A new omega-conotoxin that targets N-type voltage-sensitive calcium channels with unusual specificity.

A new specific voltage-sensitive calcium channel (VSCC) blocker has been isolated from the venom of the fish-hunting cone snail Conus consors. This peptide, named omega-Ctx CNVIIA, consists of 27 amino acid residues folded by 3 disulfide bridges. Interestingly, loop 4, which is supposed to be crucial for selectivity, shows an unusual sequence (SSSKGR). The synthesis of the linear peptide was performed using the Fmoc strategy, and the correct folding was achieved in the presence of guanidinium chloride, potassium buffer, and reduced/oxidized glutathione at 4 degrees C for 3 days. Both synthetic and native toxin caused an intense shaking activity, characteristic of omega-conotoxins targeting N-type VSCC when injected intracerebroventricularly to mice. Binding studies on rat brain synaptosomes revealed that the radioiodinated omega-Ctx CNVIIA specifically and reversibly binds to high-affinity sites with a K(d) of 36.3 pM. Its binding is competitive with omega-Ctx MVIIA at low concentration (K(i) = 2 pM). Moreover, omega-Ctx CNVIIA exhibits a clear selectivity for N-type VSCCs versus P/Q-type VSCCs targeted respectively by radioiodinated omega-Ctx GVIA and omega-Ctx MVIIC. Although omega-Ctx CNVIIA clearly blocked N-type Ca(2+) current in chromaffin cells, this toxin did not inhibit acetylcholine release evoked by nerve stimuli at the frog neuromuscular junction, in marked contrast to omega-Ctx GVIA. omega-Ctx CNVIIA thus represents a new selective tool for blocking N-type VSCC that displays a unique pharmacological profile and highlights the diversity of voltage-sensitive Ca(2+) channels in the animal kingdom.

Stereoselective synthesis of 7 alpha- and 7 beta-aminocholestanol as potent fungicidal drugs.

Potentially lymphotropic 7 alpha- and 7 beta-aminocholestanol were stereoselectively synthesized. In vitro bioassay studies have shown that these fungicidal lipidic derivatives possess strong antifungal activity against Candida spp resistant strains to amphotericin B, 5-fluorocytosine and azoles.

A new conotoxin isolated from Conus consors venom acting selectively on axons and motor nerve terminals through a Na+-dependent mechanism.

A novel conotoxin was isolated and characterized from the venom of the fish-hunting marine snail Conus consors. The peptide was identified by screening chromatography fractions of the crude venom that produced a marked contraction and extension of the caudal and dorsal fins in fish, and noticeable spontaneous contractions of isolated frog neuromuscular preparations. The peptide, named CcTX, had 30 amino acids and the following scaffold: X11CCX7CX2CXCX3C. At the frog neuromuscular junction, CcTx at nanomolar concentrations selectively increased nerve terminal excitability so that a single nerve stimulation triggered trains of repetitive or spontaneous synaptic potentials and action potentials. In contrast, CcTx had no noticeable effect on muscle excitability even at concentrations 100 x higher than those that affected motor nerve terminals, as revealed by direct muscle stimulation. In addition, CcTx increased miniature endplate potential (MEPP) frequency in a Ca2+-free medium supplemented with ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA). Blockade of voltage-dependent sodium channels with tetrodotoxin (TTX) either prevented or suppressed the increase of MEPP frequency induced by the toxin. CcTx also produced a TTX-sensitive depolarization of the nodal membrane in single myelinated axons giving rise, in some cases, to repetitive and/or spontaneous action potential discharges. In addition, CcTx increased the nodal volume of myelinated axons, as determined using confocal laser scanning microscopy. This increase was reversed by external hyperosmolar solutions and was prevented by pretreatment of axons with TTX. It is suggested that CcTx, by specifically activating neuronal voltage-gated sodium channels at the resting membrane potential, produced Na+ entry into nerve terminals and axons without directly affecting skeletal muscle fibres. CcTx belongs to a novel family of conotoxins that targets neuronal voltage-gated sodium channels.

The strategy used by some piscivorous cone snails to capture their prey: the effects of their venoms on vertebrates and on isolated neuromuscular preparations.

