholinesterase inhibition, the most widely used metric for OP insecticide toxicity. Overall, the available evidence does not establish that low-level exposures to OP insecticides cause adverse birth outcomes or neurodevelopmental problems in humans.
BACKGROUND: Exposure to chlorpyrifos (CPF), an organophosphorus (OP) anticholinesterase insecticide, occurs typically in settings where multiple agents are present (e.g., agriculture) and quantitative dose measures may be absent (e.g., pesticide application). Such exposures allow few opportunities to study potential neurobehavioral effects of CPF alone. We studied the relationship between CPF exposure and behavioral function among CPF manufacturing workers, which allowed identification, measurement, and estimation of exposure and important non-exposure variables that potentially could affect study findings. METHODS: A prospective longitudinal study design was used to compare neurobehavioral function over a one-year period among 53 CPF workers and 60 referent workers. Quantitative and qualitative measures were used, and potential confounders were identified and tested for possible inclusion in our statistical models. Neurobehavioral function was assessed by neuropsychological tests covering various behavioral domains that may be adversely affected by exposure to CPF in sufficient amount. RESULTS: CPF workers had significantly greater CPF exposures during the study period than did referents at levels where physiologic effects on plasma butyrylcholinesterase (BCHE) activity were apparent and with higher 3,5,6-trichloro-2-pyridinol (TCPy/Cr) urinary excretion (p<0.0001) and lower average BCHE activity (p<0.01). No evidence for impaired neurobehavioral domains by either group of workers was observed at baseline, on repeat examination, or between examinations. CPF workers scored higher than referent workers on the verbal memory domain score (p=0.03) at baseline, but there were no significant changes in verbal memory over time and no significant group-by-time interactions. CONCLUSIONS: The study provides important information about CPF exposure in the workplace by not supporting our working hypothesis that CPF exposure associated with various aspects of the manufacturing process would be accompanied by adverse neurobehavioral effects detectable by quantitative neurobehavioral testing. Some aspects making this workplace site attractive for study and also present limitations for the generalization of results to other situations that might have exposures that vary widely between and within different facilities and locations. For example, these results might not apply to occupations such as applicators with higher exposure or to workers with low educational levels.
Inhibition and aging of neuropathy target esterase (NTE) by neuropathic organophosphorus (OP) compounds triggers OP compound-induced delayed neuropathy (OPIDN), whereas inhibition of acetylcholinesterase (AChE) produces cholinergic toxicity. The neuropathic potential of an OP compound is defined by its relative inhibitory potency toward NTE vs. AChE assessed by enzyme assays following dosing in vivo or after incubations of direct-acting compounds or active metabolites with enzymes in vitro. The standard animal model of OPIDN is the adult hen, but its large size and high husbandry costs make this species a burdensome model for assessing neuropathic potential. Although the mouse does not readily exhibit clinical signs of OPIDN, it displays axonal lesions and expresses brain AChE and NTE. Therefore, the present research was performed as a further test of the hypothesis that inhibition of mouse brain AChE and NTE could be used to assess neuropathic potential using mouse brain preparations in vitro or employing mouse brain assays following dosing of OP compounds in vivo. Excellent correlations were obtained for inhibition kinetics in vitro of mouse brain enzymes vs. hen brain and human recombinant enzymes. Furthermore, inhibition of mouse brain AChE and NTE after dosing with OP compounds afforded ED50 ratios that agreed with relative inhibitory potencies assessed in vitro. Taken together, results with mouse brain enzymes demonstrated consistent correspondence between in vitro and in vivo predictors of neuropathic potential, thus adding to previous studies supporting the validity of a mouse model for biochemical assessment of the ability of OP compounds to produce OPIDN. Copyright (c) 2014 John Wiley & Sons, Ltd.
Title: Crystal Structure of Patatin-17 in Complex with Aged and Non-Aged Organophosphorus Compounds Wijeyesakere SJ, Richardson RJ, Stuckey JA Ref: PLoS ONE, 9:e108245, 2014 : PubMed
Patatin is a non-specific plant lipase and the eponymous member of a broad class of serine hydrolases termed the patatin-like phospholipase domain containing proteins (PNPLAs). Certain PNPLA family members can be inhibited by organophosphorus (OP) compounds. Currently, no structural data are available on the modes of interaction between the PNPLAs and OP compounds or their native substrates. To this end, we present the crystal structure of patatin-17 (pat17) in its native state as well as following inhibition with methyl arachidonyl fluorophosphonate (MAFP) and inhibition/aging with diisopropylphosphorofluoridate (DFP). The native pat17 structure revealed the existence of two portals (portal1 and portal2) that lead to its active-site chamber. The DFP-inhibited enzyme underwent the aging process with the negatively charged phosphoryl oxygen, resulting from the loss of an isopropyl group, being within hydrogen-binding distance to the oxyanion hole. The MAFP-inhibited pat17 structure showed that MAFP did not age following its interaction with the nucleophilic serine residue (Ser77) of pat17 since its O-methyl group was intact. The MAFP moiety is oriented with its phosphoryl oxygen in close proximity to the oxyanion hole of pat17 and its O-methyl group located farther away from the oxyanion hole of pat17 relative to the DFP-bound state. The orientation of the alkoxy oxygens within the two OP compounds suggests a role for the oxyanion hole in stabilizing the emerging negative charge on the oxygen during the aging reaction. The arachidonic acid side chain of MAFP could be contained within portals 1 or 2. Comparisons of pat17 in the native, inhibited, and aged states showed no significant global conformational changes with respect to their Calpha backbones, consistent with observations from other alpha/beta hydrolases such as group VIIA phospholipase A2.
Certain organophosphorus compounds (OPCs) inhibit various serine esterases (EOHs) via phosphorylation of their active site serines. We focused on 4 EOHs of particular toxicological interest: acetylcholinesterase (AChE: acute neurotoxicity; cognition enhancement), butyrylcholinesterase (BChE: inhibition of drug metabolism and/or stoichiometric scavenging of EOH inhibitors; cognition enhancement), carboxylesterase (CaE: inhibition of drug metabolism and/or stoichiometric scavenging of EOH inhibitors), and neuropathy target esterase (NTE: delayed neurotoxicity, OPIDN). The relative degree of inhibition of these EOHs constitutes the "esterase profile" of an OPC and serves as a major determinant of its net physiological effects. Thus, understanding and controlling the esterase profile of OPC activity and selectivity toward these 4 target enzymes is a significant undertaking. In the present study, we analyzed the inhibitor properties of 52 OPCs against the 4 EOHs, along with pairwise and multitarget selectivities between them, using 2 QSAR approaches: Hansch modeling and Molecular Field Topology Analysis (MFTA). The general formula of the OPCs was (RO)2P(O)X, where R=alkyl, X=- SCH(Hal)COOEt (Hal=Cl, Br), -SCHCl2, -SCH2Br, -OCH(CF3)R(1) (R(1)=C6H5, CF3, COOEt, COOMe). The Hansch model showed that increasing neuropathic potential correlated with rising R hydrophobicity; moreover, OPC binding to scavenger EOHs (BChE and CaE) had different effects on potential acute and delayed neurotoxicity. Predicted protective roles of BChE and CaE against acute toxicity were enhanced with increasing hydrophobicity, but projected protection against OPIDN was decreased. Next, Molecular Field Topology Analysis (MFTA) models were built, considering atomic descriptors, e.g., effective charge, van der Waals radius of environment, and group lipophilicity. Activity/selectivity maps confirmed predictions from Hansch models and revealed other structural factors affecting activity and selectivity. Virtual screening based on multitarget selectivity MFTA models was used to design libraries of OPCs with favorable esterase profiles for potential application as selective inhibitors of CaE without untoward side effects.
