Effects of new fluorinated analogues of GABA, pregabalin bioisosters, on the ambient level and exocytotic release of [3H]GABA from rat brain nerve terminals.

Recently, we have shown that new fluorinated analogues of γ-aminobutyric acid (GABA), bioisosters of pregabalin (ß-i-Bu-GABA), i.e. ß-polyfluoroalkyl-GABAs (FGABAs), with substituents: ß-CF3-ß-OH (1), ß-CF3 (2); ß-CF2CF2H (3), are able to increase the initial rate of [3H]GABA uptake by isolated rat brain nerve terminals (synaptosomes), and this effect is higher than that of pregabalin. So, synthesized FGABAs are structural but not functional analogues of GABA. Herein, we assessed the effects of synthesized FGABAs (100µM) on the ambient level and exocytotic release of [3H]GABA in nerve terminals and compared with those of pregabalin (100µM). It was shown that FGABAs 1-3 did not influence the ambient level of [3H]GABA in the synaptosomal preparations, and this parameter was also not altered by pregabalin. During blockage of GABA transporters GAT1 by specific inhibitor NO-711, FGABAs and pregabalin also did not change ambient [3H]GABA in synaptosomal preparations. Exocytotic release of [3H]GABA from synaptosomes decreased in the presence of FGABAs 1-3 and pregabalin, and the effects of FGABAs 1 &3 were more significant than those of FGABAs 2 and pregabalin. FGABAs 1-3/pregabalin-induced decrease in exocytotic release of [3H]GABA from synaptosomes was not a result of changes in the potential of the plasma membrane. Therefore, new synthesized FGABAs 1 &3 were able to decrease exocytotic release of [3H]GABA from nerve terminals more effectively in comparison to pregabalin. Absence of unspecific side effects of FGABAs 1 &3 on the membrane potential makes these compounds perspective for medical application.

Synthesis of new fluorinated analogs of GABA, Pregabalin bioisosteres, and their effects on [(3)H]GABA uptake by rat brain nerve terminals.

Fluorinated analogs of natural substances take an essential place in the design of new biologically active compounds. New fluorinated analogs of γ-aminobutyric acid, that is, ß-polyfluoroalkyl-GABAs (FGABAs), were synthesized with substituents: ß-CF3-ß-OH (1), ß-CF3 (2); ß-CF2CF2H (3). FGABAs are bioisosteres of Pregabalin (Lyrica®, Pfizer's blockbuster drug, ß-i-Bu-GABA), and have lipophilicity close to this medicine. The effects of synthesized FGABAs on [(3)H]GABA uptake by isolated rat brain nerve terminals (synaptosomes) were assessed and compared with those of Pregabalin. FGABAs 1-3 (100µM) did not influence the initial velocity of [(3)H]GABA uptake when applied acutely, whereas an increase in this parameter was found after preliminary incubation of FGABAs with synaptosomes. Pregabalin after preliminary incubation with synaptosomes caused unidirectional changes in the initial velocity of [(3)H]GABA uptake. Using specific inhibitors of GAT1 and GAT3, NO-711 and SNAP5114, respectively, the ability of FGABAs 1-3 to influence non-GAT1 and non-GAT3 uptake activity of nerve terminals was analyzed, but no specificity was found. Therefore, new synthesized FGABAs are structural but not functional analogs of GABA (because they did not inhibit synaptosomal [(3)H]GABA uptake). Moreover, FGABAs are able to increase the initial velocity of [(3)H]GABA uptake by synaptosomes, and this effect is higher than that of Pregabalin.

Thermodynamic theory of strain-mediated direct magnetoelectric effect in multiferroic film-substrate hybrids.

A nonlinear thermodynamic theory is developed for the strain-mediated direct magnetoelectric (ME) effect displayed by ferroelectric-ferromagnetic nanostructures. This effect results from transmission of magnetic-field-induced deformations of a thick ferromagnetic substrate to a thin ferroelectric overlayer, where the polarization changes due to lattice strains. The strain-dependent polarization and permittivity of an epitaxial nanolayer (few tens of nm thick) are calculated using the thermodynamic theory of single-domain ferroelectric films. The substrate magnetostrictive deformations are described phenomenologically, taking into account their nonlinear variation with magnetic field. The calculations show that ME polarization and voltage coefficients strongly depend on the initial strain state of the film. For BaTiO(3) and PbTiO(3) films deposited on Co(0.8)Zn(0.2)Fe(2)O(4), the out-of-plane polarization and related ME coefficients are calculated numerically as a function of magnetic field parallel to the interface. For films stabilized in the monoclinic phase, this transverse ME response depends on the orientation of magnetic field relative to their in-plane crystallographic axes. The longitudinal ME coefficient is also evaluated and, for a substrate geometry minimizing the demagnetizing field, predicted to be comparable to the transverse one. For BaTiO(3) and PbTiO(3) films deposited on Terfenol-D, the calculations yield high ME polarization coefficients approximately 10(-7) s m(-1) and giant ME voltage coefficients approximately 50 V cm(-1) Oe(-1).

