Non-competitive Inhibition of Nicotinic Acetylcholine Receptors by Ladybird Beetle Alkaloids.

Ladybird beetles (Family Coccinellidae) secrete an alkaloid rich venom from their leg joints that protects them from predators. Coccinellines, the major venom constituents, are alkaloids composed of three fused piperidine rings that share a common nitrogen atom. Although many coccinellines have been isolated and chemically characterized, their pharmacological properties are essentially unknown. Using radioligand binding and functional assays we investigated the actions of several coccinellines on skeletal muscle and α7 nicotinic acetylcholine receptors (nAChRs). The alkaloids were shown to displace the specific binding of tritiated piperidyl-N-(1-(2-thienyl)cyclohexyl)-3,4-piperidine ([(3)H]-TCP), which has been shown to bind deep within the ion channel of the electric fish (Torpedo) muscle nAChR. The stereoisomers precoccinelline and hippodamine (whose nitrogens are predicted to be ionized at physiological pH) and their respective analogs N-methyl-precoccinelline and N-methyl-hippodamine (whose quaternary nitrogens are permanently charged) displayed similar IC50s for inhibition of [(3)H]-TCP binding. However, the corresponding precoccinelline and hippodamine N-oxides, coccinelline and convergine (which have an electronegative oxygen bonded to an electropositive nitrogen) displayed significantly higher binding IC50s. Finally, exochomine, a dimeric coccinelline containing the hippodamine structure, displayed the highest IC50 (lowest affinity) for displacing specific [(3)H]-TCP binding. The presence of a desensitizing concentration (10(-3) M) of carbachol (CCh) had little or no effect on the affinity of the Torpedo nAChR for the three coccinellines tested. High concentrations of the coccinellid alkaloids did not affect binding of [(3)H]-cytisine to Torpedo receptor ACh binding sites. Inhibition of the alpha7 nAChR with pre-equilibrated precoccinelline was insurmountable with respect to ACh concentration. We conclude that the coccinellines bind to one or more allosteric sites rather than to the ACh binding sites, and inhibit nAChR responses to ACh through a non-competitive mechanism. Future chemical and pharmacological investigations of other ladybird beetle alkaloids are likely to reveal other interesting alkaloids affecting ligand-gated receptors.

Ferulaldehyde and lupeol as direct and indirect antimicrobial compounds from Cordia gilletii (Boraginaceae) root barks.

Cordia gilletii De Wild (Boraginaceae), a medicinal plant used against infectious diseases in the Democratic Republic of Congo, was investigated for direct and indirect antimicrobial properties. On one hand, the methanol extract is active against many pathogenic bacteria, including resistant strains. Its bio-guided fractionation led to the isolation of ferulaldehyde; this compound showed antimicrobial and antioxidant properties that may support the activity we observed for the methanol extract and some of the traditional uses of C. gilletii. On the other hand, the n-hexane extract of root barks possesses indirect antimicrobial properties, enhancing the activity of antibiotics against methicillin-resistant Staphylococcus aureus (MRSA). The fractionation of this extract led to the isolation of lupeol, which decreases the minimum inhibitory concentration of several antibiotics (4 to 8 fold) against MRSA and contributes to the effects observed for the raw n-hexane extract.

Triterpene saponin hemi-biosynthesis of a leaf beetle's (Platyphora kollari) defensive secretion.

The adults of the leaf beetle Platyphora kollari (Chrysomelidae) are able to metabolise the oleanane triterpene beta-amyrin (1) into the glycoside 3-O-beta-D: -glucopyranosyl-(1-->4)-beta-D: -glucuronopyranosyl-hederagenin (2) that is stored in their defensive glands. The aim of this study was to test the hypothesis that oleanolic acid (3) is an intermediate in the conversion of 1 into 2 and to check whether the sequestration of pentacyclic triterpenes is selective in favour of beta-amyrin (1). To this end, adults of P. kollari were fed with Ipomoea batatas leaf disks painted with a solution of [2,2,3-(2)H(3)]oleanolic acid or [2,2,3-(2)H(3)]alpha-amyrin and the secretion of their defensive glands analysed by HPLC-ESIMS. The data presented in this work indicated that the first step of the transformation of beta-amyrin (1) into the sequestered glycoside 2 is its oxidation into oleanolic acid (3) and that this conversion is selective but not specific in favour of beta-amyrin (1).