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.
Title: Combined QSAR studies of inhibitor properties of O-phosphorylated oximes toward serine esterases involved in neurotoxicity, drug metabolism and Alzheimer's disease Makhaeva GF, Radchenko EV, Baskin II, Palyulin VA, Richardson RJ, Zefirov NS Ref: SAR QSAR Environ Research, 23:627, 2012 : PubMed
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 Alzheimer's disease.
Chlorpyrifos is an organophosphorus (OP) anticholinesterase insecticide. Paraoxonase (PON1) is an enzyme found in liver and plasma that hydrolyzes a number of OP compounds. PON1 polymorphisms include a glutamine (Q)/arginine (R) substitution at position 192 (PON1(Q192R)) that affects hydrolysis of OP substrates, with the PON1(192Q) allotype hydrolyzing chlorpyrifos oxon less efficiently than the PON1(192R) allotype, a variation potentially important in determining susceptibility to chlorpyrifos. We studied 53 chlorpyrifos workers and 60 referents during 1 year and estimated chlorpyrifos exposure using industrial hygiene and employment records and excretion of the chlorpyrifos metabolite 3,5,6-trichloro-2-pyridinol (TCP). Plasma butyrylcholinesterase (BuChE) activity, which may by inhibited by chlorpyrifos exposure, was measured monthly. In addition, plasma samples were assayed for paraoxonase (PONase), diazoxonase (DZOase), and chlorpyrifosoxonase (CPOase) activity to determine PON1 status (inferred genotypes and their functional activity). Linear regression analyses modeled BuChE activity as a function of chlorpyrifos exposure and covariates. We postulated that the level of CPOase activity and the inferred PON1(192) genotype (together reflecting PON1 status) would differ between groups and that PON1 status would modify the models of chlorpyrifos exposure on BuChE activity. Chlorpyrifos workers and referents had a 100-fold difference in cumulative chlorpyrifos exposure. Contrary to our hypotheses, mean CPOase activity was similar in both groups (P=0.58) and PON1(192Q) showed a slight overrepresentation, not an underrepresentation, in the chlorpyrifos group compared with referents (PON1(192QQ), 51% chlorpyrifos, 40% referent; PON(192QR), 43% chlorpyrifos, 40% referent; PON(192RR), 6% chlorpyrifos, 20% referent, P=0.08). In our models, BuChE activity was significantly inversely associated with measures of interim chlorpyrifos exposure, but the biological effects of chlorpyrifos exposure on BuChE activity were not modified by PON1 inferred genotype or CPOase activity.
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.
This paper reviews our previously published data and presents new results on biosensor assay of blood esterases. Tyrosinase and choline oxidase biosensors based on nanostructured polyelectrolyte films were developed for these purposes. Experiments were performed on the quantitative determination of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), carboxylesterase (CaE), and neuropathy target esterase (NTE) in samples of whole blood of rats, mice, and humans. Good agreement was found between biosensor and spectrophotometric assays for AChE, BChE, and CaE. No direct comparison could be made for NTE because its activity cannot be measured spectrophotometrically in whole blood. A new method of simultaneous quantitative determination of AChE and BChE in test mixtures is also described. This method represents a bifunctional biosensor for the simultaneous analysis of choline and phenol based on integration of individual sensors. Algorithms for calculation of separate concentrations of AChE and BChE in the mixture were developed. The mean error of calculated component concentrations was approximately 6% for binary test mixtures. The present work provides a foundation for building multiplexed systems for the simultaneous determination of multiple esterases with applications to biomonitoring for exposures to organophosphorus compounds.
Title: Synthesis of organophosphates with fluorine-containing leaving groups as serine esterase inhibitors with potential for Alzheimer disease therapeutics Makhaeva GF, Aksinenko AY, Sokolov VB, Serebryakova OG, Richardson RJ Ref: Bioorganic & Medicinal Chemistry Lett, 19:5528, 2009 : PubMed
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.
The title compound, C(13)H(16)F(6)NO(5)PS, is of inter-est with respect to inhibition of serine hydro-lases. Its structure contains a 1.8797 (13) A P-C bond and two inter-molecular N-Hcdots, three dots, centeredO=P hydrogen bonds, resulting in centrosymmetric dimers. An intra-molecular N-Hcdots, three dots, centeredO=P hydrogen bond is also present.
Title: Mechanism of aging of mipafox-inhibited butyrylcholinesterase Kropp TJ, Richardson RJ Ref: Chemical Research in Toxicology, 20:504, 2007 : PubMed
Elucidating mechanisms of aging of esterases inhibited by organophosphorus (OP) compounds is important for understanding toxicity and developing biomarkers of exposure to these agents. Aging has classically been thought to involve net loss of a single side group from the OP moiety of phosphylated esterases, rendering the enzyme refractory to reactivation. However, recent evidence has shown that acetylcholinesterase (AChE) and the catalytic domain of human neuropathy target esterase (NEST) undergo aging by alternative mechanisms following their inhibition with N,N'-diisopropylphosphorodiamidofluoridate (mipafox, MIP). This study was performed to determine whether MIP-inhibited butyrylcholinesterase (BChE) ages conventionally, by net loss of a single side group, or by an alternate route, e.g., reversible deprotonation or displacement of both isopropylamine groups, as recently observed for MIP-inhibited NEST and AChE, respectively. Diisopropylphosphorofluoridate (DFP), the phosphate analogue of the phosphoroamidate MIP, was used for comparison. Kinetic values for MIP against BChE were as follows: ki = (1.28 +/- 0.053) x 10(6) M-1 min-1; k3 = 0.004,15 +/- 0.000,27 min-1; k4 = 0.008,49 +/- 0.000,99 min-1. Kinetic values for DFP against BChE were as follows: ki = (1.83 +/- 0.18) x 10(6) M-1 min-1; k3 = 0.004,88 +/- 0.000,24 min-1; k4 = 0.0121 +/- 0.0028 min-1. Mass spectrometric studies revealed a mass shift of 123.4 +/- 0.7 Da for the active-site peptide peak of aged DFP-inhibited BChE, corresponding to a monoisopropylphosphate adduct. Similarly, the analogous mass shift for aged MIP-inhibited BChE was 122.4 +/- 0.7 Da, corresponding to a monoisopropylphosphoroamido adduct. Therefore, we conclude that the MIP-BChE conjugate ages by loss of a single isopropylamine group, in contrast to MIP-inhibited AChE or NEST.
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 microL), 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.
