N. P. Boltneva

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.

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

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

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

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

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

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

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.

Novel conjugates of aminoadamantanes with carbazole derivatives as potential multitarget agents for AD treatment

A new group of compounds, promising for the design of original multitarget therapeutic agents for treating neurodegenerative diseases, based on conjugates of aminoadamantane and carbazole derivatives was synthesized and investigated. Compounds of these series were found to interact with a group of targets that play an important role in the development of this type of diseases. First of all, these compounds selectively inhibit butyrylcholinesterase, block NMDA receptors containing NR2B subunits while maintaining the properties of MK-801 binding site blockers, exert microtubules stabilizing properties, and possess the ability to protect nerve cells from death at the calcium overload conditions. The leading compound C-2h has been shown the most promising effects on all analyzed parameters. Thus, these compounds can be regarded as promising candidates for the design of multi-target disease-modifying drugs for treatment of AD and/or similar neuropathologies.

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

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.

N,N-disubstituted 2-aminothiazolines as new inhibitors of serine esterases

209 Recently, N,SScontaining heterocyclic comm pounds, especially the derivatives of 22aminoo1,33thii azole, draw increasing attention due to their chemical properties as well as a wide spectrum of biological activity. It is known that many compounds containing the thiazoline (4,55dihydroo1,33thiazole) fragment exhibit antibacterial, anticancer, antifungal, and other types of activities [1–3]. However, information about the inhibition of serine esterases by compounds conn taining the thiazoline fragment is practically absent in the literature. The ability to reversibly inhibit cholinestt erases was shown for only some 22substituted thiazoo lines and imidazo[2,11b]thiazole derivatives [4, 5]. Since it is known that the inhibition of serine esterases can have different pharmacological effects, the search and design of new inhibitors of these enzymes is of interest. In particular, inhibitors of acee tylcholinesterase (AChE, EC 3.1.1.7) contribute to the improvement of cognitive functions in dementia of various origins [6]. Inhibitors of butyrylcholinesterase (BChE, EC 3.1.1.8) also improve cognitive function, which is especially important for severe stages of Alzheimers disease, when the activity of AChE is decreased and its function (hydrolysis of acetylchoo line) is implemented by BChE. In contrast to AChE inhibitors, BChE inhibitors do not have a dangerous side effect such as acute cholinergic toxicity and other adverse effects characteristic of AChE inhibitors [7]. Carboxylesterase (CaE, EC 3.1.1.1) and BChE hydroo lyze many therapeutically important drugs containing ester groups; in view of this, selective inhibitors of these enzymes are important for modulating the metabolism and pharmacokinetics of these drugs [8]. Researchers of the Institute of Physiologically Active Compounds, Russian Academy of Sciences, have developed and widely use an approach including the determination of the esterase profile of compounds with a comparative evaluation of their inhibitory activv ity with respect to several esterases [9–11]. This approach provides a more comprehensive picture of the biological effects of compounds and thus makes it possible to assess the therapeutic potential and possii ble side effects of test compounds. The purpose of this study was to search for new effective inhibitors of serine esterases in the 22amii nothiazoline series. We synthesized both the comm pounds described in the literature and the original N,NNdisubstituted 55iodomethyll22aminothiazolines 1–8 of the general formula and analyzed their esterase profile. For this purpose, we studied the inhibitory activity of these compounds with respect to human erythrocyte AChE, horse serum BChE, and porcine liver CaE. 55Iodomethyll22aminothiazolines 1–8 were syn thesized by the reaction of allyl isothiocyanate with appropriate amines …

Alkyl 2-arylhydrazinylidene-3-oxo-3-polyfluoroalkylpropionates as new effective and selective inhibitors of carboxylesterase.

A series of alkyl 2-Arylhydrazinylidene-3-oxo-3-polyfluoroalkylpropionates was synthesized and their inhibitory activity with respect to porcine liver carboxylesterase (CaE, EC 3.1.1.1), human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7), and horse serum butyrylcholinesterase (BChE, EC 3.1.1.8) was studied. The molecular docking method was used to study the binding mode of the compounds in the active site of CaE. It was found that compounds containing the trifluoromethyl group in the third position of carbonyl chain are highly effective and selective inhibitors of CaE with nanomolar IC50 values, which agrees well with the results of molecular docking.