Three piscivorous Conus species, C. ermineus, C. consor and C. catus were acclimatized in aquaria. The study of their strategy to capture the prey and details of their radula's morphology revealed that all of them used a 'hook and line' strategy which consists of immobilizing the prey rapidly before engulfing it. The venoms from these piscivorous species clearly elicit, when injected into fish, an excitotoxic shock characterized by a sudden tetanus of the prey. In mammals, the venoms induce both flaccid paralysis via i.p. injection and seizures via i.c.v. injection. Intracellular recordings from frog nerve-muscle preparations revealed that the venoms from these Conus species first caused spontaneous synaptic potentials which in turn triggered muscle action potentials. Such spontaneous activity is due to an increased nerve terminal excitability. In addition, the venoms suppressed neuromuscular transmission probably by blocking postsynaptic nicotinic acetylcholine receptors. No direct effect of these Conus venoms was observed on the membrane of skeletal muscle fibres. In conclusion, C. ermineus, C. consor and C. catus, which have not securely tethered their prey used a mixture of toxins which target both pre-and postsynaptic elements of the neuromuscular junction and which produce rapid immobilization of their prey.

Synthesis of 25-aminosterols, new antifungal agents.

25-aminolanostenol 1 and 25-aminocholesterol 2 were hemisynthesized from natural sterols and tested in vitro against Candida albicans. The biological activity of compound 1 was rather weak, whereas 2 exhibited in vitro antifungal activity with MIC value of 4 microM.

Synthesis and cytotoxicity of aminosterols: activity studies on a non-small-cell bronchopulmonary carcinoma line (NSCLC-N6).

Various new aminosterols were synthesized. The antiproliferative activity of these compounds (I-IV) was studied in vitro on a continuous human non small-cell bronchopulmonary carcinoma line (NSCLC-N6) at the cell cycle level. The histograms indicate cell blockage in Phase Gl (compound I-III) associated with a reduction in the number of cells phases S and G2M and appearance of cellular debris derived from cells in Phase G1.

Antifungal metabolites from the marine sponge Pachastrissa sp.: new bengamide and bengazole derivatives.

This paper reports the studies of components of an undescribed sponge in the genus Pachastrissa sp., collected along the Djibouti coast. The extract showed activity against Candida albicans. Six new bengazoles (1-6) and a new bengamide, named bengamide L (16), in addition to the known bengazoles (7-11), bengamides A (12), B (13), E (14), and F (15), and a lactone (17) are described in this paper. All structures were determined on the basis of spectroscopic studies.

Biochemical characterization and nuclear magnetic resonance structure of novel alpha-conotoxins isolated from the venom of Conus consors.

Two novel alpha-conotoxins were purified and characterized from the venom of the fish-hunting cone snail Conus consors. These peptides were identified by screening HPLC fractions of the crude venom and by binding experiments with Torpedo nicotinic acetylcholine receptor. The toxins named alpha-CnIA and alpha-CnIB exhibited sequences of 14 and 12 amino acids, respectively. The alpha-CnIA represents the main alpha-conotoxin contained in the venom, whereas alpha-CnIB is present in a relatively small amount. Chemical synthesis of alpha-CnIA was carried out using the Fmoc methodology by selective disulfide bond formation. The biological activity of the toxin was assessed in fish and mice. The alpha-CnIA inhibited the fixation of iodinated alpha-bungarotoxin to Torpedo nicotinic acetylcholine receptors with an IC50 of 0.19 microM which can be compared to the IC50 of 0.31 microM found for the previously characterized alpha-MI isolated from the piscivorous Conus magus. The synthetic alpha-CnIA blocked spontaneous and evoked synaptic potentials in frog and mouse isolated neuromuscular preparations at sub-micromolar concentrations. Solution NMR of this toxin indicated a conformational heterogeneity with the existence of different conformers in solution, at slow and intermediate exchange rates relative to the NMR chemical shift time scale, similar to that reported for alpha-GI and alpha-MI. NMR structures were calculated for the major NMR signals representing more than 80% of the population at 5 degrees C.

Cell binding, uptake and cytosolic partition of HIV anti-gag phosphodiester oligonucleotides 3'-linked to cholesterol derivatives in macrophages.

