Thiosemicarbazones of formyl benzoic acids as novel potent inhibitors of estrone sulfatase.

Thiosemicarbazones of the microbial metabolite madurahydroxylactone, a polysubstituted benzo[a]naphthacenequinone, have been previously reported by us as potent nonsteroidal inhibitors of the enzyme estrone sulfatase (cyclohexylthiosemicarbazone 1, IC50 0.46 microM). The active pharmacophore of 1 has now been identified to be 2-formyl-6-hydroxybenzoic acid cyclohexylthiosemicarbazone (25, IC50 4.2 microM). The active partial structure was derivatized in the search for novel agents against hormone-dependent breast cancer. Further substantial increases in activity were achieved by reversal of functional groups leading to the cyclohexylthiosemicarbazones of 5-formylsalicylic acid (35, IC50 0.05 microM) and 3-formylsalicylic acid (34, IC50 0.15 microM) as the most potent analogues identified to date. Both compounds were shown to be noncompetitive inhibitors of estrone sulfatase with Ki values of 0.13 microM and 0.12 microM, respectively. The compounds showed low acute toxicity in the hen's fertile egg screening test.

Microbiaeratin, a new natural indole alkaloid from a Microbispora aerata strain, isolated from Livingston Island, Antarctica.

A new sulphur-containing natural alkaloid named microbiaeratin (1a) was isolated, together with the known microbiaeratinin (2, bacillamide) from the culture filtrate of Microbispora aerata strain IMBAS-11A. The organism was isolated from penguin excrements collected on the Antarctic Livingston Island. The structure was elucidated by one- and two-dimensional NMR experiments and mass spectrometric investigations.

The occurrence of peptaibols and structurally related peptaibiotics in fungi and their mass spectrometric identification via diagnostic fragment ions.

Peptaibols and related peptide antibiotics (peptaibiotics) display diagnostically useful fragmentation patterns during mass spectrometry (FAB-MS, ESI-CID-MS/MS and CID-MSn]. The paper compiles fragmentation data of pseudo-molecular ions reported in the literature as a guide to the rational identification of recurrently isolated and new peptaibols and peptaibiotics. Taxonomic and ecological aspects of microorganisms producing peptaibols and peptaibiotics are discussed.

Circular dichroism studies of ampullosporin-A analogues.

Ampullosporin A (AmpA), a 15mer peptalbol containing seven Aib residues is able to induce pigmentation on Phoma destructiva and hypothermia in mice, as well as to exhibit a neuroleptic effect. A circular dichroism study of ampullosporin A and its analogues was carried out in organic solvents with different polarities and detergent micelles to determine the relationship between their conformational flexibility and biological activities. The analogues were obtained by modifying the N- and C-termini of ampullosporin A. Furthermore, Gln and Leu were systematically substituted by Ala and Aib residues were replaced by Ala and/or Ac6c. To estimate the helicity of the analogues, the CD spectrum of AmpA recorded in acetonitrile was correlated to its crystal structure. All analogues displayed similar CD curve shapes in organic solvents with the ratio between two negative band intensities R = [theta]n-pi*/[theta]pi-pi* < 1. In acetonitrile, most of the analogues adopted a 70%-85% helical structure, which was higher than the average of 40%-60% obtained in TFE. In detergent micelles, the analogues were distinguishable by their CD profiles. For most of the biologically active analogues, the CD spectra in detergent micelles were characterized by a R ratio > 1 and increased helicity compared with those recorded in TFE, suggesting that the interaction of the peptides with the membrane and peptide association was necessary for their hypothermic effect.

Crystal structure and conformational analysis of ampullosporin A.

Ampullosporin A is a 15-mer peptaibol type polypeptide that induces pigment formation by the fungus Phoma destructiva, forms voltage-dependent ion channels in membranes and exhibits hypothermic effects in mice. The structure of ampullosporin A has been determined by x-ray crystallography. This is the first three-dimensional (3D) structure of the peptaibol subfamily SF6. From the N-terminus to residue 13 the molecule adopts an approximate right-handed alpha-helical geometry, whereas a less regular structure pattern with beta-turn characteristics is found in the C-terminus. Even though ampullosporin A does not contain a single proline or hydroxyproline it is significantly bent. It belongs to both the shortest and the most strongly bent peptaibol 3D structures. The straight structure part encompasses residues Ac-Trp(1)-Aib(10) and is thus less extended than the alpha-helical subunit. The 3D structure of ampullosporin A is discussed in relation to other experimentally determined peptaibol structures and in the context of its channel-forming properties. As a part of this comparison a novel bending analysis based on a 3D curvilinear axis describing the global structural characteristics has been proposed and applied to all 3D peptaibol structures. A sampling of 2500 conformations using different molecular dynamics protocols yields, for the complete ampullosporin A structure, an alpha-helix as the preferred conformation in vacuo with almost no bend. This indicates that solvent or crystal effects may be important for the experimentally observed peptide backbone bending characteristics of ampullosporin A.

Differences in membrane pore formation by peptaibols.

