Inhibition of acetylcholinesterase by an alkylpyridinium polymer from the marine sponge, Reniera sarai.

Large polymeric 3-alkylpyridinium salts have been isolated from the marine sponge Reniera sarai. They are composed of N-butyl(3-butylpyridinium) repeating subunits, polymerized head-to-tail, and exist as a mixture of two main polymers with molecular weights without counterion of about 5520 and 18900. The monomer analogue of the inhibitor, N-butyl-3-butylpyridinium iodide has been synthesized. This molecule shows mixed reversible inhibition of acetylcholinesterase. The polymers also act as acetylcholinesterase inhibitors and show an unusual inhibition pattern. We tentatively describe it as quick initial reversible binding, followed by slow binding or irreversible inhibition of the enzyme. This kinetics suggests that there are several affinity binding sites on the acetylcholinesterase molecule where the polymer can bind. The first binding favors binding to other sites which leads to an apparently irreversibly linked enzyme-inhibitor complex.

A putative kinetic model for substrate metabolisation by Drosophila acetylcholinesterase.

Insect acetylcholinesterase, an enzyme whose catalytic site is located at the bottom of a gorge, can metabolise its substrate in a wide range of concentrations (from 1 microM to 200 mM) since it is activated at low substrate concentrations. It also presents inhibition at high substrate concentrations. Among the various rival kinetic models tested to analyse the kinetic behaviour of the enzyme, the simplest able to explain all the experimental data suggests that there are two sites for substrate molecules on the protein. Binding on the catalytic site located at the bottom of the gorge seems to be irreversible, suggesting that each molecule of substrate which enters the active site gorge is metabolised. Reversible binding at the peripheral site of the free enzyme has high affinity (2 microM), suggesting that this binding increases the probability of the substrate entering the active site gorge. Peripheral site occupation decreases the entrance rate constant of the second substrate molecule to the catalytic site and strongly affects the catalytic activity of the enzyme. On the other hand, catalytic site occupation lowers the affinity of the peripheral site for the substrate (34 mM). These effects between the two sites result both in apparent activation at low substrate concentration and in general inhibition at high substrate concentration.

Biosynthetic pathway of citrinin in the filamentous fungus monascus ruber as revealed by 13C nuclear magnetic resonance

Carbon isotope distribution of [13C]citrinin from Monascus ruber incubated with [13C]acetate revealed that the biosynthesis of the toxin originated from a tetraketide, instead of a pentaketide as has been shown for Penicillium and Aspergillus species. The production of polyketide red pigments and citrinin by M. ruber may therefore be regulated at the level of the tetraketide branch point.

Structural similitudes between cytotoxic antiestrogen-binding site (AEBS) ligands and cytotoxic sigma receptor ligands. Evidence for a relationship between cytotoxicity and affinity for AEBS or sigma-2 receptor but not for sigma-1 receptor.

1-Benzyl-4-(N-2-pyrrolidinylethoxy)benzene (PBPE) is a cytotoxic derivative of the antitumoral drug tamoxifen. PBPE binds with high-affinity and specificity to the microsomal antiestrogen-binding site (AEBS). PBPE, as well as some other high-affinity AEBS ligands, shares structural features with high-affinity and selective sigma receptor ligands in the N-(arylethyl)-N-alkyl-2-(1-pyrrolidinyl)ethylamine class, such as BD1008, which are cytotoxic against tumoral cells. Based on these structural and pharmacological similitudes, we set out to examine whether AEBS and sigma receptors could be related binding sites. We showed that BD1008 had a high affinity for AEBS. However, prototypical sigma receptor ligands were very low-affinity competitors on AEBS. Surprisingly, AEBS ligands displayed a high affinity for sigma-1 and sigma-2 receptor subtypes, showing that AEBS and sigma receptor-binding sites were not mutually exchangeable. Moreover, phenytoin, which is an allosteric modulator of sigma-1 receptor, was a competitive inhibitor of [3H]tamoxifen on AEBS. These results suggest that the tamoxifen-binding site on AEBS and the sigma ligand-binding site on sigma receptors were not identical but related entities. We also showed here that the high-affinity and specific AEBS ligands also bound sigma receptors with high affinity. Moreover, the compounds that were capable of displacing tamoxifen from AEBS were cytotoxic against tumoral cells but not against the AEBS-deficient cell line Rtx-6. These results confirm that AEBS and sigma receptors might belong to the same family of proteins, and that the tamoxifen-binding site might be involved in the cytotoxicity of AEBS ligands and some classes of sigma compounds.

