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Research on cholinesterases and effects of their inhibition in the USSR and Russia since 1930-1940s till present is exposed in historical aspects. The first physiological and toxicological effects of cholinesterase inhibition were reported by Alexander Ginetsinsky during World War II, when academic institutions were evacuated from Leningrad to Kazan. The main scientific schools that initiated research on chemistry, enzymology and physiology of cholinesterases and their inhibitors were leaded by Alexandr and Boris Arbuzovs, Victor Rozengart, Viktor Yakovlev, Michael Michelson, Martin Kabachnik, Mikhail Voronkov, Ivan Knunyants, Alexandr Bretskin and others. They investigated the main physiological effects of cholinesterase inhibitors, and analyzed the catalytic mechanisms of cholinesterases and related enzymes. Their contributions are landmarks in the history of cholinesterase research. At the present time revival of research on cholinesterases in different universities and institutes is vivid, in particular at the Moscow State University, research institutes of Russian Academy of Sciences and Kazan Scientific Center.

The data on sensitivity of cholinesterases (ChE) of different insects to irreversible inhibitors, as well as the data on physicochemical parameters of amino acids constituting their active centers, were treated by factor analysis and compared. These both characteristics have been shown to be connected with the taxonomical position of the insects. There is revealed the “material substrate” of the factors responsible for the action specificity, which are specific sites in the ChE active center.

Using the methods of factor and cluster analysis, the statistical treatment is performed of data on interaction of seven cholinesterases (ChE)—human acetylcholinesterase, horse butyrylcholinesterase, cholinesterases of frog brain and of different squid species (Todarodes pacificus and Berrytheutis magister, in the latter case, individuals from three different habitats are compared)—with 141 reversible inhibitors of various structures. Statistically significant differences between ChE of squids and vertebrates are shown. The previously revealed intraspecies peculiarities of ChE in the Commander squid B. magister are statistically confirmed.

Analytical review of the literature data on interaction constant of cholinesterases from different animal (vertebrates and squids) with 45 bis-onium reversible inhibitors forming homologous series with regularly varied structure has been carried out. Values of the competitive, uncompetitive and generalized inhibitor constants are compared. Interspecies and intraspecies differences in sensitivity of ChE are revealed. Results of conformational analysis of the investigated ligand molecules are presented. The data on population of individual conformations are compared with the data on anticholinesterase potency. Conclusions are made on the action mechanism of the investigated compounds and predominant place of their sorption. The presented data are considered from the point of view of comparative biochemistry and in the light of current information about the active center structure of cholinesterases.

Analytical review is performed of literature data on inhibitor analysis of structure of cholinesterases from different animals (vertebrates, insects, molluscs) by studying rate of their interaction with 190 dialkoxyphosphates of 54 homologous series with regularly changed structure of their phosphoryl group. The presented data are discussed from the point of view of comparative biochemistry and in the light of current concepts of structure of cholinesterase active center in animals at different levels of evolutionary development.

Analytical review of literature data is carried out on inhibitor analysis of structure of cholinesterases in various animals (vertebrates, insects, and molluscs) by studying their interaction with 68 alkoxyalkylthiophosphonates of 10 homologous series with regularly changed structure of phosphoryl part. The presented data are discussed in the comparative-enzymological aspect of current concepts of structure of active center of cholinesterases.

There is presented an analytic review of literature data on inhibitory analysis of structure of cholinesterases of different animals (vertebrates, insects, and molluscs) performed by studying rate of their interaction with 64 organophosphorus compounds of 13 series with different structure of leaving group. The presented data are considered from the point of view of comparative biochemistry and in the light of current data on structure of the cholinesterase active center.

The analytical review is presented of literature data obtained with use of different substrates, on sensitivity of more than 90 preparations of cholinesterases from 65 different animals (vertebrate, insects, molluscs) to the most studied 6 irreversible inhibitors (eserine and 5 organophosphorus compounds). The considered data are discussed from the point of view of comparative biochemistry and in the light of current information about structure of active center of cholinesterases.