The purpose of this study is to evaluate the cell interactions of a new class of compounds composed of phosphodiester oligonucleotides linked to the cholesterol group at position 3, 7, or 22 of the steroid structure. The resulting conjugates were assessed for their capacity to bind, penetrate and partition in the cytoplasmic compartment of murine macrophages. The results showed that lipophilic conjugates bind to cells much faster (t(1/2) < or = 10 min) than do underivatized oligomers. Oligomers tethered to the cholesterol at positions 3 and 7 (PO-GEM-3-Chol and PO-GEM-7-Chol) interacted more efficiently with cell membranes and were better internalized than oligomers attached to the cholesterol moiety at position 22 (PO-GEM-22-Chol). The cytosolic fraction of internalized oligomers was studied by a digitonin-based membrane permeabilization method. The recovered fraction of oligomers that can freely diffuse from the cytosol was comparable for GEM-91, a phosphorothioate congener, and for PO-GEM-7-Chol (50-60% of the internalized oligomers), while that of PO-GEM-3-Chol was less (30% of the internalized oligomers) indicating a higher membrane affinity of the latter derivative as compared to the other investigated compounds. Membrane binding and cell internalization correlated well with the hydrophobicity of the conjugates as characterized by their partition coefficients in a water-octanol system. Due to their capacity of rapid binding and cytosolic partition in cells, cholesterol-derivatized oligonucleotides at position 3 or 7 of the steroid molecule appeared as good candidates for systemic delivery of anti-HIV antisense compounds.

Studies on lipidomimetic derivatives of alpha-difluoromethylornithine (DFMO) to enhance the bioavailability in a Trypanosoma B. brucei murine trypanosomiasis model.

DFMO, a trypanostatic drug, presents a satisfactory intestinal absorption but its elimination from the blood is rapid so that high doses are necessary to obtain to therapeutic effect. In this study, we propose a strategy to enhance the bioavailability of DFMO by using lipidomimetic derivatives. Three lipidomimetic DFMO derivatives called O-DFMO, S-DFMO and Chol-DFMO were designed to reach easily the plasma and to be cleaved preferentially by plasma esterases progressively liberating free DFMO. Chol-DFMO only could be cleaved partially whereas the other compounds appeared to be stable in reconstituted intestinal medium and mouse plasma. Nevertheless, the use of DFMO derivatives in T. b. brucei experimental chemotherapy appeared as an interesting approach. Thus, O-DFMO was trypanocidal in vitro whereas DFMO, the active principle, was only trypanostatic. Nevertheless, this compound did not release DFMO in mouse blood as expected and acted therefore not as a prodrug. Oral treatment using low doses of compound O-DFMO was able to cure 40% mice while the active principle (eflornithine) administered at 50 fold higher molarity failed to cure any mice. This indicates that compound O-DFMO acts by a specific mechanism which remains to be investigated. S-DFMO was less active and Chol-DFMO had no in vitro activity but released small amounts of DFMO in mice, however, too slight to obtain a therapeutic effect.

Secreted and intracellular phospholipases A2 inhibition by 1-decyl 2-octyl-glycerophosphocholine in rat peritoneal macrophages.