Title: Aging of mipafox-inhibited human acetylcholinesterase proceeds by displacement of both isopropylamine groups to yield a phosphate adduct Kropp TJ, Richardson RJ Ref: Chemical Research in Toxicology, 19:334, 2006 : PubMed
Aging of phosphylated serine esterases, e.g., acetylcholinesterase (AChE) and neuropathy target esterase (NTE), renders the inhibited enzymes refractory to reactivation. This process has been considered to require postinhibitory side group loss from the organophosphorus moiety. Recently, however, it has been shown that the catalytic domain of human NTE inhibited by N,N'-diisopropylphosphorodiamidofluoridate (mipafox, MIP) ages by deprotonation. For mechanistic understanding and biomarker development, it would be important to know the identity of the MIP adduct on target esterases after inhibition and aging occurred. Accordingly, the present study was performed to determine if MIP-inhibited human AChE ages by side group loss or an alternate method, e.g., deprotonation. Diisopropylphosphorofluoridate (DFP), the oxygen analogue of MIP, was used for comparison, because DFP-inhibited AChE is known to age by net loss of an isopropyl group. Kinetics experiments were done with DFP and MIP against AChE to follow the time course of inhibition, reactivation, and aging for each inhibitor. MS studies of tryptic digests from kinetically aged DFP-inhibited AChE revealed a mass shift of 122.8 +/- 0.7 Da for the active site peptide (ASP) peak, corresponding to the expected monoisopropylphosphoryl adduct. In contrast, the analogous mass shift for kinetically aged MIP-inhibited AChE was 80.7 +/- 0.9 Da, corresponding to a phosphate adduct. Because this finding was unexpected, the identity of the phosphoserine-containing ASP was confirmed by immunoprecipitation followed by MS. The results indicate that aging of MIP-inhibited AChE proceeds by displacement of both isopropylamine groups. Further research will be required to elucidate the detailed mechanism of formation of a phosphate conjugate from MIP-inhibited AChE; however, knowledge of the identity of this adduct will be useful in biomarker studies.
A graphite-paste tyrosinase biosensor was improved by adding 1-methoxyphenazine methosulfate as a mediator. Mediator modification enhanced sensitivity to phenol 4-fold and long-term stability 3-fold. Phenol could be detected at 25 nM (S/N = 2) using an Ag/AgCl reference electrode. The biosensor was used to measure the activity of a toxicologically significant enzyme, neuropathy target esterase (NTE), which yields phenol by hydrolysis of the substrate, phenyl valerate. Using the new biosensor, blood and brain NTE inhibition by organophosphorus (OP) compounds with different neuropathic potencies were well correlated (r = 0.990, n = 7), supporting the use of blood NTE as a biochemical marker of exposure to neuropathic OP compounds.
Questions persist about adverse effects such as impaired cognition and attention, incoordination, spasticity, or parkinsonism from chronic, low-level exposures to organophosphate (OP) compounds. In a prospective cohort study, we evaluated chlorpyrifos-manufacturing workers and a referent group on 2 occasions, 1 year apart, to determine whether occupational exposure to chlorpyrifos produced clinically evident central nervous system (CNS) dysfunction. Chlorpyrifos subjects had significantly higher TCP excretion and lower average BuChE activity than referents in a range in which physiological effects on B-esterases exist. Few subjects had neurologic symptoms or signs, and there were no significant group differences in terms of signs at baseline or second examinations. Chronic chlorpyrifos exposure produced no clinical evidence of cortical, pyramidal tract, extrapyramidal, or other CNS dysfunction among chlorpyrifos subjects compared with referents, either at baseline or after 1 year of additional chlorpyrifos exposure.
Several studies have reported the occurrence of sensory neuropathy with exposure to chlorpyrifos and other organophosphorus insecticides, at levels not associated with overt toxicity. We evaluated 113 chemical workers, including 53 of 66 (80%) eligible chlorpyrifos workers and 60 of 74 (81%) randomly selected referent workers, to identify evidence of sensory neuropathy or subclinical neuropathy. Compared to referents, chlorpyrifos subjects had significantly longer duration of work in chlorpyrifos-exposed areas (9.72 vs. 0.01 years; P < 0.0001), greater cumulative chlorpyrifos exposure (64.16 vs. 0.69 mg/m(3). day; P < 0.0001), higher urine 3,5,6-trichloro-2-pyridinol (TCP) excretion (108.6 vs. 4.3 microg/g creatinine; P < 0.0001), and lower plasma butyrylcholinesterase (BuChE) activity (7281 vs. 8176 mU/ml; P = 0.003). Despite exposures among chlorpyrifos subjects to levels at which well-described physiological effects on B-esterases exist, the frequency of symptoms or signs of neuropathy did not differ significantly between groups, and the only 2 subjects fulfilling criteria for confirmed neuropathy were both in the referent group. Mean nerve conduction study results were comparable to established control values and did not differ significantly between groups. We found no evidence of sensory neuropathy or isolated peripheral abnormalities among subjects with long-term chlorpyrifos exposure at levels known to be associated with the manufacturing process.
AIMS: To determine whether chronic occupational exposure to chlorpyrifos at levels associated with various aspects of manufacturing produced a clinically evident or subclinical peripheral neuropathy. METHODS: Clinical and quantitative nerve conduction study (NCS) examinations were performed on two occasions on chlorpyrifos manufacturing workers who had measurable chlorpyrifos exposure and a referent group. Baseline evaluations were performed on 53 of 66 eligible chlorpyrifos subjects and on 60 of 74 eligible referent subjects; one-year evaluations were completed on 111 of the 113 subjects evaluated at baseline. RESULTS: Chlorpyrifos and referent groups differed significantly in measures of 3,5,6 trichloro-2-pyridinol excretion and plasma butyrylcholinesterase (BuChE) activity, indicating substantially higher exposures among chlorpyrifos subjects. Few subjects had clinically important neurological symptoms or signs. NCS results were comparable to control values, and there were no significant group differences in NCS results at baseline, one year, or change over one year. No chlorpyrifos subject fulfilled conventional criteria for confirmed peripheral neuropathy at baseline or one-year examinations. The odds ratios for developing any diagnosable level of peripheral neuropathy among the chlorpyrifos subjects was not increased at baseline or at one year compared to referents at baseline. Mixed regression models used to evaluate subclinical group-by-time interactions showed numerous significant NCS differences attributable to near-nerve temperature differences among all subjects between the baseline and one-year examinations, but only a few disparate effects related to group. CONCLUSIONS: Chronic chlorpyrifos exposure during the manufacturing process sufficient to produce biological effects on BuChE activity was not associated with clinically evident or subclinical peripheral neuropathy at baseline or with measurable deterioration among chlorpyrifos subjects compared to referents after one year of additional exposure.
Title: The mipafox-inhibited catalytic domain of human neuropathy target esterase ages by reversible proton loss Kropp TJ, Glynn P, Richardson RJ Ref: Biochemistry, 43:3716, 2004 : PubMed
Aging of organophosphorus (OP)-compound-inhibited neuropathy target esterase (NTE) is the critical event that initiates OP-compound-induced delayed neurotoxicity (OPIDN). Aging has classically been considered to involve side-group loss from phosphylated NTE, rendering the enzyme refractory to reactivation. N,N'-Diisopropylphosphorodiamidofluoridate (mipafox, MIP)-inhibited NTE has been thought to age quickly; however, it can be reactivated under acidic conditions. The present study was undertaken to determine whether MIP-inhibited human recombinant NTE esterase domain (NEST) ages classically by isopropylamine loss. Diisopropylphosphorofluoridate (DFP), the oxygen analogue of MIP, was used for comparison. Kinetic values for DFP against NEST were as follows: k(i) = 17 200 +/- 180 M(-1) min(-1); reactivation t(1/2) approximately 90 min at pH 8.0 and approximately 60 min at pH 5.2; k(4) = 0.108 +/- 0.041 min(-1) at pH 8.0 and 0.181 +/- 0.034 min(-1) at pH 5.2. Kinetic values for MIP against NEST were as follows: k(i) = 1880 +/- 61 M(-1) min(-1); reactivation t(1/2) = 0 min at pH 8.0 and approximately 60 min at pH 5.2; aging was complete at all time points tested at pH 8.0, but no aging occurred at pH 5.2. Mass spectrometry revealed a mass shift of 123.0 +/- 0.6 Da for the active site peptide peak of aged DFP-inhibited NEST, corresponding to a monoisopropyl phosphate adduct. In contrast, the analogous mass shift for aged MIP-inhibited NEST was 162.8 +/- 0.6 Da, corresponding to the intact N,N'-diisopropylphosphorodiamido adduct. Thus, MIP-inhibited NEST does not age by isopropylamine loss. However, because kinetically aged MIP-inhibited NEST yields an intact adduct capable of reversible deprotonation, aging could occur by proton loss. Indeed, MIP-inhibited NEST does not age at pH 5.2 but ages immediately and completely at pH 8.0. Therefore, we conclude that the MIP-NEST conjugate ages by deprotonation rather than classical side-group loss.