Insecticidal properties of mushroom and toadstool carpophores.

In order to find compounds with insecticidal or antifeedant properties from mushrooms and toadstools, a wide screening was undertaken using the non-mycophagous Drosophila melanogaster as a model insect. Powdered fruit bodies of edible and poisonous mushrooms were incorporated with the Drosophila's rearing medium, and their development was observed. Among the 175 different species of fungi tested, 79 were found to inhibit insect development, hence making the isolation of new compounds look hopeful.

Identification and quantification of Tuber melanosporum Vitt. sterols.

The sterol composition of Tuber melanosporum was examined by medium-pressure liquid and high-performance liquid chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry. Ergosterol (ergosta-5,7,22-trienol) and brassicasterol (ergosta-5,22-dienol) were identified as the major components (90%). Their quantities and their relative concentrations, probably good indicators for differentiating mature truffles from immature colored ones, were determined during the maturation of the fungus. The results show that the total quantity of sterols decreased with the age of the fungus, but that the ergosterol/brassicasterol ratio remained relatively constant.

Further evidence for a biological role of anti-estrogen-binding sites in mediating the growth inhibitory action of diphenylmethane derivatives.

Several diphenylmethane derivatives have been synthesized with variable affinities for Anti-estrogen Binding Sites (ABS) but not for the estrogen receptor. Using these molecules as probes it is shown that their binding affinities for ABS correlate with their abilities to inhibit the growth of MCF-7 human breast cancer cells. In contrast they have no influence on the proliferation of tamoxifen-resistant variant cells (RTx6) in which ABS are undetectable. These data support the conclusion that ABS has a functional role in the anti-proliferative effect of triphenylethylene anti-estrogens and structurally related compounds.

Inhibition by sodium thiophosphate and L-p-bromotetramisole of neutrophil alkaline phosphatase in normal and trisomy 21 pregnancies.

Alkaline phosphatase concentrations are known to increase in blood neutrophils of normal pregnant women. The main kinetic parameters of this enzyme were analysed and compared in a group of 30 women with normal pregnancies and a group of 11 women whose fetuses had trisomy 21 (Down's syndrome = DS). The subjects were studied at an identical stage of gestation. Significant changes occurred in thermal stability and urea resistance in cases of DS pregnancies. We also investigated the inactivation constants for two chemicals: L-p-bromotetramisole, an uncompetitive inhibitor, and sodium thiophosphate, a competitive inhibitor. Ki measured for the two inhibitors were found to be significantly lower in cases of pathological pregnancies. The patterns observed in inhibition constants extend the biochemical characteristics of the atypical isoenzyme expressed in neutrophils of women with DS pregnancies.

Exploration of the Drosophila acetylcholinesterase substrate activation site using a reversible inhibitor (Triton X-100) and mutated enzymes.

Cholinesterases are activated at low substrate concentration, and this is followed by inhibition as the level of substrate increases. However, one of these two components is sometimes lacking. In Drosophila acetylcholinesterase, the two phases are present, allowing both phenomena to be studied. Several kinetic schemes can explain this complex kinetic behavior. Among them, one model assumes that activation results from the binding of a substrate molecule to a non-productive site affecting the entrance of a substrate molecule into the active site. To test this hypothesis, we looked for an inhibitor competitive for activation and we found Triton X-100. Using organophosphates or carbamates as hemisubstrates, we showed that Triton X-100 inhibits or increases phosphorylation or carbamoylation of the enzyme. In vitro mutagenesis of the residues lining the active site gorge allowed us to locate the Triton X-100 binding site at the rim of the gorge with glutamate 107 playing the major role. These results led to the hypothesis that substrate binding at this site affects the entrance of another substrate molecule into the active site cleft.