Compounds able to inhibit phospholipases A2 can be considered as potential anti-inflammatory drugs. In this respect, the inhibitory effect of the phospholipid analogue 1-decyl 2-octyl-rac-glycero-3-phosphocholine (decyloctyl-GPC) added to the culture medium of rat peritoneal macrophages stimulated with ionophore A23187 was determined. (a) The substrate of phospholipase A2 1-octadecanoyl 2-[14C]eicosatetraenoyl-sn-glycero-3-phosphocholine ([14C]20:4-GPC) was added to the culture medium. In macrophages + extracellular fluids, its hydrolysis at the 2-position, produced [14C]non-phosphorous lipids which reached 12% of the dose at 0.14 microM, 73% at 0.9 and > 90% at 1.6 microM; in experiments where macrophages and extracellular fluids were analyzed separately, decyloctyl-GPC initially added at 4 microM, significantly inhibited the release of [14C]fatty acids and the eicosanoid synthesis, demonstrating its ability to inhibit secreted and/or intracellular phospholipases A2. (b) Extracellular fluids were separated from the macrophages and incubated with [14C]20:4-GPC: 48% of the dose was hydrolyzed by extracellular fluid-associated secreted phospholipase A2 and decyloctyl-GPC at 3 microM, reduced this hydrolysis by 50%. (c) [3H]arachidonic acid ([3H]20:4) was added to the culture medium and was esterified in the macrophage membrane phospholipids. Activation of intracellular phospholipase A2 induced the release of [3H] fatty acids and eicosanoid synthesis. These releases were inhibited by 50% with decyloctyl-GPC added at 4 microM. (d) [3H]20:4 and [14C]20:4-GPC were added to the culture medium of the macrophages. [3H] and [14C] fatty acids and eicosanoids were released in macrophages or extracellular fluids. They were significantly reduced by the phospholipid analogue added at 4 microM. It is concluded that secreted and intracellular phospholipases A2 were both inhibited by decyloctyl-GPC which extensively reduced the 20:4 release from exogenous and membrane phospholipids and therefore eicosanoid synthesis.

A new strong inhibitor of beta-mannosidase.

N-phenyl-carbamate of D-mannonohydroxymolactone (I) was synthesized from mannose and was shown to be the best competitive inhibitor of beta-mannosidase so far reported (Ki = 25 nM).

Synthesis of 6(alpha, beta)-aminocholestanols as ergosterol biosynthesis inhibitors.

delta 7-5-Desaturase catalyses one of the last steps in ergosterol biosynthesis in fungi. Moreover delta 5-unsaturation is necessary for the sparking function. Synthesis of three pairs of C-6 epimeric cholestanol derivatives are described as potential growth inhibitors. Preliminary results suggest that 6 beta-aminocholestanol is a potent antifungal agent.

High rate of intestinal absorption of the phospholipid anlaogue 1-dodecyl 2-[1-14C] octanamido-sn-2-deoxy-glycero-3-phosphocholine in the rat.

The phospholipid analogue with two short fatty chains, 1-dodecyl-2-[1-14C] octanamido-sn-2-deoxy-glycero-3-phosphocholine ([14C] phospholipid analogue), with a non-hydrolyzable bond at position 2 of the glycerol, is an inhibitor of phospholipase A2. It was obtained after chemical synthesis and 0.5 micromol was solubilized in Na+ taurocholate with an equimolar amount of 1-octadecanoyl 2-[3H]eicosatetraenoyl-sn- glycero-3-phosphocholine which is the current substrate of phospholipases A2. Both molecules were introduced into the duodenum of rats in order to follow their captations by intestinal mucosa cells for 30, 60 or 90 min. The [14C] phospholipid analogue was poorly split by phospholipases A2 (pancreatic juice and intracellular enzymes). It disappeared from the intestinal contents (87% of the dose gone in 90 min) as rapidly as the tritiated lecithin (81%) but this was later split by the phospholipases at a higher rate.

Uptake of the phospholipase A2 inhibitor 1-dodecyl 2-[1-14C] octanamido-sn-2-deoxy glycero-3-phosphocholine by peritoneal macrophages.

The [14C] phospholipid analogue 1-dodecyl-2-[1-14C] octanamido-sn-2-deoxy glycero-3-phosphocholine was synthetized. With 2 short fatty chains linked by alkyl and amido bonds to positions 1 and 2 of the glycerophosphate backbone, it was an inhibitor of phospholipase A2 in ionophore A23187-stimulated macrophages. Its uptake by rat peritoneal macrophages and its resistance towards phospholipases A2 were determined at nanomolar or micromolar concentrations in the culture medium. A control substrate for phospholipases A2 activity was established with the lecithin 1-octadecanoyl 2-[3H] eicosatetraenoyl-sn-glycero-3-phosphocholine ([3H] 20:4-GPC), a source of [3H] arachidonic acid after cleavage at position 2. Non-stimulated- or ionophore A23187-stimulated macrophages incorporated extensively the [14C] phospholipid analogue added at 30-4000 nM. At 4000 nM which induced 50% inhibition of the phospholipase, 40% of the dose was found associated with the [14C] phospholipids of 2 x 10(6) stimulated macrophages after 120 min incubation, while only low amounts of [14C] non-phosphorous lipids were detected. It is concluded that the [14C] phospholipid analogue was readily taken up by the macrophages with limited hydrolysis.