Title: Stereoselective inactivation of Torpedo californica acetylcholinesterase by isomalathion: inhibitory reactions with (1R)- and (1S)-isomers proceed by different mechanisms Doorn JA, Thompson CM, Christner RB, Richardson RJ Ref: Chemical Research in Toxicology, 16:958, 2003 : PubMed
The present study was undertaken to test the hypothesis that acetylcholinesterase (AChE) inhibition by isomalathion stereoisomers proceeds with different primary leaving groups for (1R)- and (1S)-isomers. Consistent with results obtained with enzyme from other species, AChE from Torpedo californica (TcAChE) was stereoselectively inhibited by isomalathion isomers with the (1R,3R)-isomer exhibiting greater potency than (1S,3S)-isomalathion. TcAChE modified by (1R)-isomers readily reactivated in the presence of 2-pralidoxime methiodide (2-PAM), whereas enzyme inhibited by (1S)-isomalathions was intractable toward reactivation. Computer-based molecular modeling showed that the ligand positioned as the primary leaving group was diethyl thiosuccinyl for (1R)-isomers and thiomethyl for (1S)-isomalathions. Mass spectral analysis revealed that inhibition of TcAChE by (1R)-isomers resulted in an O,S-dimethyl phosphate adduct, as expected from expulsion of the diethyl thiosuccinyl ligand. In contrast, inactivation of the enzyme by (1S)-isomalathions yielded an O-methyl phosphate adduct, consistent with initial loss of thiomethyl followed by displacement of the diethyl thiosuccinyl group. The findings demonstrate that the inhibitory reactions of TcAChE with (1R)- and (1S)-isomalathions proceed by different mechanisms involving distinct primary leaving groups.
Title: Relative inhibitory potencies of chlorpyrifos oxon, chlorpyrifos methyl oxon, and mipafox for acetylcholinesterase versus neuropathy target esterase Kropp TJ, Richardson RJ Ref: J Toxicol Environ Health A, 66:1145, 2003 : PubMed
The relative inhibitory potency (RIP) of an organophosphorus (OP) inhibitor against acetylcholinesterase (AChE) versus neuropathy target esterase (NTE) may be defined as the ratio [k(i)(AChE)/k(i)(NTE)], where k(i) is the bimolecular rate constant of inhibition for a given inhibitor against each enzyme. RIPs greater than 1 correlate with the inability of ageable OP inhibitors or their parent compounds to produce OP compound-induced delayed neurotoxicity (OPIDN) at doses below the LD50. The RIP for chlorpyrifos oxon (CPO) is >>1 for enzymes from hen brain homogenate, and the parent compound, chlorpyrifos (CPS), cannot produce OPIDN in hens at sublethal doses. This study was carried out to test the hypothesis that the RIP for the methyl homologue of CPO, chlorpyrifos methyl oxon (CPMO), is >>1 and greater than the RIP for CPO. Mipafox (MIP), an OP compound known to produce OPIDN, was included for comparison. Hen brain microsomes were used as the enzyme source, and k(i) values (mean +/- SE, microM(-1) min(-1)) were determined for AChE and NTE (n = 3 and 4 separate experiments, respectively). The k(i) values for CPO, CPMO, and MIP against AChE were 17.8 +/- 0.3, 10.9 +/- 0.1, and 0.00429 +/- 0.00001, respectively, and for NTE were 0.0993 +/- 0.0049, 0.0582 +/- 0.0013, and 0.00498 +/- 0.00006, respectively. Corresponding RIPs for CPO, CPMO, and MIP were 179 +/- 9, 187 +/- 4, and 0.861 +/- 0.011, respectively. The results demonstrate that RIPs for CPO and CPMO are comparable, markedly different from that for MIP, and >>1, indicating that CPS methyl, like CPS, could not cause OPIDN at sublethal doses.
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 degrees C) for N,N'-di-2-propylphosphorodiamidofluoridate (mipafox) against hen lymphocyte NTE was 6.94 +/- 0.28 microM amperometrically and 6.02 +/- 0.71 microM colorimetrically. For O,O-di1-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 +/- 14 nM, 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 highly 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.
Title: Quantitative structure-activity relationships predict the delayed neurotoxicity potential of a series of O-alkyl-O-methylchloroformimino phenylphosphonates Malygin VV, Sokolov VB, Richardson RJ, Makhaeva GF Ref: J Toxicol Environ Health A, 66:611, 2003 : PubMed
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 phenylphosphonates (RO)C6H5P(O)ON = CCICH3 (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 i.m. injection of hens with increasing doses of methyl and butyl derivatives. Analysis of dose-response curves yielded ED50(AChE)/ED50(NTE) ratio 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.
Title: Probing the active sites of butyrylcholinesterase and cholesterol esterase with isomalathion: conserved stereoselective inactivation of serine hydrolases structurally related to acetylcholinesterase Doorn JA, Talley TT, Thompson CM, Richardson RJ Ref: Chemical Research in Toxicology, 14:807, 2001 : PubMed
Previous work has shown that acetylcholinesterase (AChE), a member of the alpha/beta-hydrolase superfamily, is stereoselectively inhibited by the four stereoisomers of isomalathion. Recent kinetic and mass spectral data demonstrated that a difference in mechanism of inactivation exists for AChE treated with (1R)- versus (1S,3S)-stereoisomers. This study sought to determine whether other alpha/beta-hydrolases are stereoselectively inhibited by isomalathion and if the difference in mechanism of AChE inactivation between (1R)- and (1S,3S)-isomers is conserved for other alpha/beta-hydrolases. Bimolecular rate constants of inhibition (k(i)) were measured for human and equine butyrylcholinesterase (HBChE and EBChE, respectively) and bovine cholesterol esterase (BCholE) with all four isomers. Isomalathion isomers inhibited these enzymes with the following order of potency: (1R,3R) > (1R,3S) > (1S,3R) > or = (1S,3S). Ratios of k(i) values for the most potent to the least potent isomer were 10.5 (HBChE), 11.9 (EBChE), and 68.6 (BCholE). Rate constants of reactivation (k(3)) were measured for enzyme inhibited by isomalathion isomers. HBChE, EBChE, and BCholE inactivated by the (1R)-isomers readily reactivated. However, enzymes modified by (1S)-isomalathions were refractory toward reactivation, and k(3) values were not significantly different from zero for HBChE and BCholE treated with the (1S,3S)-isomer. Computer-based docking experiments were performed for BCholE with (1R,3R)- and (1S,3S)-enantiomers. Calculated structures predicted a difference in primary leaving group: diethyl thiosuccinate for (1R,3R)-isomalathion and thiomethyl for the (1S,3S)-isomer. The data demonstrate that the alpha/beta-hydrolases used in this study are stereoselectively inhibited by isomalathion. Furthermore, the results suggest that the mechanistic shift demonstrated to occur for inhibition of AChE by (1R)- versus (1S,3S)-isomers is conserved for butyrylcholinesterase and cholesterol esterase.
