Novel natural vanilloid receptor agonists: new therapeutic targets for drug development.

The discovery that compounds lacking a recognizable vanillyl-like motif might act as vanilloids has given new impetus to a search for novel vanilloid receptor agonists and antagonists in compound libraries. The availability of cell lines transfected with a cloned human vanilloid receptor will further expedite this search. In this article, the pharmacological properties of unsaturated dialdehydes and triprenyl phenols that represent two newly discovered chemical classes of vanilloids will be discussed. The existence of vanilloid receptors in several brain nuclei as well as in non-neuronal tissues predicts novel, innovative therapeutic indications for vanilloids. However, these findings also suggest that vanilloids might cause side-effects. An exploration of the uses of unsaturated dialdehydes in indigenous medicine might help identify new therapeutic targets for vanilloids and avoid unwanted actions.

Isolation and characterization of the naphthocyclinone gene cluster from Streptomyces arenae DSM 40737 and heterologous expression of the polyketide synthase genes.

Streptomyces arenae produces the aromatic polyketide naphthocyclinone, which exhibits activity against Gram-positive bacteria. A cosmid clone containing the putative naphthocyclinone gene cluster was isolated from a genomic library of S. arenae by hybridization with a conserved region from the actinorhodin PKS of S. coelicolor. Sequence analysis of a 5.5-kb DNA fragment, which hybridizes with the actI probe, revealed three open reading frames coding for the minimal polyketide synthase. A strong sequence similarity was found to several previously described ketosynthases, chain length factors and acyl carrier proteins from other polyketide gene clusters. An additional open reading frame downstream of the PKS genes of S. arenae showed 53% identity to act VII probably encoding an aromatase. Another open reading frame was identified in a region of 1.436 bp upstream of the PKS genes, which, however, had no similarity to known genes in the database. Approximately 8 kb upstream of the PKS genes, a DNA fragment was identified that hybridizes to an actVII--actIV specific probe coding for a cyclase and a putative regulatory protein, respectively. Disruption of the proposed naphthocyclinone gene cluster by insertion of a thiostrepton resistance gene completely abolished production of naphthocyclinones in the mutant strain, showing that indeed the naphthocyclinone gene cluster had been isolated. Heterologous expression of the minimal PKS genes in S. coelicolor CH999 in the presence of the act ketoreductase led to the production of mutactin and dehydromutactin, indicating that the S. arenae polyketide synthase forms a C-16 backbone that is subsequently dimerized to build naphthocyclinone. The functions of the proposed cyclase and aromatase were examined by coexpression with genes from different polyketide core producers.

Structure-activity relationships and molecular modeling analysis of flavonoids binding to the benzodiazepine site of the rat brain GABA(A) receptor complex.

The affinities for the benzodiazepine binding site of the GABA(A) receptor of 21 flavonoids have been studied using [(3)H]flumazenil binding to rat cortical membranes in vitro. We show that flavonoids with high affinity for the benzodiazepine receptor in vitro spanning the whole efficacy range from agonists (1q) to inverse agonists (1l) can be synthesized. The receptor binding properties of the flavonoids studied can successfully be rationalized in terms of a comprehensive pharmacophore model recently developed by Cook and co-workers (Drug Des. Dev. 1995, 12, 193-248), supporting the validity of this model. However, in contrast to the requirement by the model that an interaction with the hydrogen bond-accepting site A2 is necessary for compounds to display inverse agonistic activity, 6-methyl-3'-nitroflavone (1l), which cannot engage in such an interaction, nevertheless displays inverse agonism. The analysis of the binding affinities of 3'- and 4'-substituted flavones in terms of the pharmacophore model has yielded new information for the further development of the pharmacophore model.

The stimulation of capsaicin-sensitive neurones in a vanilloid receptor-mediated fashion by pungent terpenoids possessing an unsaturated 1,4-dialdehyde moiety.

