Antimicrobial activity of ergokonin A from Trichoderma longibrachiatum.

AIMS: Natural fungal products were screened for antifungal compounds. The mode of action of one of the hits found and the taxonomy of the producing organism were analysed. METHODS AND RESULTS: An extract from a Trichoderma species showed a more potent activity in an agar-based assay against the null mutant fks1::HIS strain than against the wild-type strain, suggesting that it could contain a glucan synthesis inhibitor. The active component was identified as the known compound ergokonin A. The compound exhibited activity against Candida and Aspergillus species, but was inactive against Cryptococcus species. It induced alterations in the hyphal morphology of Aspergillus fumigatus. The identification of the producing isolate was confirmed by sequencing of the rDNA internal transcribed spacers and comparison with the sequences of other Trichoderma species. The analysis showed that the producing fungus had a high homology with other strains classified as Trichoderma longibrachiatum and its teleomorph Hypocrea schweinitzii. CONCLUSIONS: The antifungal activity spectrum of ergokonin A and the morphology alterations induced on A. fumigatus are consistent with glucan synthesis as the target for ergokonin A. The production of ergokonin A is not uncommon, but is probably restricted to Trichoderma species. SIGNIFICANCE AND IMPACT OF THE STUDY: The discovery that ergokonin A could be an inhibitor of glucan synthesis, having a structure very different to other inhibitors, increases the likelihood that orally active agents with this fungal-specific mode of action may be developed.

Discovery of novel antifungal (1,3)-beta-D-glucan synthase inhibitors.

The increasing incidence of life-threatening fungal infections has driven the search for new, broad-spectrum fungicidal agents that can be used for treatment and prophylaxis in immunocompromised patients. Natural-product inhibitors of cell wall (1,3)-beta-D-glucan synthase such as lipopeptide pneumocandins and echinocandins as well as the glycolipid papulacandins have been evaluated as potential therapeutics for the last two decades. As a result, MK-0991 (caspofungin acetate; Cancidas), a semisynthetic analogue of pneumocandin B(o), is being developed as a broad-spectrum parenteral agent for the treatment of aspergillosis and candidiasis. This and other lipopeptide antifungal agents have limited oral bioavailability. Thus, we have sought new chemical structures with the mode of action of lipopeptide antifungal agents but with the potential for oral absorption. Results of natural-product screening by a series of newly developed methods has led to the identification of four acidic terpenoid (1,3)-beta-D-glucan synthase inhibitors. Of the four compounds, the in vitro antifungal activity of one, enfumafungin, is comparable to that of L-733560, a close analogue of MK-0991. Like the lipopeptides, enfumafungin specifically inhibits glucan synthesis in whole cells and in (1,3)-beta-D-glucan synthase assays, alters the morphologies of yeasts and molds, and produces a unique response in Saccharomyces cerevisiae strains with point mutations in FKS1, the gene which encodes the large subunit of glucan synthase.

Ophiobolin M and analogues, noncompetitive inhibitors of ivermectin binding with nematocidal activity.

A series of ophiobolins were isolated from a fungal extract based on their nematocidal activity. These compounds are non-competitive inhibitors of ivermectin binding to membranes prepared from the free-living nematode, Caenorhabditis elegans, with an inhibition constant of 15 microM. The ophiobolins which were most potent in the biological assays, ophiobolin C and ophiobolin M, were also the most potent compounds when evaluated in a C. elegans motility assay. These data suggest that the nematocidal activity of the ophiobolins is mediated via an interaction with the ivermectin binding site. The isolation, structure and biological activity of ophiobolins have been described.

Photoaffinity labeling of bacteriorhodopsin.

14C-Labeled optically pure 3S- and 3R-(diazoacetoxy)-all-trans-retinals were incorporated separately into bacterioopsin to reconstitute functional bacteriorhodopsin (bR) analogues, 3S- and 3R-diazo-bRs. UV irradiation at 254 nm generated highly reactive carbenes, which cross-linked the radiolabeled retinals to amino acid residues in the vicinity of the beta-ionine ring. The 3S- and 3R-diazo analogues were found to cross-link, respectively, to cyanogen bromide fragments CN 7/CN9 and CN 8/CN 9. More specifically, Thr121 and Gly122 in fragment CN 7 were found to be cross-linked to the 3S-diazo analogue. The identification of cross-linked residues and fragments favors assignments of the seven helices A-G-F-E-D-C-B or B-C-D-E-F-G-A to helices 1-2-3-4-5-6-7 in the two-dimensional electron density map (Henderson et al., 1975, 1986; Mogi et al., 1987). The present results show that the chromophore chain is oriented with the ionone ring inclined toward the outside of the membrane (the 9-methyl group also faces the extracellular side of the membrane).

Liquid chromatographic separation of enantiomers of beta-amino acids using a chiral stationary phase.

The enantiomers of both alpha-substituted beta-alanines and beta-substituted beta-alanines may be chromatographically separated using silica-bonded chiral stationary phases derived from N-acetylated alpha-arylalkylamines. The amino acids are chromatographed as alkyl esters of N-3,5-dinitrobenzoyl derivatives; separability factors range from 1.11 to 1.65 for nine alpha-substituted beta-alanines and from 1.08 to 1.20 for nine beta-substituted beta-alanines. The enantiomers of beta-aminoisobutyrate and beta-leucine, chiral beta-amino acids occurring in animal tissues and physiological fluids, are among those resolved. The enantiomers of R,S-beta-aminoisobutyrate and several related alpha-alkyl-beta-alanines were prepared by chromatographic resolution of diastereomeric dipeptides.

A rational approach to the design of highly effective chiral stationary phases for the liquid chromatographic separation of enantiomers.

The design and rationale of some novel chiral stationary phases (CSPs) are discussed with respect to methods for determining enantiomeric purity, absolute configuration and for obtaining enantiomerically pure materials by liquid chromatography. The commercially-available dinitrobenzoylamino CSP type 1 is discussed with respect to the chiral recognition mechanisms which may operate in the resolution of some polycyclic and heterocyclic aromatic molecules and some benzodiazepines. N-Acyl alpha-arylalkylamines are also employed as models to formulate mechanisms for the chiral properties of type 1 CSPs in terms of enantiomeric stacking of the most stable conformations in solution. The properties of new types of 'reciprocal' CSPs are discussed and illustrated by enantiomeric separation of some amino acid and amino phosphoric acid derivatives, and by the separation of the following enantiomeric drugs as their 3,5-dinitrobenzoyl derivatives: metoprolol, oxoprenolol, ephedrine and alprenolol.