Title: Identification of Butyrylcholinesterase Adducts after Inhibition with Isomalathion Using Mass Spectrometry: Difference in Mechanism between (1R)- and (1S)-Stereoisomers Doorn JA, Schall M, Gage DA, Talley TT, Thompson CM, Richardson RJ Ref: Toxicol Appl Pharmacol, 176:73, 2001 : PubMed
Previous kinetic studies found that butyrylcholinesterase (BChE) inhibited by (1R)-isomalathions readily reactivated, while enzyme inactivated by (1S)-isomers did not. This study tested the hypothesis that (1R)- and (1S)-isomers inhibit BChE by different mechanisms, yielding distinct adducts identifiable by peptide mass mapping with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Equine BChE (EBChE) was inhibited to <10% of control activity with each isomer of isomalathion and the reference compound isoparathion methyl. Control and treated enzyme was digested with trypsin, and peptides were fractionated with HPLC. Separated and unseparated peptides were analyzed with MALDI-TOF-MS. Identity of an organophosphorus peptide adduct was confirmed by fragmentation using postsource decay analysis. EBChE inhibited by (1R)-isomalathions or (S)-isoparathion methyl readily reactivated after oxime treatment with 30-40% activity recovered. Enzyme inactivated by (1S)-isomalathions or (R)-isoparathion methyl recovered <2% and <5% activity, respectively, after oxime treatment. MALDI-TOF-MS analysis revealed that inhibition of EBChE by (1R)-isomalathions and (R)- or (S)-isoparathion methyl yielded O,S-dimethyl phosphate adducts. Enzyme inactivated by (1S)-isomalathions produced only O-methyl phosphate adduct. EBChE modified by (1R)-isomalathions or either enantiomer of isoparathion methyl yielded an O-methyl phosphate adduct as well. The results indicate that EBChE inhibition by (1R)-isomalathions proceeds with loss of diethyl thiosuccinate, but inactivation by (1S)-isomers occurs with loss of thiomethyl as the primary leaving group followed by rapid expulsion of diethyl thiosuccinate to yield an aged enzyme. Furthermore, the data suggest that aging of the O,S-dimethyl phosphate adduct occurs via an S(N)2 process with loss of thiomethyl.
Title: Inhibition of acetylcholinesterase by (1S,3S)-isomalathion proceeds with loss of thiomethyl: kinetic and mass spectral evidence for an unexpected primary leaving group Doorn JA, Gage DA, Schall M, Talley TT, Thompson CM, Richardson RJ Ref: Chemical Research in Toxicology, 13:1313, 2000 : PubMed
Previous work demonstrated kinetically that inhibition of mammalian acetylcholinesterase (AChE) by (1S)-isomalathions may proceed by loss of thiomethyl instead of the expected diethyl thiosuccinate as the primary leaving group followed by one of four possible modes of rapid aging. This study sought to identify the adduct that renders AChE refractory toward reactivation after inhibition with the (1S, 3S)-stereoisomer. Electric eel acetylcholinesterase (EEAChE) was inhibited with the four stereoisomers of isomalathion, and rate constants for spontaneous and oxime-mediated reactivation (k(3)) were measured. Oxime-mediated k(3) values were >25-fold higher for enzyme inhibited by (1R)- versus (1S)-stereoisomers with the greatest contrast between the (1R,3R)- and (1S,3S)-enantiomers. EEAChE inactivated by (1R,3R)-isomalathion reactivated spontaneously and in the presence of pyridine-2-aldoxime methiodide (2-PAM) with k(3) values of 1.88 x 10(5) and 4.18 x 10(5) min(-)(1), respectively. In contrast, enzyme treated with the (1S,3S)-enantiomer had spontaneous and 2-PAM-mediated k(3) values of 0 and 6.05 x 10(3) min(-)(1), respectively. The kinetic data that were measured were consistent with those obtained for mammalian AChE used in previous studies. Identification of the adduct that renders EEAChE stable toward reactivation after inhibition with (1S,3S)-isomalathion was accomplished using a peptide mass mapping approach with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). A peak with a mass corresponding to the active site peptide containing the catalytic Ser with a covalently bound O-methyl phosphate adduct was found in the mass spectra of (1S, 3S)-treated EEAChE but not control samples. Identities of the modified active site peptide and adduct were confirmed by fragmentation in MALDI-TOF-MS post-source decay (PSD) analysis, and peaks corresponding to the loss of an adduct as phosphorous/phosphoric acid methyl ester were observed. The results demonstrate that inhibition of EEAChE by (1S,3S)-isomalathion proceeds with loss of thiomethyl as the primary leaving group followed by rapid expulsion of diethyl thiosuccinate as the secondary leaving group to yield an aged enzyme.
Title: Brainstem axolemmal protein phosphorylation in vitro in hens dosed with di-1-butyl-2,2-dichlorovinyl phosphate Huggins DJ, Richardson RJ Ref: J Toxicol Environ Health A, 56:263, 1999 : PubMed
Neuropathy target esterase (neurotoxic esterase, NTE), a protein thought to be involved in the production of organophosphorus compound-induced delayed neurotoxicity (OPIDN), has been postulated to be a component of endogenous neuronal protein phosphorylation systems. The purpose of this work was to test this hypothesis as well as to investigate further the role of endogenous protein phosphorylation in toxic neuropathies. White Leghorn hens were dosed with the neuropathic compounds di-1-butyl-2,2-dichlorovinyl phosphate (dibutyl dichlorvos, DBDCV), tri-o-cresyl phosphate (TOCP), or acrylamide, and regions from brain were fractionated into axolemmal, synaptosomal, and microsomal preparations. Radiolabeling of NTE or endogenously phosphorylated proteins was carried out by incubation with [14C]-DFP or gamma-[32P]-ATP, respectively. Radiolabeled proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by autoradiography. Relative amounts of phosphoproteins were quantified by densitometry of the autoradiographs. Changes in endogenous phosphorylation of a protein exhibiting the characteristics of NTE were not observed in these experiments. However, levels of a [32P]-labeled 50-kDa brainstem axolemmal protein were decreased significantly on d 15, but not on d 1, 3, 7, or 10 after dosing with 2.8 mg/kg DBDCV. Clinical signs of ataxia and histopathological findings of axonal degeneration in the spinocerebellar tracts of the brainstem were evident on d 10-15, and hens were unable to perch on a horizontal wooden rod from d 12 after dosing with DBDCV. The decrease in the 50-kDa phosphoprotein was not observed on d 15 after the production of clinically evident neuropathy with either 14 daily doses of 50 mg/kg acrylamide or with a single dose of 500 mg/kg TOCP. These results suggest that NTE is not an endogenously phosphorylated protein under the conditions of these experiments. However, an effect on endogenous phosphorylation limited to a 50-kDa axolemmal protein was selectively produced by treatment with a neuropathic dose of DBDCV that was in evidence only after clinical signs and histopathological findings of axonopathy were apparent.