A lactosylated steroid contributes in vivo therapeutic benefits in experimental models of mouse lymphoma and human glioblastoma.

Various mono- and disaccharides were grafted onto a steroid backbone. Whereas in vitro these glycosylated steroids had no cytotoxic effects on six different human cancer cell lines, several of the glycosylated steroids under study did significantly modify the levels of in vitro migration of the human U373 glioblastoma, the A549 non-small-cell-lung cancer (NSCLC), and the PC-3 prostate cancer cells, with more pronounced effects in the case of a monosubstituted beta-L-fucopyranosyl-steroid (19), a monosubstituted beta-D-isomaltosyl-steroid (22), and a monosubstituted beta-D-lactosyl-steroid (24). These three compounds significantly increased the survival of conventional mice grafted subcutaneously with the P388 lymphoma, a lymphoma that metastasizes toward the liver. In vivo, the monosubstituted beta-D-lactosyl-steroid (24) also increased the antitumor effectiveness of cisplatin, a cytotoxic pro-apoptotic drug, in the case of the P388 lymphoma model. This compound also increased the survival of immunodeficient mice into whose brains human U373 glioblastoma cells had been orthotopically grafted.

Identification of a novel cardenolide (2''-oxovoruscharin) from Calotropis procera and the hemisynthesis of novel derivatives displaying potent in vitro antitumor activities and high in vivo tolerance: structure-activity relationship analyses.

Analysis of the methanolic extract of Calotropis procera root barks enabled the identification of a novel cardenolide (2''-oxovoruscharin) to be made. Of the 27 compounds that we hemisynthesized, one (23) exhibited a very interesting profile with respect to its hemisynthetic chemical yield, its in vitro antitumor activity, its in vitro inhibitory influence on the Na+/K+-ATPase activity, and its in vivo tolerance. Compound 23 displayed in vitro antitumor activity on a panel of 57 human cancer cell lines similar to taxol, and higher than SN-38 (the active metabolite of irinotecan), two of the most potent drugs used in hospitals to combat cancer.

Nicotinic receptor inhibition by Tetraponera ant alkaloids.

1. Tetraponerines are a group of alkaloids occurring in the venoms of ants belonging to the genus Tetraponera. Eight compounds had been isolated and their structures elucidated, but their mechanisms of action had not yet been reported. We have studied the actions of several of these tetraponerines on vertebrate neuromuscular, ganglionic, and brain nicotinic acetylcholine receptors (nAChRs) using a variety of techniques including muscle contracture, cultured cell functional assays, neuronal patch clamping, and radioligand binding methods. 2. Potency for inhibition of the frog muscle carbachol-elicited contracture increased as the carbon 9 side chain alkyl group was increased in length to 10-12 carbons, then decreased when the chain was 18-carbons long. Potency differences between T-7 and T-8, which differ only in the stereochemistry of the carbon pentyl side chain were rather small. Quaternization of either N atom in a T-8 analog bearing a 10-carbon length alkyl substituent did not greatly affect potency for inhibition of the muscle response; thus the ionized form is an active form of this tetraponerine. 3. T-7 inhibited the nicotine-stimulated efflux of 86Rb from cultured PC12 cells, which primarily express alpha3-beta4 ganglionic type nicotinic receptors. T-8 blockade of BTX-sensitive and insensitive neuronal nAChRs, as studied by patchclamp recordings from cultured rat brain neurons, was also consistent with a noncompetitive type of inhibition. 4. T-7 displaced binding of the nAChR ion channel binding ligand thienylcyclophenidyl (TCP), an analog of PCP, to Torpedo neuromuscular type receptors. The affinity of the TCP binding site for T-7 did not depend upon the desensitization state of the receptor. 5. We conclude that the tetraponerines act at a site on nAChRs different from the ACh binding site which is probably located within the ion channel.

Recycling plant wax constituents for chemical defense: hemi-biosynthesis of triterpene saponins from beta-amyrin in a leaf beetle.