[Inhibition of alkaline phosphatase by thioureido derivatives of methylenebisphosphonic acid].

A series of thioureido derivatives of methylenebisphosphonic acid were synthesized by the reaction of aminomethylenebisphosphonic acid with the corresponding isothiocyanates, and their effect on the activity of alkaline phosphatases from bovine small intestine mucosa (BSIM) and human placenta was studied. It was found that (3-phenylthioureido)methylenebisphosphonate is approximately one order of magnitude more effective in inhibiting the activity of alkaline phosphatase from BSIM than the alkyl derivatives of thioureidomethylenebisphosphonic acid with methyl, ethyl, tert-butyl, or cyclohexyl substituents. The introduction of substituents into the benzene ring of (3-phenylthioureido)methylenebisphosphonate decreased the effect of the inhibitor on the activity of the enzyme. The affinity of (3-phenylureido)methylenebisphosphonate to the alkaline phosphatase of BSIM was also weaker as compared with the corresponding thioureidomethylenebisphosphonate. The insertion of thioureidobisphosphonates into the active site of alkaline phosphatase of human placenta by the method of molecular docking indicated that the methylenebisphosphonate residue and the substituted amino groups of the inhibitor are involved in the mechanisms of complex formation with the enzyme. It is supposed that the improvement of the inhibitory activity of (3-phenylthioureido)methylenebisphosphonate toward alkaline phosphatase of BSIM is due to the additional fixation of the phenyl substituent in the active site of the enzyme.

[Antithrombic activity of N(alpha)-lauroyl-L-arginine ethyl ester (LAE) and 9-fluorenylmethoxycarbonyl-L-arginine methyl ester (Fmoc-L-Arg-OMe)].

Simple synthetic compounds of lauroyl-arginine ethyl ester (LAE) and 9-fluorenylmethoxycarbonyl-L-agrinine methyl ester (Fmoc-Arg-OMe) were studied for their inhibitory effect on the hydrolysis of chromogenic substrate Tos-Gly-Pro-Arg-pNA (Chromozym TH) by thrombin with K(i) for LAE 1.92 microM and 77 microM for Fmoc Arg-OMe. It was shown that LAE inhibits thrombin activity almost 20 times more strongly than trypsin (K(i) = 18.9 microM). At the same time LAE preserves the ability to be hydrolyzed by thrombin at pH 8.5 (k(cat) = 3.6 c(-1)) and trypsin (k(cat) = 56 c(-1)). It is suggested, that LAE ability to suppress growth of some microorganisms is conditioned to some extent by its ability to inhibit the activity of trypsin-like serine proteases, participating in the infection process.

[Calix[4]arene methylenebisphosphonic acids: the features of alkaline phosphatases inhibition].

The inhibition of alkaline phosphatases by calix[4]arenes functionalysed at the macrocyclic upper rim by one or two methylenebisphosphonic acid fragments has been investigated. It is established, that calix[4]arene bismethylenebisphosphonic acid displayed stronger inhibition of alkaline phosphatase from bovine intestine mucosa than calix[4]arene methylenebisphosphonic acid. At the same time, the both inhibitors showed almost similar levels of inhibitory activities in respect of bovine kidney alkaline phosphatase or E. coli alkaline phosphatase. The tested compounds were docked computationally to the active site of the E. coli alkaline phosphatase. On the basis of results obtained the possible binding modes of inhibitors were analysed.

[Inhibiting properties of stable nitroxyl radicals in reactions of linoleic acid and linoleyl alcohol oxidation catalyzed by 5-lipoxygenase].

The inhibiting effects of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and its 4-substituted derivatives in reactions of linoleyl acid or linoleyl alcohol oxidation catalyzed by potato tuber 5-lipoxygenase were investigated. Inhibiting properties of stable nitroxyl radicals in presence of lubrol and SDS were reduced at the transition from TEMPO to 4-hydroxy-TEMPO or 4-amino-TEMPO and increased at use of adamantane-1-carboxylic or 3-methyladamantane-1-carboxylic acid 1-oxyl-2,2,6,6-tetramethylpiperidine-4-yl esters. Enzyme activity at saturating concentrations of inhibitor was not suppressed completely, and decreased up to the certain level determined by the substrate nature. The dependence of partial inhibition efficiency on rotational correlation time of stable nitroxides in model micellar systems were analysed. It was supposed that 5-lipoxygenase inhibition includes the interaction of hydrophobic nitroxide with radical intermediate formed in enzymatic process.