Inhibition of platelet type II phospholipase A2 by an acylamino phospholipid does not alter arachidonate liberation.

An acylamino phospholipid analogue (2-(R)-N-palmitoylnorleucinol-1-phosphoglycol or (R)-PNPG) was examined for its inhibitory effects against type II phospholipase A2 (PLA2) acting on membranes from Escherichia coli. Using two enzyme sources (rat platelet membranes or recombinant human type II PLA2), (R)-PNPG inhibited phospholipid hydrolysis to a maximal value of 80-85%, half-maximal effect being attained at a substrate/inhibitor molar ratio of 80-250. In contrast, (S)-PNPG was 12-fold less potent and thus provided a control for possible non-specific effects of these polar lipids. However, both analogues exerted only marginal effects on the liberation of [3H]arachidonic acid from rat platelets challenged with calcium ionophore A23187. Since, among various animal species, rat platelets contain by far the highest amounts of this enzyme, our data rule out any possible involvement of secretory PLA2 in arachidonic acid liberation from platelet phospholipids, cytosolic PLA2 appearing in this case as the best candidate able to regulate eicosanoid biosynthesis.

Production and Identification of N-Glucosylrubropunctamine and N-Glucosylmonascorubramine from Monascus ruber and Occurrence of Electron Donor-Acceptor Complexes in These Red Pigments.

The filamentous fungus Monascus ruber produces water-soluble red pigments in a submerged culture when grown in a chemically defined medium containing glucose as a carbon source and monosodium glutamate as a nitrogen source. Two new molecules with polyketide structures, N-glucosylrubropunctamine and N-glucosylmonascorubramine, constituting under some conditions 10% of the total extracellular coloring matter when glucose as a carbon source was in excess (20 g/liter), were isolated and structurally characterized by high-pressure liquid chromatography, Dionex methods, (sup1)H and (sup13)C nuclear magnetic resonance spectroscopy, and mass spectrometry. The occurrence of the electron donor-acceptor complex effect was demonstrated by UV spectroscopy, polarography, and thin-layer voltammetry. The use of n-butanol as an extraction solvent stabilized the pigments against the effects of daylight for several months, promoting the stability of this type of complex.

Synthetic hispidin, a PKC inhibitor, is more cytotoxic toward cancer cells than normal cells in vitro.

The trypanocidal activity of naturally occurring 6-(3,4-dihydroxystyryl)-4-hydroxy-2-pyrone (hispidin) prompted us to examine its cytotoxic activity toward normal and cancerous cells in culture. Hispidin synthesized in our laboratory to a high degree of purity (checked by 1H and 13C NMR spectroscopy) was shown to be cytotoxic (between 10(-3) mol/L and 10(-7) mol/L) toward normal human MRC-5 fibroblasts, human cancerous keratinocytes (SCL-1 cell line), and human cancerous pancreatic duct cells (Capan-1 cell line). Interestingly, addition of hispidin in three successive doses (between 10(-5) mol/L and 10(-7) mol/L) led to a 100-fold increase in activity with an enhanced activity on cancer cells compared to normal cells (50%). Synthetic hispidin was found to inhibit isoform beta of protein kinase C (IC50 of 2 x 10(-6) mol/L), but not E. coli and placental type XV alkaline phosphatases. The enhanced activity of hispidin toward the cancerous cell lines is discussed.

Medium-chain fatty acids affect citrinin production in the filamentous fungus Monascus ruber.