1. The irritant fungal terpenoid isovelleral caused protective eye-wiping movements in the rat upon intraocular instillation and showed cross-tachyphylaxis with capsaicin, the pungent principle in hot pepper. 2. Isovelleral induced a dose-dependent calcium uptake by rat dorsal root ganglion neurones cultured in vitro with an EC50 of 95 nM, which was fully inhibited by the competitive vanilloid receptor antagonist capsazepine. 3. Isovelleral inhibited specific binding of [3H]-resiniferatoxin (RTX), an ultrapotent capsaicin analogue, to rat trigeminal ganglion or spinal cord preparations with an IC50 of 5.2 microM; in experiments in which the concentration of [3H]-RTX was varied, isovelleral changed both the apparent affinity (from 16 pM to 37 pM) and the co-operativity index (from 2.1 to 1.5), but not the Bmax. 4. The affinity of isovelleral for inducing calcium uptake or inhibiting RTX binding was in very good agreement with the threshold dose (2.2. nmol) at which it provoked pungency on the human tongue. 5. For a series of 14 terpenoids with an unsaturated 1,4-dialdehyde, a good correlation was found between pungency on the human tongue and affinity for vanilloid receptors on the rat spinal cord. 6. The results suggest that isovelleral-like compounds produce their irritant effect by interacting with vanilloid receptors on capsaicin-sensitive sensory neurones. Since these pungent diterpenes are structurally distinct from the known classes of vanilloids, these data provide new insights into structure-activity relations and may afford new opportunities for the development of drugs targeting capsaicin-sensitive pathways.

A non-pungent triprenyl phenol of fungal origin, scutigeral, stimulates rat dorsal root ganglion neurons via interaction at vanilloid receptors.

1. A [3H]-resiniferatoxin (RTX) binding assay utilizing rat spinal cord membranes was employed to identify novel vanilloids in a collection of natural products of fungal origin. Of the five active compounds found (scutigeral, acetyl-scutigeral, ovinal, neogrifolin, and methyl-neogrifolin), scutigeral (Ki=19 microM), isolated from the edible mushroom Albatrellus ovinus, was selected for further characterization. 2. Scutigeral induced a dose-dependent 45Ca uptake by rat dorsal root ganglion neurons with an EC50 of 1.6 microM, which was fully inhibited by the competitive vanilloid receptor antagonist capsazepine (IC50=5.2 microM). 3. [3H]-RTX binding isotherms were shifted by scutigeral (10-80 microM) in a competitive manner. The Schild plot of the data had a slope of 0.8 and gave an apparent Kd estimate for scutigeral of 32 microM. 4. Although in the above assays scutigeral mimicked capsaicin, it was not pungent on the human tongue up to a dose of 100 nmol per tongue, nor did it provoke protective wiping movements in the rat (up to 100 microM) upon intraocular instillation. 5. In accord with being non-pungent, scutigeral (5 microM) did not elicit a measurable inward current in isolated rat dorsal root ganglion neurons under voltage-clamp conditions. It did, however, reduce the proportion of neurons (from 61 to 15%) that responded to a subsequent capsaicin (1 microM) challenge. In these neurons, scutigeral both delayed (from 27 to 72 s) and diminished (from 5.0 to 1.9 nA) the maximal current evoked by capsaicin. 6. In conclusion, scutigeral and its congeners form a new chemical class of vanilloids, the triprenyl phenols. Scutigeral promises to be a novel chemical lead for the development of orally active, non-pungent vanilloids.

Synthesis of (+)-galiellalactone. Absolute configuration of galiellalactone.

[reaction: see text]. (+)-Galiellalactone was synthesized starting from (R)-(+)-pulegone. Natural and synthetic galiellalactone have opposite optical rotations, demonstrating that the structure of the natural product is 1a.

Unnatural natural products from the transannular cyclization of lathyrane diterpenes.

The potential of macrocyclic diterpenoids to afford natural product-like polycyclic compounds was demonstrated by the conversion of two lathyrane Euphorbia factors into a series of densely functionalized diterpenoids of unnatural skeletal type. Apparently, Nature is far from having fully exploited the built-in reactivity of these compounds to generate chemical diversity.

Dialdehyde sesquiterpenes and other terpenoids as vanilloids.

