Comparative analysis of sensitivity of cholinesterases of different origin to monoonium reversible inhibitors

The analytic review of the literature data on constants of interaction of cholinesterases of different animal (vertebrates and squids) with 89 onium (ammonium, phosphonium, sulfonium) reversible inhibitors constituting homologous series with regularly varied structure is carried out. Values of the competitive, uncompetitive and generalized inhibitor constants are compared. On the basis of that, conclusions about the mechanism of action of the studied compounds and primary place of their sorption—in “anionic” or peripheral “anionic” sites of enzymes—are made. The presented data are considered from the point of view of comparative biochemistry and in light of current concepts of cholinesterase active center structure.     

Comparative analysis of sensitivity of cholinesterases of various origin to monoonium reversible inhibitors

Analytical review of literature data has been presented about constants of interaction of cholinesterases of various animals (verterbrates and squids) with 89 onium (ammonium, phosphonium, and sulfonium) reversible inhibitors forming homologous series with regularly changed structure. Values of competitive, uncompetitive, and generalized inhibition constants have been compared. On this basis, conclusions are made about mechanism of action of the studied compounds and the predominant areas of their sorption--in the or peripheral sites of the enzymes. The presented data are discussed from the point of view of comparative biochemistry and in the light of the current information about structure of the cholinesterase active center.     

Reversible lupininin inhibitors of cholinesterases of mammalian blood and of optical ganglia of individuals of the commander squid Berryteuthis magister from different zones of species areal

Arylsulfoesters and carbonic lupinin esters are studied for the first time as reversible inhibitors of mammalian blood cholinesterases. Studied in detail is sensitivity of cholinesterases to mono- and bislupinin inhibitors in Commander squid individuals from different habitation zones.     

Reversible lupinin inhibitors of cholinesterases of mammalian blood and of optical ganglia of individuals of the Commander squid <Emphasis Type="Italic">Berryteuthis magister</Emphasis> from different zones of species areal

Arylsulfoesters and carbonic lupinin esters are studied for the first time as reversible inhibitors of mammalian blood cholinesterases. Studied in detail is sensitivity of cholinesterases to mono- and bilupinin inhibitors in Commander squid individuals from different habitation zones.     

Facile synthesis of sulfonium ion derivatives of 1,5-anhydro-5-thio-L-fucitol as potential alpha-L-fucosidase inhibitors

Five sulfonium ion derivatives with 1,5-anhydro-5-thio-L-fucitol as a core structure were efficiently synthesized as potential alpha-L-fucosidase inhibitors. The key unit, the tri-O-benzyl derivative of L-fucitol, was readily synthesized from methyl alpha-D-mannopyranoside. Alkylation with methyl iodide or 5-methoxycarbonyl-1-pentyl iodide in acetonitrile containing AgBF4 afforded the corresponding alkylated sulfonium tetrafluoroborates. Alternatively, ring opening of three 1,3-cyclic sulfates in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) containing K2CO3 afforded the corresponding zwitterionic sulfonium sulfates.     

Derivatives of lupinin and epilupinin as ligands of various cholinesterases

Literature data are summarized on cholinesterases of some mammals and arthropods with a group of isomer derivatives of alkaloid lupinin and its epimer epilupinin. As substrates of cholinesterases of some mammals there are studied 8 acetates containing in their molecule chinolysidin bicycle with different structure of N-alkyl radical, which showed certain elements of action specificity. For 2 isomer esters that are derivatives of protonated base of lupinin and epilupinin, differences are revealed in their substrate characteristics. Polyenzyme analysis of anticholinesterase efficiency is performed for 30 organophosphorus inhibitors that are dialcoxyphosphorus derivatives of lupinin and epilupinin; as a result, we managed to find out quite a few peculiarities of their action depending on structure. Several tested compounds were revealed to be specific inhibitors of cholinesterases of several mammals and arthropods.     

Derivatives of lupinin and epilupinin as ligands of various cholinesterases

Literature data have been summarized on interaction of cholinesterases of some mammals and arthropods with a group of isomer derivatives of alkaloid lupini and its epimer epilupinin. As substrates of cholinesterases of several mammals there are studied 8 acetates containing in their molecules the chinolysidin bicycle with different structure of N-alkyl radical, which showed certain elements of specificity of action. For 2 isomer esters that are derivatives of the protonated base of the lupinin and epilupinin structures, differences in their substrate characteristics were revealed. The polyenzyme analysis if anticholinesterase efficiency was performed for 30 organophosphorus inhibitors that are dialkoxyphosphorus derivatives of lupinin and epilupinin; as a result, quite a few peculiarities of their action depending on their structure were revealed. Several tested compounds turned out to act as specific inhibitors of cholinesterases of some mammals and arthropods.     