[Hydrophobic nitroxyl radicals inhibit linoleyl alcohol oxidation by 5-lipoxygenase]. / Gidrofobnye nitroksil'nye radikaly--ingibitory okisleniia linolevogo spirta 5-lipoksigenazoi.

The linoleyl alcohol oxidation catalyzed by potato tuber 5-lipoxygenase was found to be efficiently inhibited by stable nitroxyl radicals: 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl 1-bicyclo[2,2,2]octane-1-carboxylate, 1-adamantylacetate, dodecanoate, and octadecanoate. The dependence of apparent IC50 values on the rotational correlation times of times of 4-hydroxy-1-oxyl-2,2,6,6-tetramethylpiperidine and its derivatives in model micellar systems was analyzed. The inhibition mechanism was proposed; it involves the interaction of hydrophobic nitroxyl radical with the intermediate radical enzyme-substrate complex.

[Synthesis and antithrombin activity of phosphoalanine-containing peptides]. / Sintez i uzuchenie antitrombinovoi aktivnosti pepidov, soderzhashchikh fosfoalanin.

Boc/Tos-L-Phe-L-Arg-Xaa tripeptides (where Xaa = L-Ala-OBut, L-Ala, or DL-AlaP (OC2H5)2) were synthesized by conventional methods of peptide synthesis in solution. Special features of their interaction with thrombin and trypsin were studied. Unlike trypsin, thrombin did not catalyze the hydrolysis of the L-Arg-L-AlaP-(OC2H5)2 bond. The Tos-L-Phe-L-Arg-DL-AlaP(OC2H5)2 peptide was the most active inhibitor of thrombin among all the compounds studied. The relationship between the structure and inhibitory action of the synthesized peptides is discussed. A part of this study was reported in the Augustusburg Conference of Advanced Science: Nucleic Acids--Targets and Tools, September 17-19, 2000, Germany.

[Inhibition of penicillin acylase from Escherichia coli by benzylalkylketones and benzylalkylcarbinols]. / Ingibirovanie benzilalkilketonami i benzilalkilkarbinolami penitsillinatsilazy iz Escherichia coli.

A number of benzylalkylketones and benzylalkylcarbinols have been synthesized as non-hydrolizable substrate analogues of penicillin acylase (EC 3.5.1.11), and their affinity to the enzyme has been studied. The compounds with plane trigonal carbonyl group (ketones) were established to has bind to the enzyme 20-40 times more tightly than their tetrahedral counterparts with a hydroxyl function (carbinols). 4-Oxo-5-phenylpentanoic acid was found to be one of the most potent reversible competitive inhibitors of penicillin acylase with Ki-31 microM.

[Phosphorus-containing inhibitors of penicillin acylase. 4. Irreversible inhibition of penicillin acylase from Escherichia coli by monoaryl esters of benzylphosphonic acid]. / Fosfor-soderzhashchie ingibitory penitsillinatsilazy. 4. Neobratimoe ingibirovanie penitsillinatsilazy iz Escherichia coli monoarilovymi éfirami benzilfosfonovoi kisloty.

Monoaryl of benzylphosphonic acid have been synthesized and studied as the inhibitors of penicillin acylase. These compounds were found to be effective and selective irreversible inhibitors of the enzyme. The kinetic parameters of enzyme inactivation are determined, and possible mechanism of the inhibition is discussed. These phosphonates should be useful as both penicillin acylase active site titrants and the tools for the enzyme function study. Benzylchloromethyl keton has been also prepared and it is an irreversible inhibitor of penicillin acylase.

HIV-1 reverse transcriptase inhibitor design using artificial neural networks.

Artificial neural networks were used to analyze and predict the human immunodeficiency virus type 1 reverse transcriptase inhibitors. The training and control sets included 44 molecules (most of them are well-known substances such as AZT, dde, etc.). The activities of the molecules were taken from literature. Topological indices were calculated and used as molecular parameters. The four most informative parameters were chosen and applied to predict activities of both new and control molecules. We used a network pruning algorithm and network ensembles to obtain the final classifier. Increasing of neural network generalization of the new data was observed, when using the aforementioned methods. The prognosis of new molecules revealed one molecule as possibly very active. It was confirmed by further biological tests.