Several species of Doryphorina leaf beetles from Central- and South America produce oleanane triterpene glycosides in their defensive glands. The presence of pentacyclic triterpenes in insects is intriguing since they lack the key enzymes necessary to synthesize these compounds. Since beta-amyrin is a common constituent of leaf waxes, we hypothesized that these leaf beetles use this compound as a precursor to their oleanane glycosides. To test this hypothesis we first confirmed the presence of beta-amyrin in Ipomoea batatas, the food plant of Platyphora kollari. Next, adults of P. kollari were fed for 10 days with I. batatas leaf disks painted with a solution of [2,2,3-(2)H(3)]beta-amyrin ([2,2,3-(2)H(3)]-1). The secretion from their defensive glands was collected and analyzed by HPLC-ESIMS. The results demonstrated that the secretion of beetles fed with an amount of [2,2,3-(2)H(3)]beta-amyrin corresponding to the quantity of unlabeled (natural) beta-amyrin present in the leaf disks contained on average 5.1% of [2,2,3-(2)H(3)]-3- O-beta- d-glucopyranosyl-(1-->4)-beta- d-glucuronopyranosyl-hederagenin ([2,2,3-(2)H(3)]-2), whereas the secretions of beetles fed with 10 times this amount of [2,2,3-(2)H(3)]beta-amyrin contained on average 23.9% of [2,2,3-(2)H(3)]-2. In both series of experiments, the percentage of labeled versus unlabeled triterpene glycoside in the secretion was positively correlated with the amount of deuterated beta-amyrin ingested. These results demonstrate for the first time that some leaf beetles are able to metabolize a widespread triterpenic constituent of leaf wax into more complex glycosides that are stored in their defensive glands.

Primary cultures from the marine sponge Xestospongia muta (Petrosiidae, Haplosclerida).

In the context of the investigations on the origin and in vitro production of bioactive compounds, primary cultures were developed from ectosomal and choanosomal cell suspensions from the sponge Xestospongia muta. Dissociated cells aggregated and reorganized into a striking reticulated network of cells, typical for X. muta. Moreover, in some cultures an isotropic reticulation of small spicules, very similar to that found in the ectosome of adult sponges, was observed. Phytohaemagglutinin promoted aggregation and the reorganization of the cells. HPLC analyses revealed that straight-chain acetylenic compounds were recovered from short-term cultures and that they were synthesized during culture. Heterotrophic bacteria were assumed to be involved in the process. Together our results established that X. muta would be an excellent experimental model to study, in laboratory conditions, the differentiation of the skeleton and the in vitro biosynthesis of straight-chain acetylenic compounds.

In vitro production of adaline and coccinelline, two defensive alkaloids from ladybird beetles (Coleoptera: Coccinellidae).

In vitro experiments using [1-(14)C] and [2-(14)C]acetate were devised to study the biosynthesis of the defensive coccinellid alkaloids adaline and coccinelline in Adalia 2-punctata and Coccinella 7-punctata, respectively. The labelled alkaloids obtained in these experiments had a specific activity about ten times higher than that of the samples obtained in feeding experiments. This in vitro assay has enabled us to demonstrate that these two alkaloids are most likely biosynthesised through a fatty acid rather than a polyketide pathway, that glutamine is the preferred source of the nitrogen atom and that alkaloid biosynthesis takes place in the insect fat body.

5-hydroxytryptamine-derived alkaloids from two marine sponges of the genus Hyrtios.

Indonesian specimens of the marine sponges Hyrtios erectus and H. reticulatus were found to contain 5-hydroxytryptamine-derived alkaloids. Their structures were determined on the basis of their spectral properties. H. erectus contained hyrtiosulawesine (4), a new beta-carboline alkaloid, together with the already known alkaloids 5-hydroxyindole-3-carbaldehyde (1), hyrtiosin B (2), and 5-hydroxy-3-(2-hydroxyethyl)indole (3). H. reticulatus contained the novel derivative 1,6-dihydroxy-1,2,3,4-tetrahydro-beta-carboline (11) together with serotonin (5), 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-beta-carboline (7), and 6-hydroxy-3,4-dihydro-1-oxo-beta-carboline (9).