An antienzyme potency of 18 organophosphorus inhibitors (OPI), S-alkyl derivatives of thiophosphoric and thiophosphonic acids, to acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) of the spring grain aphid and corresponding mammalian enzymes is studied. It is shown that dependence of the inhibitory potency on length of alkyl radicals differs in human and aphid AChE. Smaller differences were observed in the case of aphid and horse BuChE. The obtained data are compared to the data obtained at studying other series of OPI and cholinesterases of other animals. It is suggested that the revealed peculiarity of the inhibitory specificity of aphid AChE is due to the presence of serine instead of phenylalanine in the position 331 (the numeration according to AChE of the electric ray Torpedo californica).

Study of spatial structure of biologically active guanidine derivatives by the method of molecular mechanics has shown that in an anticoccidial drug, 1,3-bis ( p-chlorobenzylidenamino)guanidine (Cl-BAG) the most preferable are convolute conformations, in which the chlorine atoms that are distant in the valent chain are approached to each other at a distance of 3.7 A. This indicates predisposition of the optimal conformations to form chelate complexes with ions of metals, which is confirmed by comparative spectrophotometric studies of the second derivative of differential UV-spectra of Cl-BAG in the presence and absence of calcium ions. Its derivative without chlorine (BAG) is unable to bind Ca2+ and has been shown to have no anticoccidial action, which associates the biological potency with the presence of calcium-binding ability of the compounds. The capability of Cl-BAG for chelation depends essentially on nature of the chelated metal ion. The antienzyme testing of inhibiting action of the guanidine derivatives toward cholinesterases of human erythrocytes, horse blood serum, mink brain and serum, optic ganglia of the Pacific squid Todarodes pacificus has revealed difference between the enzymes due to possibility of redistribution of the positive charge between the guanidinium fragment and amino groups and a change of the degree of charge delocalization.

Comparative enzymologic study of catalytic properties of cholinesterase (ChE) in blood serum of the American mink Mustela vison Schr. has revealed several peculiarities of this enzyme. First, using the method of substrate–inhibitor analysis, homogeneity of the ChE preparation has been established, i.e. only one ChE has been found in mink serum. Second, the rate of acetylcholine hydrolysis was higher than of thiocholine substrates, among which propionylthiocholine was hydrolyzed at the highest rate. Third, propionylthiocholine had the highest V/K M value that reflects to a degree affinity of the substrate to enzyme. Fourth, the phenomenon of “substrate inhibition,” which is not inherent for mammalian serum cholinesterases, is revealed and kinetically analyzed. Fifth, study of inhibitory specificity has not revealed differences of the mink serum ChE from other serum ChE.

Statistical Analysis of Data on Specificity of Action of Oligopeptides and Amino Acids on Tissues with Different Genesis and Function N. I. Chalisovaa and S. N. Moralev †b a Pavlov Institute of Physiology, Russian Academy of Sciences, St. Petersburg, Russia b Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia E-mail: ni_chalisova@mail .ru

The cluster and factor analyses of the data on variability of active center structure of 41 cholinesterases from different animals were carried out. As characteristics of 32 variable amino acid residues, their hydrophobicity, isoelectric point, polarity, and volume were chosen. As a result of the factor analysis, seven factors were extracted (which, based on the factor scores analysis are called the factors of insect AChE, of nematode AChE-3,4, of nematode AChE-1, of nematode AChE-2,B,C, of mammalian BuChE, of squid AChE, and of tick AChE); they are responsible for 79% of the observed variability. The areas of the active center and the characteristics of the residues, which are the “material substrate” of these factors, are specified. The hierarchical cluster analysis has shown that dendrograms reflecting similarity in “summarized” reactivity of the active center do not coincide with the phylogenetic “tree” of the animals. The conclusion has been made that the direction of evolution of the ChE active center, on one hand, does not have a gradual and regular character and, on the other hand, does not correlate with evolution of ChE protein as a whole.

An analytical review is performed of the literature data on the hydrolysis rate, “affinity” of substrate to active center, and constants of the “substrate inhibition” (K ss) at hydrolysis of the choline (acetyl-, propyonyl-, butyrylcholine, acetyl-β-methylcholine) and/or of corresponding thiocholine substrates by 59 cholinesterases from 49 different animals (chordate, insects, molluscs, nematodes). The characteristic peculiarities of enzymes from different groups of animals are revealed. The absence of regular changes of parameters of the cholinesterase substrate specificity in the course of evolutionary development is shown.