Inhibition of acetylcholinesterase (AChE) by isomalathion has been assumed to proceed by expulsion of diethyl thiosuccinyl to produce O, S-dimethyl phosphorylated AChE. If this assumption is correct, AChE inhibited by (1R)- or (1S)-isomalathions should reactivate at the same rate as AChE inhibited by configurationally equivalent (S)- or (R)-isoparathion methyl, respectively, which are expected to inhibit AChE by loss of 4-nitrophenoxyl to yield O,S-dimethyl phosphorylated AChEs. Previous work has shown that rat brain AChE inhibited by (1R)-isomalathions reactivates at the same rate as the enzyme inhibited by (S)-isoparathion methyl. However, although rat brain AChE inhibited by (R)-isoparathion methyl reactivates at a measurable rate, the enzyme inhibited by (1S)-isomalathions is intractable to reactivation. This surprising finding suggests the hypothesis that (1R)- and (1S)-stereoisomers of isomalathion inhibit AChE by different mechanisms, yielding enzymatic species distinguishable by their postinhibitory kinetics. The present study was carried out to test this hypothesis by comparing kinetic constants of reactivation (k+3) and aging (k+4) of hen brain AChE and bovine erythrocyte AChE inhibited by the four stereoisomers of isomalathion and the two stereoisomers of isoparathion methyl. Both AChEs inhibited by either (1R,3R)- or (1R,3S)-isomalathion had comparable corresponding k+3 values (spontaneous and oxime-mediated) to those of AChEs inhibited with (S)-isoparathion methyl. However, spontaneous and oxime-mediated k+3 values comparable to those of (R)-isoparathion methyl could not be obtained for AChEs inhibited by (1S,3R)- and (1S,3S)-isomalathion. Comparison of k+4 values for hen brain AChE inhibited by each stereoisomer of isomalathion and isoparathion methyl corroborated that only the (1S)-isomalathions failed to produce the expected O,S-dimethyl phosphoryl-conjugated enzymes. The results for (1R)-isomalathions suggest that the mechanism of inhibition of AChE by these isomers is the expected one involving diethyl thiosuccinyl as the primary leaving group. In contrast, the results for (1S)-isomalathions are consistent with an alternative mechanism of inhibition by these isomers implicating loss of thiomethyl as the primary leaving group.
The Food Quality Protection Act of 1996 (FQPA) requires the EPA to consider "available information concerning the cumulative effects of such residues and other substances that have a common mechanism of toxicity ... in establishing, modifying, leaving in effect, or revoking a tolerance for a pesticide chemical residue." This directive raises a number of scientific questions to be answered before the FQPA can be implemented. Among these questions is: What constitutes a common mechanism of toxicity? The ILSI Risk Science Institute (RSI) convened a group of experts to examine this and other scientific questions using the organophosphorus (OP) pesticides as the case study. OP pesticides share some characteristics attributed to compounds that act by a common mechanism, but produce a variety of clinical signs of toxicity not identical for all OP pesticides. The Working Group generated a testable hypothesis, anticholinesterase OP pesticides act by a common mechanism of toxicity, and generated alternative hypotheses that, if true, would cause rejection of the initial hypothesis and provide criteria for subgrouping OP compounds. Some of the alternative hypotheses were rejected outright and the rest were not supported by adequate data. The Working Group concluded that OP pesticides act by a common mechanism of toxicity if they inhibit acetylcholinesterase by phosphorylation and elicit any spectrum of cholinergic effects. An approach similar to that developed for OP pesticides could be used to determine if other classes or groups of pesticides that share structural and toxicological characteristics act by a common mechanism of toxicity or by distinct mechanisms.
Title: Isomalathion Stereoisomers Talley TT, Jianmongkol S, Richardson RJ, Radic Z, Thompson CM Ref: In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases, (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp.:531, 1998 : PubMed
Carbamyl sulfonate (CS) compounds are a novel class of carbamates derived from amino acid methyl esters. They have the general structure RCH(COOCH3)NH(CO)SO-3K+, where R is the sidechain of the parent amino acid. These compounds were developed as active site-directed inhibitors of human leukocyte elastase (HLE). The purpose of this study was to characterize the inhibition of hen brain neurotoxic esterase (neuropathy target esterase, NTE), horse serum butyrylcholinesterase (BCHE), and bovine erythrocyte acetylcholinesterase (AChE) by CS analogs derived from the methyl esters of L-ala, D-norval, L-norval, L-phe, L-val, L-norleu, D-met, and L-met. Bimolecular rate constants of inhibition (ki) for NTE ranged from 0.571 for L-ala-CS to 17.7 mM-1 min-1 for L-norleu-CS (10-min I50 values of 123 and 3.92 microM, respectively). Potency against NTE increased with chain length for straight-chain R-groups of L-CS compounds. Unlike HLE, NTE was only weakly stereoselective for CS compound enantiomers. The L-isomers were weaker inhibitors of BCHE than NTE (10-min I50 range of 742 to 35.6 microM). In contrast to the L-enantiomers, the I50 plots of D-met-CS and D-norval-CS were not linear for BCHE, suggesting a possible stereospecific mechanistic shift for inhibition of this enzyme, AChE was not effectively inhibited by any of the CS compounds (I50 values > 750 microM). The specificity and charged nature of CS compounds give these unusual NTE inhibitors potential advantages for mechanistic studies of organophosphorus compound-induced delayed neurotoxicity (OPIDN) and its protection or potentiation.
Title: Potentiation of organophosphorus compound-induced delayed neurotoxicity (OPIDN) in the central and peripheral nervous system of the adult hen: distribution of axonal lesions Randall JC, Yano BL, Richardson RJ Ref: J Toxicol Environ Health, 51:571, 1997 : PubMed
Clinical manifestations of mild organophosphorus compound-induced delayed neurotoxicity (OPIDN) produced by diisopropylphosphorofluoridate (DFP) in adult hens are potentiated by posttreatment with phenylmethanesulfonyl fluoride (PMSF). The purpose of this study was to assess whether potentiation of mild OPIDN produces a pattern of axonal lesions in the central and peripheral nervous system similar to that seen in severe OPIDN. Groups of 6 hens each were given the following priming/challenge doses sc at 0 and 4 h, respectively: 0.20 ml/kg corn oil/0.50 ml/kg glycerol formal (GF) (control); 0.50 mg/kg DFP/GF (low-dose DFP); 0.50 mg/kg DFP/60 mg/kg PMSF (potentiated DFP); 60 mg/kg PMSF/GF (PMSF alone); 60 mg/kg PMSF/1.5 mg/kg DFP (protected DFP); and 1.5 mg/kg DFP/GF (high-dose DFP). Two hens from each group were used to assay brain neurotoxic esterase (NTE) 24 h after the challenge dose, and the remaining hens were scored for deficits in walking, standing, and perching ability on d 18. Three hens from each group were perfusion-fixed on d 22 and neural tissues were prepared for histologic evaluation. DFP and/or PMSF caused > 88% brain NTE inhibition in all treated groups, compared to control. Protected DFP yielded no clinical deficits and a distribution and frequency of axonal lesions similar to control. PMSF alone produced a small increase in the frequency of lesions in the cervical spinal cord and peripheral nerves compared to control. Low-dose DFP caused minimal ataxia and increased frequency of axonal lesions in dorsal and lateral cervical spinal cord, ventral lumbar spinal cord, and inferior cerebellar peduncles (ICP) compared to control. Potentiated DFP and high-dose DFP produced maximal ataxia and essentially identical increases in the frequency of lesions in dorsal and ventral thoracic spinal cord, lateral lumbar spinal cord, and peripheral nerves compared to low-dose DFP. The results indicate that PMSF potentiation of mild OPIDN induced in adult hens by low-dose DFP results in an overall pattern of axonal degeneration like that produced by a threefold higher dose of DFP alone, and support the hypothesis that potentiation causes an increase in the frequency of axonal lesions in central and peripheral loci normally affected by OPIDN.