During submerged culture in the presence of glucose and glutamate, the filamentous fungus Monascus ruber produces water-soluble red pigments together with citrinin, a mycotoxin with nephrotoxic and hepatoxic effects on animals. Analysis of the (13)C-pigment molecules from mycelia cultivated with [1-(13)C]-, [2-(13)C]-, or [1, 2-(13)C]acetate by (13)C nuclear magnetic resonance indicated that the biosynthesis of the red pigments used both the polyketide pathway, to generate the chromophore structure, and the fatty acid synthesis pathway, to produce a medium-chain fatty acid (octanoic acid) which was then bound to the chromophore by a trans-esterification reaction. Hence, to enhance pigment production, we tried to short-circuit the de novo synthesis of medium-chain fatty acids by adding them to the culture broth. Of fatty acids with carbon chains ranging from 6 to 18 carbon atoms, only octanoic acid showed a 30 to 50% stimulation of red pigment production, by a mechanism which, in contrast to expectation, did not involve its direct trans-esterification on the chromophore backbone. However, the medium- and long-chain fatty acids tested were readily assimilated by the fungus, and in the case of fatty acids ranging from 8 to 12 carbon atoms, 30 to 40% of their initial amount transiently accumulated in the growth medium in the form of the corresponding methylketone 1 carbon unit shorter. Very interestingly, these fatty acids or their corresponding methylketones caused a strong reduction in, or even a complete inhibition of, citrinin production by M. ruber when they were added to the medium. Several data indicated that this effect could be due to the degradation of the newly synthesized citrinin (or an intermediate in the citrinin pathway) by hydrogen peroxide resulting from peroxisome proliferation induced by medium-chain fatty acids or methylketones.

Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity.

The substrate specificity of a calcium-independent, 97-kDa phospholipase B purified from guinea pig intestine was further investigated using various natural and synthetic lipids. The enzyme was equally active toward enantiomeric phosphatidylcholines under conditions allowing a strict phospholipase A activity. The lysophospholipase activity declined with the following substrates: 1-acyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-propanediol-3-phosphocholine greater than 1-palmitoyl-glycol-2-phosphocholine, suggesting some influence of the polar residue vicinal to the cleavage site. The enzyme also acted on various neutral lipids including triacylglycerol, diacylglycerol, and monoacylglycerol, whereas cholesteryl oleate remained refractory to enzymatic hydrolysis. The lipase hydrolyzed sequentially the sn-2 and sn-1 acyl ester bonds of diacylglycerol, although some direct cleavage of the external acyl ester bond could also occur, as shown with diacylglycerol analogues bearing a nonhydrolyzable alkyl ether or amide bond in the sn-1 or sn-2 position. The three main activities of the enzyme (phospholipase A2, lysophospholipase, and diacylglycerol lipase) were resistant to 4-bromophenacyl bromide, but they were inhibited by N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), and diisopropyl fluorophosphate, suggesting the possible involvement of both cysteine and serine residues in a single active site. It is concluded that guinea pig intestinal phospholipase B, which was also detected in rat and rabbit, is actually a glycerol ester lipase with broad substrate specificity and some unique enzymatic properties.

Involvement of deacylation in activation of substrate hydrolysis by Drosophila acetylcholinesterase.

Insect acetylcholinesterase (AChE), an enzyme whose catalytic site is located at the bottom of a gorge-like structure, hydrolyzes its substrate over a wide range of concentrations (from 2 microm to 300 mm). AChE is activated at low substrate concentrations and inhibited at high substrate concentrations. Several rival kinetic models have been developed to try to describe and explain this behavior. One of these models assumes that activation at low substrate concentrations partly results from an acceleration of deacetylation of the acetylated enzyme. To test this hypothesis, we used a monomethylcarbamoylated enzyme, which is considered equivalent to the acylated form of the enzyme and a non-hydrolyzable substrate analog, 4-oxo-N,N,N-trimethylpentanaminium iodide. It appears that this substrate analog increases the decarbamoylation rate by a factor of 2.2, suggesting that the substrate molecule bound at the activation site (K(d) = 130 +/- 47 microm) accelerates deacetylation. These two kinetic parameters are consistent with our analysis of the hydrolysis of the substrate. The location of the active site was investigated by in vitro mutagenesis. We found that this site is located at the rim of the active site gorge. Thus, substrate positioning at the rim of the gorge slows down the entrance of another substrate molecule into the active site gorge (Marcel, V., Estrada-Mondaca, S., Magné, F., Stojan, J., Klaébé, A., and Fournier, D. (2000) J. Biol. Chem. 275, 11603-11609) and also increases the deacylation step. This results in an acceleration of enzyme turnover.