For the second half of the XX century, Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences was the center of the Russian cholinesterase investigations ("the Russian cholinesterase club"). The close cooperation with chemists-syntheticians of different scientific schools provided success and fruitfulness of this scientific search. All these years, there was preserved dualism of this investigation: a study of the mechanism of functioning and kinetics of cholinesterase catalysis as well as the comparative-enzymological character of studies of cholinesterases of the animals being at different levels of evolutionary development.

The data on sensitivity of cholinesterases (ChE) of different insects to reversible inhibitors, as well as the data on physico-chemical parameters of amino acids constituting their active centers, were treated by factor analysis and juxtaposed. It is shown that both these characteristics are related to taxonomical belonging of insects. It is revealed the "material substrate" of the factors determining inhibitor action specificity, which are specific sites in ChE active center.

It is known than the most potent homologues in various series of O,O-dialkylphosphates are the dibutyl or diamyl derivatives toward mammalian cholinesterases (ChEs) (both Acetyl- and Butyryl-ChEs), and the dimethyl or diethyl ones toward insect AChEs. To investigate the ChE interaction with organophosphorus inhibitors (OPIs) in more detail, we have performed in silico docking of the series of O,O-dialkylfluorophosphates into active center of different ChEs – both from mammals (human and mouse AChEs and horse BChE), and from insects (spring grain aphid AChE belonging to AChE-1 type, and housefly AChE belonging to AChE-2 type). According to the modeling results, one radical is directed to the anionic site W84, another to the acyl pocket. In addition to well-known residues 288 and 290 (Torpedo AChE sequence numbering), we showed an essential influence of residue 400 – a short alkyl residue in mammalian ChEs and phenylalanine in insect ChEs. Phenylalanine in this position creates sterical hindrance for proper orientation of the OPI molecule, which increases the distance between the catalytic serine γ-oxygen and phosphorus, and decreases the angle of nucleophylic attack. This suggestion was supported by docking of dibutylfluorophosphate into the active center of AChEs with in silico mutations. Thus, we suggest both the angle of nucleophylic attack and the distance between the catalytic serine and phosphorus atom as measures of productivity of OPI binding.

A series of alkyl N-β-hydroxypropylanabasinyl and alkyl N-but-2-ynylana-basinyl phenylphosphonates and also N-β-hydroxypropylanabasinyl and N-but-2-enylanabasinyl diphenylphosphinates possessing considerable antimonooxygenase activity, exceeding that of the standard inhibitor SKF-525A with respect both to an insecticide (paraoxon) and to a medicinal drug (aminopyrine), have been synthesized.

Analytical review of literature data has been carried out about kinetic parameters of cholinesterases (ChE) of various animals (vertebrates and squids) with 45 reversible bis-onium inhibitors forming homologous series with regularly changing structure. Values of competitive, non-competitive, and generalized inhibitory constants are compared. Interspecies and intraspecies differences are revealed in sensitivity of ChE to bis-onium inhibitors. Results of conformational analysis of molecules of the studied ligands are presented. Data on population of individual conformations are compared with values of anticholinesterase efficiency. Conclusions are made about mechanisms of action of the studied compounds and the predominant site of their sorption. The presented data are discussed from the point of view of comparative enzymology and in the light of the current information about structure of active center of cholinesterases.

Results obtained from complex medical-physiological investigations performed during 10 scientific expeditions in the Arkhangel’sk Region in 2003–2005 are presented. The effects of climatological-geographic, biogeochemical, and social conditions of the conditions obtaining in the Far North region of Russia on sexual maturation, formation of the structural-functional organization of the brain, autonomic functions, and immunological and biochemical status of schoolchildren were studied using state-of-the-art neurophysiological methods (computerized electroencephalography, computerized rheoencephalography, computerized electric dipole origin tomography, etc.), psychophysiological and psychometric methods (assessment of the state of cognitive and memory functions, Wechsler intellectual scale), along with biochemical assay of monoamine oxidase (MAO, the key enzyme in adrenergic neurotransmitter metabolism) and the liver enzyme butyrylcholinesterase (BuChE) and physicochemical analysis of the levels of macroelements and trace elements in the body.

Anticholinesterase activity of carboranyl containing thio- and selenoesters of pentavalent phosphorus acids has been studied. Insertion of the carboranyl substituents in the thioester group of phosphororganic compounds was found to increase the anticholinesterase activity as compared with the thioalkyl analogues. The compounds with B-carboranyl group are less active inhibitors of cholinesterase than their isomers with C-carboranyl group.