Title: Relative potencies of the four stereoisomers of isomalathion for inhibition of hen brain acetylcholinesterase and neurotoxic esterase in vitro Jianmongkol S, Berkman CE, Thompson CM, Richardson RJ Ref: Toxicology & Applied Pharmacology, 139:342, 1996 : PubMed
The cholinergic toxicity of malathion is exacerbated by its isomerization product, isomalathion, which inhibits detoxifying carboxylesterases as well as target acetylcholinesterase (AChE). Previous work has shown that the four stereoisomers of isomalathion, (1R, 3R), (1R, 3S), (1S, 3R), and (1S, 3S), differ in their inhibitory potencies against either rat brain or electric eel AChE. The present study examined the relative inhibitory potencies of these stereoisomers and the totally racemic mixture (1RS, 3RS) against hen brain AChE and neurotoxic esterase (NTE) to provide new data on stereoselective inhibition of neurotoxicologically significant esterases and to assess the potential of these compounds to cause organophosphorus (OP) compound-induced delayed neurotoxicity (OPIDN). The order of potencies against hen brain AChE was (1R, 3R) > (1R, 3S) > (1RS, 3RS) > (1S, 3R) > (1S, 3S), with a 15-fold difference between the strongest (ki = 388 mM-1 min-1; 20 min I50 = 89.3 nM) and weakest (ki = 25.6 mM-1 min-1; 20 min I50 = 1354 nM) inhibitors. Both asymmetric centers contributed substantially and interdependently to inhibitory potency, but the effect of changing the configuration at phosphorus alone was greater than changing the configuration at carbon alone. None of the isomalathions was an effective inhibitor of hen brain NTE (extrapolated 20 min I50 values were 1.2 to 29 mM), yielding NTE/ AChE I50 ratios (neuropathy target ratios, NTRs) of 1.5 x 10(3) to 1.5 x 10(5). NTRs of this magnitude indicate that none of the isomalathions should initiate OPIDN, even after doses greatly exceeding the LD50. Therefore, reports of OPIDN or other neuropathic sequelae associated with malathion exposures in humans cannot be explained on the basis of NTE inhibition by contaminating isomalathions.
Title: Assessment of the neurotoxic potential of chlorpyrifos relative to other organophosphorus compounds: a critical review of the literature. Richardson RJ Ref: Journal of Toxicology & Environmental Health, 44:135, 1995 : PubMed
Chlorpyrifos (diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate) is a broad-spectrum organophosphorus (OP) insecticide. Anticipated increases in the already extensive use of this compound have prompted this reassessment of its neurotoxicity. Because chlorpyrifos and other OP insecticides are designed to produce acute cholinergic effects through inhibition of acetylcholinesterase (AChE) and some OP compounds can cause OP compound-induced delayed neurotoxicity (OPIDN) via chemical modification of neurotoxic esterase (neuropathy target esterase, NTE), this review focuses on the capacity of chlorpyrifos to precipitate these and other adverse neurological consequences. Chlorpyrifos exhibits only moderate acute toxicity in many mammalian species, due largely to detoxification of the active metabolite, chlorpyrifos oxon, by A-esterases. Rats given large doses of chlorpyrifos (sc in oil) have prolonged inhibition of brain AChE, possibly due to slow release of the parent compound from a depot. Associated cognitive and motor deficits return to normal well before recovery of AChE activity and muscarinic receptor down-regulation, as expected from classic tolerance. Controlled studies of OP compound exposures in humans also indicate that cognitive dysfunction requires substantial AChE inhibition. Information is relatively sparse on neurological dysfunction that is secondary to theoretical reproductive, developmental, or immunological effects, but the best available data indicate that such effects are unlikely to result from exposures to chlorpyrifos. In accord with the much greater inhibitory potency of chlorpyrifos oxon for AChE than for NTE, clinical reports and experimental studies indicate that OPIDN from acute exposures to chlorpyrifos requires doses well in excess of the LD50, even when followed by repeated doses of the OPIDN potentiator phenylmethanesulfonyl fluoride (PMSF). Likewise, studies in hens show that subchronic exposures at the maximum tolerated daily dose do not result in OPIDN. Although exposure to chlorpyrifos as a result of normal use is unlikely to produce classical OPIDN, a recent report stated that mild reversible sensory neuropathy had occurred in eight patients who had been exposed subchronically to unknown amounts of chlorpyrifos. It is not clear whether these cases represent an incorrect linkage of cause and effect, a newly disclosed reversible sensory component of OPIDN, or an entirely new phenomenon. The question of the potential for chlorpyrifos to cause this mild sensory neuropathy could be resolved by the use of quantitative tests of sensory function in animal experiments and/or prospective studies of humans with known exposures to chlorpyrifos.
Title: Inhibition of hen brain acetylcholinesterase and neurotoxic esterase by chlorpyrifos in vivo and kinetics of inhibition by chlorpyrifos oxon in vitro: application to assessment of neuropathic risk Richardson RJ, Moore TB, Kayyali US, Fowke JH, Randall JC Ref: Fundamental & Applied Toxicology, 20:273, 1993 : PubMed
Chlorpyrifos (CPS; O,O-diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate; Dursban) is a widely used broad-spectrum organophosphorus (OP) insecticide. Because some OP compounds can cause a sensory-motor distal axonopathy called OP compound-induced delayed neurotoxicity (OPIDN), CPS has been evaluated for this paralytic effect. Early studies of the neurotoxicity of CPS in young and adult hens reported reversible leg weakness but failed to detect OPIDN. More recently, a human case of mild OPIDN was reported to result from ingestion of a massive dose (about 300 mg/kg) in a suicide attempt. Subsequent experiments in adult hens (the currently accepted animal model of choice for studies of OPIDN) showed that doses of CPS in excess of the LD50 in atropine-treated animals inhibited brain neurotoxic esterase (NTE) and produced mild to moderate ataxia. Considering the extensive use of CPS and its demonstrated potential for causing OPIDN at supralethal doses, additional data are needed to enable quantitative estimates to be made of the neuropathic risk of this compound. Previous work has shown that the ability of OP insecticides to cause acute cholinergic toxicity versus OPIDN can be predicted from their relative tendency to inhibit the intended target, acetylcholinesterase (AChE), versus the putative neuropathic target, NTE, in brain tissue. The present study was designed to clarify the magnitude of neuropathic risk associated with CPS exposures by measuring hen brain AChE and NTE inhibition following dosing in vivo and determining the bimolecular rate constant of inhibition (ki) for each enzyme by the active metabolite, CPS oxon (CPO), in vitro.