Comparative Sensitivity of Cholinesterases of Different Origin to Some Irreversible Inhibitors

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.     

Mechanism of anticholinesterase activities of cardiotoxin, protamine and polylysine.

Cardiotoxin, protamine and polylysine are potent inhibitors of various cholinesterases. CaCl2 and MgCl2 overcome the inhibition. The order of addition of the inhibitor and the protecting agent (MgCl2) influences the final degree of the inhibition observed. These findings suggest that cardiotoxin, protamine and polylysine inhibit cholinesterases by the ionic binding of their basic groups with the anionic sites of cholinesterase molecules.     

Comparative analysis of sensitivity of cholinesterases of different origin to reversible bis-onium inhibitors

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.     

Kinetic properties of butyrylcholinesterases immobilised on pH-sensitive field-effect transistor surface and inhibitory action of steroidal glycoalkaloids on these enzymes

The inhibitory action of steroid glycoalkaloids alpha-solanine, alpha-chaconine and tomatine on horse and human serum butyryl cholinesterases immobilized on the pH-sensitive field-effect transistors has been studied. Using acetyl- and butyryl choline as substrates, the optimal pH and the apparent kinetic parameters (< K(m) >, < V(max) >) of immobilized butyryl cholinesterases have been calculated in the absence of inhibitors. The affinity of each enzyme to glycoalkaloids has been estimated from calculation of apparent inhibition constants < K(i) > and inhibition coefficients i(0.5). Application of the studied cholinesterases for biosensoric determination of glycoalkaloids in the wide range of concentrations (10(-7)-10(-4) M) in different media has been discussed.     

Inhibition of monoterpene cyclases by sulfonium analogs of presumptive carbocationic intermediates of the cyclization reaction

The enzymatic cyclization of geranyl pyrophosphate to monoterpenes is thought to proceed through a series of carbocation-pyrophosphate anion paired intermediates. Sulfonium analogs of two putative carbocationic intermediates of the cyclization sequence were shown to be inhibitors of the conversion of the acyclic precursor to the bicyclic monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate by partially purified cyclase preparations from sage (Salvia officinalis). The sulfonium analog of the tertiary allylic, linalyl, intermediate (i.e. methyl-(4-methylpent-3-en-1-yl)vinyl-sulfonium perchlorate) provided respective Ki values of 2.5 microM and 3.0 microM against the cyclization to alpha-pinene and bornyl pyrophosphate at a substrate concentration of 5 microM, whereas the sulfonium analog of the monocyclic, alpha-terpinyl, intermediate (i.e. dimethyl-(4-methylcyclohex-3-en-1-yl) sulfonium iodide) exhibited respective Ki values of 3.4 microM and 3.9 microM against the same two cyclizations. The potency of inhibition in all cases increased with increasing substrate concentration, indicating that the affinity of the enzymes for the sulfonium analogs was increased by the presence of the pyrophosphate ester. Inorganic pyrophosphate at a concentration of 50 microM, which alone had little influence on the cyclizations, increased the effectiveness of inhibition of the sulfonium analogs severalfold, and the apparent Ki for inorganic pyrophosphate was reduced manyfold by the presence of either analog at 5 microM. That the combination of sulfonium analog and pyrophosphate provided synergistic inhibition of the electrophilic cyclizations indicated that the cyclases bind the paired species more tightly than either partner alone. Specificity studies suggested that inhibition by the above sulfonium ion:pyrophosphate pairs was due to both electronic and structural resemblance to intermediates of the reaction.     

On Establishment of Individuality of the Cholinesterase Enzyme in the Studied Preparation

Theoretical analysis is carried out and a reliable kinetic method for establishment of individuality of cholinesterases in studied preparation is proposed. For reliable conclusion about the presence of single cholinesterase or a mixture of cholinesterases in the biosample, it is necessary to determine values of the experimental kinetic constant of irreversible inhibition by several (three or more) inhibitors, using for evaluation of the catalytic activity of the biomaterial not less than three different substrates consecutively, for example, acetyl--methylcholine (substrate selective to typical acetylcholinesterase), butyrylcholine (substrate selective to typical butyrylcholinesterase), and acetylcholine (substrate hydrolyzed easily by cholinesterases of different types).     

Comparative analysis of sensitivity of cholinesterase inhibitors by multidimensional statistical methods

Using the methods of factor and cluster analysis, the statistical treatment is performed of data on interaction of 7 cholinesterases (ChE)--human acetylcholinesterase, horse butyrylcholinesterase, cholinesterases of frog brain and of various 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.     

