Elena V. Rudakova

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

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

Biosensor analysis of blood esterases for organophosphorus compounds exposure assessment: approaches to simultaneous determination of several esterases.

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.

Further studies toward a mouse model for biochemical assessment of neuropathic potential of organophosphorus compounds

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

Esterase profile of O-phosphorylated ethyltrifluorolactates in prediction of their therapeutic and toxic effects

81 The use of anticholinesterase compounds in medi cine, veterinary practice and agricultural chemistry is based on their common mechanism of action, which is determined by the inhibition of acetylcholinesterase (EC 3.1.1.7, AChE) [1]. Many cholinesterase inhibi tors, in addition to AChE, interact with other serine esterases, that can lead to both toxic and therapeutic effects [2]. Toxic and therapeutic effects resulting from the inhibition by organophosphorus compounds (OPCs) of four serine esterases—acetylcholinest erase, neuropathy target esterase (EC 3.1.1.5, NTE), butyrylcholinesterase (EC 3.1.1.7, BChE), and car boxylesterase (EC 3.1.1.1, CaE)—are shown in Scheme 1, where the effects that determine the toxic action of OPCs are shown in italic.

Blood esterases as a complex biomarker for exposure to organophosphorus compounds

The growing threat of international terrorism brings with it new scenarios for disaster. For example, in the case of toxic organophosphorus compounds (OPs), it possible for terrorists to use known agents or inadvertently to produce highly toxic OPs of unknown structure as the result of attacks on chemical plants or stockpiles of pesticides and other chemicals. Defending against such agents requires rapid, sensitive, and specific detection of them and their biological effects. Thus, the development of biomarkers of human exposures to OPs is a vital component of the system of prediction and early diagnosis of induced diseases. The phosphylating properties of OPs lead to their differential interactions with various serine esterases. These enzymes include primary targets, e.g., acetylcholinesterase (AChE, acute toxicity) and neuropathy target esterase (NTE, delayed neuropathy, OPIDN); as well as secondary targets, e.g., butyrylcholinesterase (BChE) and carboxylesterase (CaE), which act as scavengers of OPs. The set of activities of these esterases as well as that of paraoxonase (PON1), which can hydrolyze and detoxify OPs, constitutes the “esterase status” of an organism that largely determines indi-vidual sensitivity to OPs and that may be used as a complex biomarker of exposure. This complex biomarker is more effective and informative than the standard determination of erythrocyte AChE and total blood cholinesterases. In particular, it assists with distinguishing between acute and delayed neurotoxicity induced by OPs, as we showed in experiments on acute exposure of hens to a neuropathic compound, O,O-dipropyl-O-dichlorovinyl phosphate. In addition, measuring decreased activities of BChE and CaE, which are often more sensitive biomarkers of OP exposure, allows us to reveal exposure to low doses, as demonstrated by treating mice with low doses of phosphorylated oximes. The aim of the ISTC Project summarized here is to develop a smart biosensor system for simultaneous analysis of a set of blood esterases including AChE, BChE, NTE, CaE, and PON1. The speed, sensitivity, and integrated approach of the method will allow hazards to be assessed and appropriate interventions to be recommended before overt toxic damage has occurred.