Title: Chlorpyrifos: assessment of potential for delayed neurotoxicity by repeated dosing in adult hens with monitoring of brain acetylcholinesterase, brain and lymphocyte neurotoxic esterase, and plasma butyrylcholinesterase activities Richardson RJ, Moore TB, Kayyali US, Randall JC Ref: Fundamental & Applied Toxicology, 21:89, 1993 : PubMed
Previous work has shown that acute exposures to chlorpyrifos (CPS; diethyl 3,5,6-trichloro-2-pyridyl phosphorothionate) cannot produce > 70% inhibition of brain neurotoxic esterase (NTE) and cause organophosphorus compound-induced delayed neurotoxicity (OPIDN) unless the dose is well in excess of the LD50, necessitating aggressive therapy for cholinergic toxicity. The present study was carried out to determine if repeated doses of CPS at the maximum tolerated daily dose without prophylaxis against cholinergic toxicity could cause cumulative inhibition of NTE and OPIDN. Adult hens were dosed daily for 20 days with CPS (10 mg/kg/day po in 2 ml/kg corn oil) or corn oil (vehicle control) (2 ml/kg/day po) and observed for an additional 4 weeks. Brain acetylcholinesterase (AChE), brain and lymphocyte NTE, and plasma butyrylcholinesterase (BCHE) activities were assayed on Days 0 (control only), 4, 10, 15, 20, and 48. During Days 4-20, brain AChE and plasma BCHE activities from CPS-treated hens were inhibited 58-70% and 49-80% of contemporaneous controls, respectively. At 4 weeks after the end of dosing, brain AChE activity in treated birds had recovered to 86% of control and plasma BCHE activity was 134% of control. Brain and lymphocyte NTE activities of treated animals throughout the study were 82-99% and 85-128% of control, respectively. Neither brain nor lymphocyte NTE activities in treated hens exhibited cumulative inhibition. The 18% inhibition of brain NTE seen on days 10 and 20 was significant, but substantially below the putative threshold for OPIDN.
It has been recently reported that phenylmethanesulfonyl fluoride (PMSF) when given to hens after a neuropathic organophosphate (OP) promotes organophosphate-induced delayed polyneuropathy (OPIDP). Chicks are resistant to OPIDP despite high inhibition/aging of neuropathy target esterase (NTE), the putative target of OPIDP initiation. However, when PMSF (300 mg/kg s.c.) is given to chicks after di-butyl 2,2-dichlorovinyl phosphate (DBDCVP, 1 or 5 mg/kg s.c.), OPIDP is promoted. Inhibition/aging of at least 30% of NTE was thought to be an essential prerequisite for promotion to be elicited in adult hens. However, we observed in hens that when NTE is maximally affected (greater than 90%) by phenyl N-methyl N-benzyl carbamate (40 mg/kg i.v.), a non-ageable inhibitor of NTE, and then PMSF is given (120 mg/kg/day s.c. x 3 days) clinical signs of neuropathy become evident. Methamidophos (50 mg/kg p.o. to hens), which produces in vivo a reactivatable form of inhibited NTE, was shown either to protect from or promote OPIDP caused by DBDCVP (0.45 mg/kg s.c.), depending on the sequence of dosing. Because very high doses of methamidophos cause OPIDP, we considered this effect to be a "self-promoted" OPIDP. We concluded that NTE inhibitors might have different intrinsic activities for producing OPIDP once NTE is affected. Aging might differentiate highly neuropathic OPs, like DBDCVP, from less neuropathic OPs, like methamidophos, or from the least neuropathic carbamates, which require promotion in order for neuropathy to be expressed.(ABSTRACT TRUNCATED AT 250 WORDS)
Previous work in our laboratory indicated that di-n-butyl-2,2-dichlorovinyl phosphate (DBCV) produced electrophysiologic changes in hen peripheral nerve that coincided with the development of histopathologic changes and neurologic signs of peripheral neuropathy. The purpose of the present study was to follow the time course for the development of the electrophysiologic changes and to determine whether pretreatment with the phosphinate analog of DBCV (DBCV-P), a nonageable organophosphorus compound, prevented these effects. Although significant electrophysiologic deficits occurred in the tibial and sciatic nerve 24 h after DBCV treatment, the most marked changes coincided with the onset of clinical signs of organophosphorus-induced delayed neuropathy (14-21 d). The sciatic and tibial nerves were equally susceptible to DBCV in producing deficits characterized by changes in the relative refractory period and an increased strength-duration threshold. Pretreatment with DBCV-P prevented the clinical signs and also attenuated the electrophysiologic deficits induced by DBCV treatment. These data suggest that electrophysiologic deficits occur before clinical signs of organophosphorus-induced delayed neuropathy (OPIDN) and may be indicative of a link between neurotoxic esterase (NTE) inhibition and onset of overt clinical toxicity.
Heat inactivation was studied at 45, 50, 55, and 60 degrees for all of the phenyl valerate hydrolases (PVase), including neurotoxic esterase (NTE) and inhibitor-resistant esterase (IRE), in homogenates of hen or rat brain or in preparations of hen brain microsomal membranes. Hen and rat brain homogenates were prepared in buffer (50 mM Tris/0.20 mM EDTA, pH 8.00, at 25 degrees). Hen brain microsomes were suspended either in buffer or in aqueous dimethyl sulfoxide (DMSO, 40%, w/v), or solubilized either in aqueous Triton X-100 (0.10%, w/v) or in 40% (w/v) DMSO. Enzyme activities were measured at 37 degrees using phenyl valerate as substrate. Each enzyme activity in all of the preparations exhibited biphasic heat inactivation kinetics. Apparent rate constants were calculated for the fast (kf) and slow (ks) reactions, along with the relative amounts of activity in each component (Af, As) expressed as percentages of the total activity. For a given preparation and temperature, respective values of kf or ks were similar for PVase, NTE, and IRE, with a mean kf/ks ratio of 52 across all preparations. Af and As were a function of temperature. Mean values of the apparent activation energies (Ea) for all activities and preparations were 44 and 25 kcal/mol for the fast and slow inactivation reactions respectively. These results indicate that all phenyl valerate hydrolases in hen and rat brain undergo a common heat-induced structural change leading to loss of enzymic activity.
Title: Lymphocyte and brain neurotoxic esterase: dose and time dependence of inhibition in the hen examined with three organophosphorus esters Schwab BW, Richardson RJ Ref: Toxicol Appl Pharmacol, 83:1, 1986 : PubMed
Certain organic phosphorus esters produce sensorimotor axonopathy in man and other species. There is an excellent correlation between the capacity of an organophosphorus compound to produce axonopathy and its ability to inhibit brain neurotoxic esterase (NTE) in hens. Because NTE is present in peripheral lymphocytes of both hen and man, it has been suggested that the lymphocyte enzyme might be useful both in experimental and clinical situations as an indicator of exposure to organophosphorus compounds producing axonopathy. Diethyl 4-nitrophenyl phosphate (paraoxon), tri-2-cresyl phosphate (TOCP), methyl 2,5-dichloro-4-bromophenyl phenylphosphonothionate (leptophos), and di-n-butyl-2,2-dichlorovinyl phosphate (di-n-butyl dichlorvos, DBDCV) were used to examine the relationship between lymphocyte and brain NTE inhibition in hens. As expected, paraoxon (0.75 mg/kg) did not inhibit NTE in brain or lymphocytes. TOCP (10 to 100 mg/kg), leptophos (25 to 150 mg/kg), and DBDCV (1.0 to 4.0 mg/kg) inhibited both brain and lymphocyte NTE activity in a dose-related manner with good correlation of inhibition between tissues taken 24 hr after exposure (r2 = 0.53 to 0.67; p less than 0.020 to 0.001). However, correlation of inhibition between tissues taken from animals killed 48 hr after exposures was poor (r2 = 0.15 to 0.30; p less than 0.10 to 0.05), with consistently less inhibition of lymphocyte NTE relative to brain NTE. This study indicates that assay of lymphocyte NTE can provide a good monitor of exposure to axonotoxic organophosphorus compounds within 24 hr between exposure and measurement.
Title: Direct comparison of the distribution of neurotoxic esterase and acetylcholinesterase in rat and hen brain Hollingsworth PJ, Dudek BR, Smith CB, Richardson RJ Ref: In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases, (Brzin M, Barnard EA, Sket D, Eds) De Gruyter:483, 1984 : PubMed