Lymphotropic antifilarial agents derived from closantel and chlorambucil.

New closantel and chlorambucil prodrugs expected to accumulate in the lymphatic system were evaluated on the filaria Molinema dessetae. The prodrugs of closantel had a delayed effect in vitro on the infective larvae compared to the free drug. The closantel prodrugs were less toxic in vivo than closantel itself. The most active prodrug after treatment at 200 mumol/kg by the oral route was the 1,3-dipalmitoyl-2-succinyl-glycerol-closantel. The macrofilaricidal delayed effect of closantel prodrugs was of interest to prevent anaphylactic shock. In vitro, chlorambucil was active on M. dessetae infective larvae with an IC50 of 26 microM. 1,3-Dipalmitoyl-2-chlorambucil-glycerol was slightly active while the addition of a thioether function between the drug and the lymphotropic ligand canceled the activity. However, no activity with chlorambucil and its prodrugs was observed in vivo. The lymphotropism of these prodrugs has now to be verified using comparative pharmacokinetics in serum and lymph to quantify the increase in drug concentration in lymph.

Structure-activity of three phospholipid analogues towards inhibition of phospholipase A2 in macrophages.

At concentrations 1-20 microns in culture medium of rat peritoneal macrophages which were stimulated with ionophore A23187, the phospholipid analogues 1-decyl-2-octyl-glycerophosphocholine and 1-dodecyl-2-octanamido-2-deoxy glycerophosphocholine were found more potent inhibitors than 1-octyl-2-deoxy glycerophosphocholine to lower the phospholipase A2 activities. The inhibitory effect was measured by [3H] eicosatetraenoic acid ([3H]20:4) release in macrophages and extracellular fluids and synthesis of [3H] eicosanoids after incubation of macrophages with traces of the molecular species of lecithin 1-octadecanoyl-2-[3H] eicosatetraenoyl glycerophosphocholine. The three phospholipid analogues developed higher inhibitory effects than mepacrine, dexamethasone or bromophenacyl bromide, at corresponding concentrations in medium.

Synthesis and evaluation of the anti-inflammatory effects of niflumic acid lipophilic prodrugs in brain edema.

Five new lipophilic prodrugs of the non-steroidal anti-inflammatory drug, niflumic acid (Nifluril, CAS 4394-00-7), were synthetized and evaluated on the experimental brain edema (injection of phospholipase A2). The effect of these drugs in comparison with dexamethasone which elicits a marked effect on clinical and experimental brain edema was evaluated. Niflumic acid was vectorised by cholesterol, hexadecanol and by three 1,3-diacylglycerols. The anti-inflammatory activity of these compounds on experimental brain edema was evaluated by determination of the prostaglandin E2 (PGE2) brain tissue concentration. Niflumic acid reduced the prostaglandin E2 production more significantly than dexamethasone. Niflumic acid prodrug forms (1,3-dihexadecanoyl-2-[2-[3-(trifluoromethyl)anilino]nicotinoyl] glycerol and 1,3-dihexadecanoyl-2-[2-[3-(trifluoromethyl)anilino]nicotinoyloxybuta noyl] glycerol also showed a marked anti-inflammatory activity at low concentrations.

Characterization of the Saccharomyces cerevisiae RTA1 gene involved in 7-aminocholesterol resistance.

7-aminocholesterol has been described as being a strong inhibitor of yeast and of Gram+-bacteria proliferation. In order to determine the precise molecular target of the toxicity of this compound, we searched for yeast resistance linked to gene over-expression. We named the new yeast gene that was isolated RTA1 (EMBL X84736). This gene led to strong resistance to the inhibitor. Gene sequencing revealed that RTA1 is adjacent to the NAB1 gene which is orientated in an opposite direction and localized on chromosome VII. The RTA1 gene, which encodes a putative protein with seven potential membrane-spanning segments, was shown to be a non-essential gene in yeast.