Synthesis of a novel class of sulfonium ions as potential inhibitors of UDP-galactopyranose mutase

Two sulfonium salts of 1,4-anhydro-4-thio-D-galactitol, with structures related to the known sulfonium salt glycosidase inhibitor, salacinol, have been synthesized as potential inhibitors of UDP-galactopyranose mutase. The synthetic strategy relies on the alkylation reaction of 1,4-anhydro-2,3,5,6-tetra-O-benzyl-4-thio-D-galactitol at the sulfur atom with 2,4-O-benzylidene-D- or -L-erythritol-1,3-cyclic sulfate. In each case, the reaction proceeded stereoselectively to yield only one stereoisomer at the stereogenic sulfur atom. The effect of the polar solvent, 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), in promoting high-yielding reactions is highlighted. The target compounds are then obtained by hydrogenolysis.     

Biochemical and physiological studies of soluble esterases fromDrosophila melanogaster

Twenty-two soluble esterases have been identified inD. melanogaster by combining the techniques of native polyacrylamide gel electrophoresis and isoelectric focusing. The sensitivity of each isozyme to three types of inhibitors (organophosphates, eserine sulfate, and sulfydryl reagents) identified 10 as carboxylesterases, 6 as cholinesterases, and 3 as acetylesterases. Three isozymes could not be classified and no arylesterases were identified. The carboxyl- and cholinesterases could each be further divided into two subclasses on the basis of inhibition by organophosphates and sulfhydryl reagents, respectively. Cholineand acetylesterases have characteristic substrate preferences but both subclasses of carboxylesterases are heterogeneous in substrate utilization. Subclass 2 carboxylesterases exhibit diverse temporal expression patterns, with subclass 1 carboxylesterases generally found in larvae and subclass 1 cholinesterases and acetylesterases more characteristic of pupae and adults. Tissues showing the greatest number of isozymes are larval body wall (eight) and digestive tract (six in larvae, six in adults). Carboxylesterases are distributed across a wide range of tissues, but subclass 1 cholinesterases are generally associated with neural or neurosecretory tissues and subclass 2 cholinesterases with digestive tissues.This study was funded in part by the Rural Credits Development Fund.     

Synthesis of potent water-soluble tissue transglutaminase inhibitors

Dipeptide-based sulfonium peptidylmethylketones derived from 6-diazo-5-oxo-L-norleucine (DON) have been investigated as potential water-soluble inhibitors of extracellular transglutaminase. The lead compounds were prepared in four steps and exhibited potent activity against tissue transglutaminase.     

Stereochemical aspects of cholinesterase catalysis

Proceeding from the sterochemical regularities of the nucleophilic substitution reaction at the carbonyl group and the assumption that the spatial structure of the active center of cholinesterases is complementary to the molecule of the ester substrates for these enzymes, some general features of the stereoselectivity phenomena in the reactions of acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) with organophosphorus inhibitors are discussed. For these enzymes the models of the active center are proposed in terms of different binding sites and the catalytic center. On the basis of this model, the stereochemical pecularities and the physicochemical background of the stereoselectivity effects on enzyme inhibition, reactivation, and “aging” reactions can be understood. Knowledge of the absolute configuration of several chiral organophosphorus inhibitors also makes it possible to determine the absolute spatial arrangement of the hydrophobic binding sites on the active surface of cholinesterases.     

The specificity of the interaction of the cholinesterases in Far Eastern squid and certain vertebrates with reversible inhibitors

Studies have been made of the effect of three groups of ammonia reversible inhibitors on the activity of erythrocyte acetylcholinesterase, serum butyrylcholinesterase, cholinesterase from frog brain, as well as cholinesterases from the optical ganglia of the Pacific and three populations of the commander squids. Determination of kinetic parameters of the reversible inhibition of these enzymes revealed differences resulting from the specific structure of their catalytic centers. Tetramethylammonium assay confirmed different properties of cholinesterases in individuals of the commander squid from various habitats in the Bering Sea; this finding may be taken as an indication of intraspecific differentiation of these cephalopods. Certain similarity was noted in the inhibitory specificity of cholinesterases from the Pacific and "southern" commander squids with the overlapping habitats.     

Isolation, structure identification and SAR studies on thiosugar sulfonium salts, neosalaprinol and neoponkoranol, as potent α-glucosidase inhibitors

Two hitherto missing members of sulfonium salts family in Salacia genus plants as a new class of α-glucosidase inhibitors, neoponkoranol (7) and neosalaprinol (8), were isolated from the water extracts, and their structures were unambiguously identified. For further SAR studies on this series of sulfonium salts, several epimers of 7 and 8 were synthesized, and their inhibitory activities against rat small intestinal α-glucosidases were evaluated. Among them, 3'-epimer of 7 was found most potent in this class of molecules, and revealed as potent as currently used antidiabetics, voglibose and acarbose.