Selected naturally occurring unsaturated dialdehyde sesquiterpenes and related bioactive terpenoids were assayed for vanilloid-like activity. Out of the 25 compounds tested, eight inhibited completely the specific binding of [3H]resiniferatoxin by rat spinal cord membranes: binding affinities ranged from 0.6 microM for cinnamodial to 19.0 microM for hebelomic acid F. These values were comparable to the binding affinity of capsaicin (2.7 microM). With the exception of four ligands, compounds that inhibited resiniferatoxin binding to rat spinal cord membranes were also pungent on the human tongue where they showed cross-tachyphylaxis with capsaicin. As expected from their reactive nature, these compounds possess additional sites of action, as reflected in the complex behavior of the stimulation of calcium influx by cinnamodial and cinnamosmolide at high concentrations. This observation might explain the unexpectedly weak membrane depolarization by cinnamodial compared to capsaicin. We conclude that a range of sesquiterpene dialdehydes and related terpenoids, both pungent and non-pungent, may function as vanilloids. These compounds may represent a new chemical lead for the development of vanilloid drugs, structurally unrelated to either capsaicin or resiniferatoxin.

Characterisation of the furanocoumarin phellopterin as a rat brain benzodiazepine receptor partial agonist in vitro.

Phellopterin, a naturally occurring furanocoumarin found in the roots of Angelica dahurica, inhibits [3H]diazepam and ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4] benzodiazepine-3-carboxylate ([3H]Ro 15-1788) binding to the benzodiazepine site of the rat brain gamma-aminobutyric acidA (GABAA) receptor in vitro with IC50 values of 400 and 680 nM, respectively. Two other naturally occurring furanocoumarins, byakangelicol and imperatorin were significantly less potent, with IC50 values for inhibition of [3H]diazepam binding of 8.0 and 12.3 microM, respectively. Scatchard plot analysis showed that the inhibitory activity of phellopterin was due to competitive inhibition of the benzodiazepine ligand binding. The results of GABA- and t-butylbicyclophosphorothionate (TBPS)-shift assays suggest that phellopterin is a partial agonist of the central benzodiazepine receptors in vitro.

Isolation and identification from Salvia officinalis of two diterpenes which inhibit t-butylbicyclophosphoro[35S]thionate binding to chloride channel of rat cerebrocortical membranes in vitro.

Ethanolic extracts from dried leaves of sage (Salvia officinalis) showed inhibition of [35S]tertiary-butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain membranes in vitro. This ligand is considered to bind to the chloride channel of the GABA/benzodiazepine receptor complex in brain tissue. Substances having inhibitory activity were purified and their chemical structure identified as the diterpenes carnosic acid and carnosol (IC50 values of 33 +/- 3 microM and 57 +/- 4 microM, respectively). The two compounds did not affect binding of the ligands [3H]muscimol and [3H]diazepam to the GABA/benzodiazepine complex in vitro. Saturation experiments of [35S]TBPS binding indicated that carnosic acid decreases the binding affinity.

11-Hydroxy-4-methyl-2,4,6-dodecatrienoic acid from fermentations of a Mucor species.

11-Hydroxy-4-methyl-2,4,6-dodecatrienoic acid was isolated from fermentations of the Mucor species, strain KL 94-92 aq. The compound exhibits cytotoxic activity and the structural elucidation, as well as the biological properties of the new compound, are described.

Secondary metabolites from a Gloeophyllum species.

Six new sesquiterpenoids, four rearranged illudalanes, one rearranged protoilludane and one sterpurane, were isolated from fermentations of Gloeophyllum sp. 97022. Their structures were elucidated by spectroscopy. Gloeophyllol B and C show weak antifungal activity, while 1-hydroxy-3-sterpurene shows weak antifungal, antibacterial and cytotoxic activities.

Dermatolactone, a cytotoxic fungal sesquiterpene with a novel skeleton.

The nematicidal 5-pentyl-2-furaldehyde and the cytotoxic sesquiterpene dermatolactone were isolated from the extracts of an Ascomycete belonging to the Dermateaceae. The furan has previously been reported from the Basidiomycete Irpex lacteus, while dermatolactone is a new compound the structure of which was determined by spectrosocpic methods.

Two new bioactive diterpenes from Lepista sordida.