[Phosphorus-containing inhibitors of penicillin acylase. 3. Inhibition of Escherichia coli penicillin acylase by phosphonate analogs of substrates]. / Fosforsoderzhashchie ingibitory penitsillinatsilazy. 3. Ingibirovanie penitsillinatsilazy Escherichia coli fosfonatnymi analogami substratov.

Phosphonic analogues of penicillin acylase substrates are found to be selective reversible competitive inhibitors of the enzyme from E. coli (EC 3.5.1.11). The mode of binding of the inhibitors to the enzyme and the influence of the stereoelectronic parameters of the phosphonic inhibitors on their affinity to the enzyme are discussed.

[Phosphorus-containing inhibitors of penicillin acylase. 2. Design, synthesis and study of the hydrolytic stability of phosphonic acid derivatives as potential inhibitors of penicillin acylase]. / Fosforsoderzhashchie ingibitory penitsillinatsilazy. 2. Dizain, sintez i izuchenie gidroliticheskoi stabil'nosti proizvodnykh fosfonovykh kislot kak potentsial'nykh ingibitorov penitsillinatsilazy.

Phosphonate and phosphonoamidate derivatives of benzylphosphonic acids were synthesized as potential inhibitors of penicillin acylase (EC 3.5.1.11) proceeding from the concept of transition-state analogues. The compounds obtained are not the substrates of the enzyme and they are stable under conditions of enzyme activity testing.

Influence of phosphate ion on the fluorescence of 3-fluorotyrosine.

3-Fluorotyrosine fluorescence is quenched effectively by phosphate ions not only by a dynamic but also by a static mechanism owing to H-bond complex formation in ground state. 3-Fluorotyrosine pKa values both in the ground and first excited state (8.3 and 4, respectively) are appreciably lower than those of tyrosine, thus promoting 3-fluorotyrosinate ion formation in the excited state. Additional emission owing to 3-fluorotyrosinate ion (near 350 nm) may be taken erroneously for tryptophan fluorescence.

O-Perfluoroalkylation of 4-hydroxyphenylglycine.

Racemic N-Boc-4-difluoromethoxyphenylglycine was prepared by O-difluoromethylation of D-N-Boc-4-hydroxyphenylglycine under basic conditions, whereas the hexafluoropropylation reaction gives optically pure D-N-Boc-4-hexafluoropropoxyphenylglycine. D,L-4-difluoromethoxyphenylglycine was obtained by the action of TFA on the corresponding amino acid derivative.

[Inhibition of yeast pyruvate decarboxylase by alkyl phosphates]. / Ingibirovanie drozhzhevoi piruvatdekarboksilazy alkilfosfatami.

It is found that yeast pyruvate decarboxylase is inhibited by alkyl phosphates. Inhibition is competitive with respect to a substrate. The inhibition constants with n-butyl and n-heptyl esters of phosphoric acid are the values of the same order of magnitude. With an increase in the length of the alkyl phosphates hydrocarbon chain from 7 to 10 carbon atoms inhibition constants change drastically. For n-heptyl phosphate and n-decyl phosphate values KI are equal to 1.6 x 10(-4) M and 1.7 x 10(-6) M, respectively. A further increase in the number of carbon atoms in the alkyl substituent of phosphoric acid ester induces no reduction of the inhibition constant. Multiple-inhibitor experiments of pyruvate decarboxylase show that inorganic phosphate and n-decyl ester of phosphoric acid are mutually exclusive. It is suggested that the inhibition mechanism with alkyl phosphates includes the competition of the phosphoric acid residue with alpha-ketocarboxyl group of pyruvate as well as the interaction between a hydrocarbon radical and hydrophobic parts on the enzyme surface, one of them being outside the substrate binding site.

The unusual action of (R,S)-2-hydroxy-2-trifluoromethyl-trans-n-octadec-4-enoic acid on 5-lipoxygenase from potato tubers.

We found that (R,S)-2-hydroxy-2-trifluoromethyl-trans-n-octadec-4-enoic acid (HTFOA) is a powerful activator of 5-lipoxygenase from potato tubers. The degree of activation of the enzyme is proportional to the HTFOA concentration and is a maximum at about 0.1 mM independently of initial substrate concentration (25 microM or 0.1 mM). At greater concentrations of HTFOA, enzyme inhibition takes place. Enzyme activation is inversely proportional to the substrate (linoleic acid) concentration. The results may be explained by assuming that a regulatory center exists in the enzyme molecule, which shows affinity to both substances: activator and linoleic acid.