Synthesis, binding and structure-affinity studies of new ligands for the microsomal anti-estrogen binding site (AEBS).

New compounds have been synthesized based on the structure of the anti-tumoral drug tamoxifen and its diphenylmethane derivative, N,N-diethyl-2-[(4-phenyl-methyl)-phenoxy]-ethanamine, HCl (DPPE). These new compounds have no affinity for the estrogen receptor (ER) and bind with various affinity to the anti-estrogen binding site (AEBS). Compounds 2, 10, 12, 13, 20a, 20b, 23a, 23b, 29 exhibited 1.1-69.5 higher affinity than DPPE, and compounds 23a and 23b have 1.2 and 3.5 higher affinity than tamoxifen. Three-dimensional structure analysis, performed using the intersection of the van der Waals volume occupied by tamoxifen in its crystallographic state and the van der Waals volume of these new compounds in their calculated minimal energy conformation, correlated well with their pKi for AEBS (r = 0.84, P<0.0001, n = 18). This is the first structure-affinity relationship (SAR) ever reported for AEBS ligands. Moreover in this study we have reported the synthesis of new compounds of higher affinity than the lead compounds and that are highly specific for AEBS. Since these compounds do not bind ER they will be helpful to study AEBS mediated cytotoxicity. Moreover our study shows that our strategy is a new useful guide to design high affinity and selective ligands for AEBS.

Structure-activity analysis of the effects of lysophosphatidic acid on platelet aggregation.

Lysophosphatidic acid (1-acyl-sn-glycero-3-phosphate or LPA) is a phospholipid mediator displaying numerous and widespread biological activities and thought to act via G-protein-coupled receptors. Here we have studied the effects on human platelets of a number of LPA analogues, including two enantiomers of both N-palmitoyl-(L)-serine-3-phosphate ((L) and (D)NAPS for N-acyl-phosphoserine) and 2-(R)-N-palmitoyl-norleucinol-1-phosphate ((R) and (S)PNPA), cyclic analogues of 1-acyl-sn-glycero-3-phosphate (cPA) and of 1-O-hexadecyl-sn-glycero-3-phosphate (cAGP), sphingosine-1-phosphate (SPP), as well as two palmitoyl derivatives of dioxazaphosphocanes bearing either a P-H or a P-OH bond (DOXP-H and DOXP-OH, respectively). Nine of these compounds induced platelet aggregation with the following order of potency: SPP < cAGP < DOXP-OH < (L)NAPS = (D)NAPS < (R)PNPA = (S)PNPA < LPA < AGP, EC50 varying between 9.8 nM and 8.3 microM. Two of these compounds (SPP and cAGP) appeared as weak agonists inducing platelet aggregation to only 33% and 41%, respectively, of the maximal response attained with LPA and other analogues. In cross-desensitization experiments, all of these compounds specifically inhibited LPA-induced aggregation, suggesting that they were all acting on the same receptor(s). In contrast, cPA and DOXP-H did not trigger platelet aggregation but instead specifically inhibited the effects of LPA in a concentration-dependent manner. The inhibitory action of cPA did not vary with the acyl chain length or the presence of a double bond and did not involve an increase in cAMP. These data thus confirm the lack of stereospecificity of platelet LPA receptor(s). In addition, since the order of potency of some analogues is different from that described in other cells, our results suggest that platelets contain (a) pharmacologically distinct receptor(s) whose molecular identity still remains to be established. Finally, this unique series of compounds might be used for further characterization of other endogenous or recombinant LPA receptors.