In a screening for inducers of the differentiation of human leukaemic cells, two new active diterpenoids were isolated from fermentations of the basidiomycete Lepista sordida. The structural elucidation by spectroscopic methods and the biological activities of both metabolites are described.

Furanocoumarins with affinity to brain benzodiazepine receptors in vitro.

Eight furanocoumarins were isolated from a methanol extract of dried roots of Angelica dahurica. One of these, phellopterin, strongly (IC50 = 0.36 microM) inhibits the binding of [3H]diazepam to central nervous system benzodiazepine receptors in vitro, while the others, despite their structural similarities with phellopterin, are considerably less active.

Diarylheptanoids from Myrica arborea.

Investigations of the stem and root bark of Myrica arborea (Myricaceae) have yielded two novel diarylheptanoids, myricarborin and 11-O-beta-D-xylopyranosylmyricanol along with the known myricanol and 5-O-beta-D-glucopyranosylmyricanol. The structures of the novel compounds were determined by spectroscopic methods.

Prenylated isoflavanone from the roots of Erythrina sigmoidea.

A novel prenylated isoflavanone, sigmoidin I, has been isolated from the roots of Erythrina sigmoidea, in addition to the known isoflavones, corylin and neobavaisoflavone and the known pterocarpan, phaseollidin. Its structure was established as 7,4'-dihydroxy-3'-methoxy-5'-(3-methylbut-2-enyl) isoflavanone by means of spectroscopic analyses and chemical transformations. Neobavaisoflavone displayed antifungal potency in vitro with minimum inhibitory concentrations against Aspergillus fumigatus and Cryptococcus neoformans, of 50 mg ml-1.

Anti-plasmodial sesquiterpenoids from the African Reneilmia cincinnata.

A new isodaucane sesquiterpenoid, 6,7,10-trihydoxyisodaucane, was isolated from the fruits of Reneilmia cincinnata, together with the known sesquiterpenoids oplodiol, oplopanone, 5E,10(14)-germacradien-1 beta, 4 beta-diol, 1(10)E,5E-germacradien-4 alpha-ol and eudesman-1,4,7-triol. A large amount of 5-hydroxy-3,7,4'-trimethoxyflavone was also isolated. Their structures were established by NMR techniques using 1D and 2D experiments. Three of the known sesquirernenoids exhibited noteworthy anti-plasmodial activity against Plasmodium falciparum strains.

Inhibition of kainic acid binding to glutamate receptors by extracts of Gastrodia.

S-(4-hydroxybenzyl)glutathione was isolated as the major principle responsible for the inhibition of the in vitro binding of kainic acid to brain glutamate receptors by water extracts of the plant Gastrodia elata. The affinity (IC50 value) of the compound is slightly lower compared to glutamate and glutathione.

The effect of six sesquiterpenoid unsaturated dialdehydes on cell membrane permeability in human neuroblastoma SH-SY5Y cells.

The effect of six sesquiterpenes containing an unsaturated dialdehyde functionality, on cell membrane permeability in the human neuroblastoma cell line SH-SY5Y has been studied. The kinetics of the membrane leakage after addition of the sesquiterpenes were determined by measuring the efflux of radioactivity from cells preloaded with tritiated 2-deoxyglucose. The concentrations that gave 5% and 20% efflux of radioactivity as compared with control cells (EC5 and EC20) were determined for each compound. In spite of the structural similarities between the compounds, the effects on cell membrane permeability varied considerably. EC20 for polygodial, which is the most active compound, is 2.5 microM after 20-min incubation, but no leakage could be determined for merulidial even at concentrations as high as 4 mM. Rather, this compound seems to stabilize or fix the cell membrane and a lower efflux of radioactivity was observed as compared to the control cells. A quantitative structure-activity relationship analysis for the five active compounds showed a good correlation between the membrane leakage activity and certain chemical characteristics. Structural features strongly correlated with high activity were found to be: The geometry and the atomic charges of the unsaturated dialdehyde functionality, the dipole moment, the energy difference between the lowest unoccupied molecular orbital and the highest occupied molecular orbital and the lipophilicity.