A New Selective Inhibitor of Mouse Blood Plasma Carboxylesterase

1) are enzymes with a broad substrate specificity that catalyze hydroll ysis and esterification of a wide spectrum of structurr ally different exogenous and endogenous substances [1]. These enzymes play an important role in the metabolism of numerous therapeutically important compounds containing ester or amide groups. This accounts for therapeutic significance of CaE inhibii tors that influence the rate of hydrolysis of drugs behaving as CaE substrates and thereby determine the rate of drug conversion into active substance or proo long the halfflife of the latter in the body [2, 3]. Investigations of pharmacologically active preparaa tions, including preclinical trials, are conducted on rodents, which, unlike humans, have a high level of blood plasma CaE [4, 5]. Consequently, preparations with ester or amide groups are rapidly hydrolyzed in the rodent plasma, which decreases their effective concentration and creates difficulties in studying their pharmacological and toxicological properties as well in extrapolating the results of these studies to humans [6, 7]. Thus, it is a relevant task to search for comm pounds capable of inhibiting rodent plasma CaE in vivo and in vitro, having more prolonged effect irree versible inhibitors are more preferred. Low acute toxx icity of CaE inhibitors is also an important requiree ment when using in vivo. Application of such inhibii tors will make it possible to create an adequate rodent model for preclinical trials of pharmacological prepaa rations subject to hydrolysis by CaE [6]. Today, only one selective and irreversible CaE inhibitor, bissp nitrophenyl phosphate, is used both in tests on prepaa rations of rodent blood plasma and, in some cases, in experiments on rodents in vivo [8, 9]. Our previous kinetic studies with commercial prepaa rations of human red blood cells acetylcholinesterase (AChE, EC 3.1.1.7), horse serum butyrylcholinestt erase (BChE, EC 3.1.1.8), and porcine liver CaE showed that fluorineecontaining phosphates with a general formula of (AlkO) 2 P(O)OCH(CF 3) 2 had a selective effect on CaE, compared to AChE and BChE [10]. The leader compound (О,Оdibutyl))О 11trifluoromethyll2,2,22trifluoroethyl phosphate (nnС 4 H 9 O) 2 P(O)OCH(CF 3) 2 (DBFP) was irreversible effective and selective inhibitor of CaE: k i (CaE) = posed that DBFP may be effective as a selective inhibb itor of plasma CaE both in experiments with mouse blood preparations in vitro and upon systemic adminn istration in vivo. The purpose of this study was to test the potential of DBFP as an effective and selective …

Focused design of polypharmacophoric neuroprotective compounds: Conjugates of γ-carbolines with carbazole derivatives and tetrahydrocarbazole

Abstract Alzheimer’s disease has a complex multifactorial nature; therefore, a promising approach for the development of efficient therapeutic agents is the concept of multitarget drugs, which affect several biological targets involved in the pathogenesis of the disease. We developed a synthetic algorithm for conjugating several pharmacophoric ligands acting on the key stages of pathogenesis of several neurodegenerative diseases and synthesized hybrid structures combining the γ-carboline fragment of Dimebon with carbazole and tetrahydrocarbazole moieties. Using the complex primary screening system the structures have been revealed that combine the high inhibitory activity and selectivity towards butyrylcholinesterase with the radical-scavenging activity and the ability to potentiate tubulin polymerization to microtubules with a normal structure and/or prevent mitochondrial permeability transition. The lead compound was identified for future optimization and development of new multi-target drugs against neurodegenerative diseases combining the cognitive-stimulating and neuroprotective potentials.

1,2,4-Thiadiazoles as promising multifunctional agents for treatment of neurodegenerative diseases

Detailed studies of properties of new 3-substituted 5-anilino-1,2,4-thiadiazoles containing different substituents at position 3 of the thiadiazole ring were carried out, in particular, their esterase profile and antioxidant properties. It was found that the presence in the molecule of 2-aminopropyl fragment determines an efficient and selective inhibition of butyrylcholinesterase as compared to acetylcholinesterase and carboxylesterase, with radical-scavenging activity being weak. The compounds containing a 2-aminopropenyl fragment possess a high radicalscavenging activity, weakly inhibit cholinesterases, and exhibit anticarboxylesterase activity. A wide spectrum of activity of 3-substituted 5-anilino-1,2,4-thiadiazoles as inhibitors of cholinesterases and highly efficient scavengers of free radicals gives a basis for the optimization of structure and development in this series of original agents for therapy of neurodegenerative diseases.

Neuropathy target esterase in mouse whole blood as a biomarker of exposure to neuropathic organophosphorus compounds

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

Synthesis, molecular docking, and biological activity of 2-vinyl chromones: Toward selective butyrylcholinesterase inhibitors for potential Alzheimer's disease therapeutics

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.