Isolation and insecticidal activity of mellamide from Aspergillus melleus.

Mellamide, a novel indole amide, was isolated from a fermentation of Aspergillus melleus using silica gel and high-performance liquid chromatographic methods. This allowed its separation from three known antiparasitic compounds (ochratoxin A, viomellin and xanthomegnin) also present in the potent extract. The structure was elucidated by (1)H, (13)C, COSY, DEPT, HMQC and HMBC NMR experiments. HR-FTMS aided in the molecular weight and formula determination. Mellamide showed in vitro insecticidal activity in bioassays against larvae of Lucilia sericata and Aedes egypti with LD(90) of 1,000 and 50 micro g/ml, respectively.

Comparison of nodulisporic acid analogs in a Lucilia sericata in vitro assay and a Ctenocephalides felis membrane feeding system.

A medicinal chemistry program on the nodulisporic acid chemical class, guided by an artificial membrane flea-feeding assay, has recently identified permissive and nonpermissive regions of the pharmacophore for exploitation against fleas. This pathway was validated when several promising compounds from this program were administered orally to dogs at 15.0 mg/kg and found to have >90% flea activity for 2 wk. To determine if a surrogate insect assay would have provided the same guidance, a nodulisporic acid analog series was examined in both a Lucilia sericata larval assay and an artificial membrane flea-feeding assay using Ctenocephalides felis. Results from both insect assays were concordant in that even subtle chemical modification or substitution to the left-hand side of the nodulisporic acid pharmacophore resulted in substantial loss of insecticidal activity. Both assays were also in general agreement that the only modifications to the pharmacophore that did not result in loss of activity occurred to the C-8 side chain on the right-hand side of the molecule. Although there was good agreement between the 2 assays on the general regions of the pharmacophore, there was variance on individual compounds in the mono- and disubstituted amide series from the C-8 side chain. For example, the L. sericata assay showed several analogs from this subclass to possess similar activity to the parent acid, whereas the membrane assay indicated superior activity against fleas relative to the same parent. Consequently, although there was substantial general agreement between the assays, it was concluded that finer optimization of a lead compound should be done against the target parasite, even if it is ex vivo, as early as possible in a medicinal chemistry program.

Systemic efficacy of nodulisporic acid against fleas on dogs.

Nodulisporic acid A (NSA) is a novel natural product from a new structural class that was shown previously to have insecticidal activity against blowfly larvae. To determine if there was useful systemic efficacy against fleas (Ctenocephalides felis). NSA was evaluated in an artificial membrane flea feeding device and in dogs. In the artificial membrane flea feeding device, adult C. felis were allowed to feed on bovine blood containing various concentrations of NSA through a Parafilm membrane. NSA killed the fleas with a 50% lethal concentration of 0.68 microg/ml and was approximately 10-fold more potent than the systemic insecticide ivermectin. In the initial probe dog test, a single beagle was challenged with 100 C. felis before oral dosing with 15 mg/kg of NSA. Flea counts conducted at 72 hr postdosing showed an 88% reduction relative to control. Re-challenge of the same dog at 5 days postdosing showed 50% reduction of fleas at day 7, demonstrating some residual flea activity. In a confirmatory study, 8 dogs were challenged with 100 fleas just before oral dosing with 15 mg/kg of NSA (4 dogs) or vehicle (4 dogs). There was 99% reduction of fleas at 48 hr postdosing in the NSA-treated dogs relative to control. Additional challenges with 100 fleas were performed on these 8 dogs at 48-hr intervals to determine the duration of efficacy, and there was 97, 51, and 0% reduction of fleas relative to control on days 4, 6, and 8, respectively. No adverse effects were observed in the dogs in these studies. These data show that NSA has potent oral activity in the dog for the control of fleas, while lacking overt mammalian toxicity.

Coprophilin: an anticoccidial agent produced by a dung inhabiting fungus.

Coprophilin, a decalin pentanedienoic acid methyl ester, was isolated from an unidentified fungus by bioassay guided separation. It inhibited (MIC = 1.5 microM) the growth of Eimeria tenella in an in vitro assay. The isolation, structure elucidation, absolute stereochemistry and biology are described.

Ca2+-entry blockade by CAF603, a carotane sesquiterpene isolated from Trichoderma virens.

Isometric tension recordings and patch-clamp methods were combined to explore the functional effects and mechanisms of action of 8-daucene-3,4-diol (CAF603), a carotane sesquiterpene isolated from the fungus Trichoderma virens. CAF603 (1-100 microM) inhibited the spontaneous motility of guinea-pig portal vein, duodenum and ileum, and the Ca2+-induced tension of depolarized ileum strips. These effects were not antagonized by either iberiotoxin or glyburide. CAF603 increased the spontaneous motility of guinea-pig detrusor muscle, but inhibited the contraction induced by high-KCl, depolarizing salines. CAF603 blocked L-type Ca2+ channel currents of rabbit cardiac myocytes. It is proposed that Ca2+-entry blockade accounts for the inhibitory effects of CAF603 on smooth muscle contractility, whereas the stimulation of spontaneous motility of detrusor muscle is ascribed to blockade of Ca2+-activated K+ (BKCa) channel currents. The latter interpretation is consistent with the allosteric modulation of charybdotoxin binding to BKCa in smooth muscle membranes [Lee et al., 1995. J. Nat. Prod. 58, 1822-1828].