The efficiencies of membrane pore formation by 14 naturally occurring peptaibols and two structurally modified ampullosporins were compared using an artificial bilayer membrane model. Major differences were found in the dependence on peptide sequences and the constituting amino acids. Alamethicin F-30, chrysospermins C/D, paracelsin and texenomycin A displayed higher activity by several orders of magnitude in comparison with smaller peptaibols containing < 17 amino acids such as ampullosporins, trichofumins. bergofungins and cephaibols. Biological activities such as the induction of pigment formation by the fungus Phoma destructiva and long acting hypothermia and depression of locomotor activity in mice were correlated with moderate membrane permeabilization. No or weak membrane activities corresponded with biological inactivity. Highly membrane-active structures such as alamethicin F-30, chrysospermin C, texenomycin A and paracelsin A displayed antibiotic effects against the fungus and toxicity in mice.

Effects of the mycotoxin ochratoxin A and some of its metabolites on the human cell line THP-1.

The immunomodulatory effects of ochratoxin A (OTA) and some of its metabolites on the human monocyte/macrophage line THP-1 are described. Metabolic activity, cell proliferation, cell membrane integrity, cell differentiation, phagocytic behaviour, nitrogen oxide synthesis and cell surface markers were largely suppressed by these mycotoxins at concentrations between 10 and 1000 ng/ml, in individual cases already at 1 ng/ml. After analysis of a crude toxin, a substance designated RE2 could be isolated besides OTA, which was identified as ochratoxin C (OTC). The latter showed a stronger suppressive effect on most functions studied than all other metabolites of OTA. Because of the immunomodulatory effects of OTA and OTC, more attention should be paid to their immunopathogenic importance in addition to their known cytotoxic and genotoxic effects. The occurrence and importance of the mycotoxin OTC should be more closely examined in this context.

New and bioactive compounds from Streptomyces strains residing in the wood of Celastraceae.

Wood from three different plants of the Celastraceae growing in their natural habitats in Brazil (Maytenus aquifolia Mart.) and South Africa [Putterlickia retrospinosa van Wyk and Mostert, P. verrucosa (E. Meyer ex Sonder) Szyszyl.] was established as a source of endophytic bacteria using a medium selective for actinomycetes. Two isolates were identified as Streptomyces setonii and S. sampsonii whereas two others were not assignable to any of the known Streptomyces species. They were preliminarily named Streptomyces Q21 and Streptomyces MaB-QuH-8. The latter strain produces a new chloropyrrol and chlorinated anthracyclinone. The chloropyrrol showed high activity against a series of multiresistent bacteria and mycobacteria.

Synthesis and biological evaluation of analogues of the peptaibol ampullosporin A.

A series of analogues of the fungal peptaibol type metabolite ampullosporin A containing modifications in the C and N terminus as well as alpha-aminoisobutyric acid (Aib) substitutions in different positions of the peptide were synthesized by solid phase synthesis using the 9-fluorenylmethyloxycarbonyl strategy. Depending on the sequence position, couplings were performed with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/1-hydroxybenzotriazole and tetramethylfluoroformamidinium hexafluorophosphate, respectively. The structures of the target peptides were analyzed by electrospray ionization mass spectrometry and chromatographic methods (high-performance liquid chromatography, thin-layer chromatography). The biological activities of these compounds have been evaluated by assaying their potencies for the induction of pigment formation on the fungus Phoma destructiva as well as for the induction of hypothermia and inhibition of locomotoric activity in mice and were compared to the naturally occurring ampullosporins. Native ampullosporin A and analogues with C-terminal Leu or Leu-NH(2) showed comparable activity in the pigmentation assay. Similarly, the ampullosporin A analogues with N-terminal aromatic amino acid residues, such as D-Trp and Tic, also have high potency for pigment formation. The peptides containing structural modifications of ampullosporin A by systematic replacement of Aib by Ala (Ala scan) displayed moderate or high activity in the pigmentation assay, whereas simultaneous substitution of all Aib residues by Ala and Ile, respectively, or by insertion of nonaromatic residues into position 1 resulted in a loss of the effect on P. destructiva. Most of the compounds with no or weak activity in the microbial assay were not active in the hypothermic test, too, except the compound with 1-amino-1-cyclohexane carboxylic acid in position 4 instead of Aib. However, only a few compounds with high potency for pigmentation induction were found to produce strong hypothermia in mice. Thus, in contrast to the native ampullosporins, we succeeded to a certain degree in differentiation of the bioactivities with our synthetic analogues.

Radical scavengers from the entomogenous deuteromycete Beauveria amorpha.

The culture medium of Beauveria amorpha Hohn. exhibited strong radical scavenging properties. Activity-directed fractionation assisted by a radical scavenging assay on TLC afforded two active compounds which were identified as 6-methyl-2,4-dihydroxyphenyl 4-O-methyl-beta-D-glucopyranoside (1) and (-)-terredionol (2). Compound 1 displays radical scavenging and lipid peroxidation inhibitory activity comparable to those of TroloxC, ascorbic acid or quercetin.