L-741,494, a fungal metabolite that is an inhibitor of interleukin-1 beta converting enzyme.

gamma-Pyrone-3-acetic acid (L-741,494) is a novel metabolite produced by a culture of the fungal genus Xylaria. This substance is a water-soluble, competitive, irreversible inhibitor of Interleukin-1 beta Converting Enzyme that is inactive against papain and trypsin. It has a mol wt of 154 and an empirical formula of C7H6O4. We propose the name xylaric acid for this compound.

L-696,474, a novel cytochalasin as an inhibitor of HIV-1 protease. II. Isolation and structure.

A novel HIV-1 protease inhibitor, L-696,474 (C30H39NO4, 477), was isolated from the fermentations of the fungus Hypoxylon fragiforme (ATCC 20995, MF5511) and purified by silica gel chromatography followed by crystallization. Spectroscopic studies have shown the competitive inhibitor L-696,474 to be a novel cytochalasin. Two related novel cytochalasins were also isolated and had no effect on the enzyme.

L-696,474, a novel cytochalasin as an inhibitor of HIV-1 protease. I. The producing organism and its fermentation.

A novel cytochalasin, L-696,474, (18-dehydroxy cytochalasin H) that inhibits HIV-1 protease was discovered in fermentations of a bark-inhabiting Ascomycete, Hypoxylon fragiforme. The product was first identified from extracts of an agar medium. Fermentation studies on a number of media indicated that the product can be made on several solid and liquid media. Optimum production was obtained from growth in a complex medium composed of glycerol, glucose, citrate, Ardamine, soybean meal, tomato paste, and inorganic salts. Other Hypoxylon spp., related species of Xylariales, and other fungi known to produce cytochalasins, were also surveyed for their ability to make L-696,474. Only one other Hypoxylon fragiforme isolate was found to make this novel cytochalasin; none of the other cultures surveyed made L-696,474 or any other compounds which inhibit HIV-1 protease.

L-696,474, a novel cytochalasin as an inhibitor of HIV-1 protease. III. Biological activity.

L-696,474, an inhibitor of the HIV-1 protease, was discovered in extracts of the fungal culture Hypoxylon fragiforme (MF5511; ATCC 20995). L-696,474 is a novel cytochalasin with a molecular weight of 477 and an empirical formula of C30H39NO4. L-696,474 inhibited HIV-1 protease activity with an IC50 of 3 microM and the mode of inhibition was competitive with respect to substrate (apparent Ki = 1 microM). Furthermore, L-696,474 was not a slow-binding inhibitor. The inhibition due to L-696,474 was also independent of the HIV-1 protease concentration. L-696,474 was inactive against pepsin, another aspartyl protease; stromelysin, a zinc-metalloproteinase; papain, a cysteine-specific protease or human leucocyte elastase, a serine-specific protease. Two other novel cytochalasins (L-697,318 and L-696,475) isolated from the same culture were inactive against the HIV-1 protease. Commercially available cytochalasins B, C, D, E, F, H and J were inactive while cytochalasin A was as active as L-696,474 against the HIV-1 protease.

[3H]paraherquamide binding to Caenorhabditis elegans. Studies on a potent new anthelmintic agent.

Paraherquamide was identified recently as a potent anthelmintic agent. In this paper we describe the identification and characterization of a specific, high-affinity paraherquamide binding site in a membrane preparation isolated from the free-living nematode, Caenorhabditis elegans. [3H] Paraherquamide bound specifically to C. elegans membranes with an apparent dissociation constant, Kd, of 263 nM. A series of paraherquamide analogs were examined, and their relative affinity for the paraherquamide binding site correlated with their nematocidal activity. Phenothiazines were the only other class of anthelmintics tested which inhibited specific [3H]paraherquamide binding. These results suggest that the anthelmintic activity of paraherquamide and phenothiazine is mediated via an interaction with a common binding site.

Novel antinematodal and antiparasitic agents from Penicillium charlesii. I. Fermentation, isolation and biological activity.

Paraherquamide and six novel analogs were isolated from the fermentation of Penicillium charlesii (ATCC 20841). All seven natural products displayed potent antinematodal activity against Caenorhabditis elegans. None of the novel analogs were more potent than paraherquamide.

Novel cholecystokinin antagonists from Aspergillus alliaceus. I. Fermentation, isolation, and biological properties.

The discovery and biological properties of four novel cholecystokinin antagonists produced by Aspergillus alliaceus is described. One of these was seven times more potent than the previously reported asperlicin.

On the biosynthesis of asperlicin and the directed biosynthesis of analogs in Aspergillus alliaceus.

Feeding of 14C-labeled amino acids to resting cells of Aspergillus alliaceus strongly supported the intuitive hypothesis that asperlicin is biosynthesized from tryptophan, anthranilate and leucine. The resting cell system was used also to prepare 25 asperlicin analogs via directed biosynthesis in presence of analogs of tryptophan and leucine.