Search

In our screening program for new biologically active compounds, a new polyene macrolide, lavencidin (1), along with known compound RKGS-A2215A (2), was isolated from the fermentation broth of Streptomyces lavendulae FRI-5 by changing the composition of liquid medium normally used for the strain. Their structures were elucidated by spectral methods (high-resolution fast-atom bombardment mass spectrometry (HRFABMS) and nuclear magnetic resonance (NMR)). Compound 1 includes a conjugated pentaene moiety together with six hydroxy groups and a carboxylic acid as a side chain. Lavencidin (1) showed moderate growth-inhibitory activity against yeast and was cytotoxic against human cancer cell lines with low-micromolar IC 50 values.

Secondary metabolites of microorganisms have proven to be an excellent source of drugs. We isolated a new antibiotic, named pestiocandin (1), from a culture broth of a filamentous fungus, Pestalotiopsis humus FKI-7473, using a multidrug-sensitive budding yeast, S. cerevisiae 12geneΔ0HSR-iERG6. The structure of 1 was elucidated by various NMR studies. All geometric isomerisms of 1 were shown to be the E-form and two pyranose units of 1 were found to be glucose and galactose types. Compound 1 showed weak growth inhibition against Gram-positive and Gram-negative bacteria, yeasts and a filamentous fungus. It displayed more potent growth inhibition against multidrug-sensitive yeasts than wild-type yeasts.

The alarming issue of antibiotic resistance expansion requires a continuous search for new and efficient antibacterial agents. Here we describe the design of new tools to screen for target-specific inhibitors of the bacterial Rho factor directly inside eukaryotic cells. Rho factor is a global regulator of gene expression which is essential to most bacteria, especially Gram-negative. Since Rho has no functional or structural homolog in eukaryotes, it constitutes a valuable and well known bacterial target as evidenced by its inhibition by the natural antibiotic, Bicyclomycin. Our screening tools are based on perturbation of mRNA processing and packaging reactions in the nucleus of eukaryotic cells by the RNA-dependent helicase/translocase activity of bacterial Rho factor leading to a growth defect phenotype. In this approach, any compound that impedes Rho activity should restore growth to yeast or human cells expressing Rho protein, providing valuable means to screen for target-specific antibacterial agents within the environment of a eukaryotic cell. The yeast tool expressing E. coli Rho factor was validated using Bicyclomycin as the control antibacterial agent. The validation of the screening tool was further extended with a stable human cell line expressing Rho factor conditionally. Finally, we show that Rho factors from different bacterial pathogens can also be designed as yeast-based screening tools which can reveal subtle variations in the functional features of the proteins.

Crotalicidin (Ctn), a 34-residue cathelicidin from a South American rattlesnake, and its fragment (Ctn[15-34]) have shown anti-infective and cytotoxic activities against Gram-negative bacteria and certain tumor lines, respectively. The extent of such effects has been related to physicochemical characteristics such as helicity and hydrophobicity. We now report the anti-fungal activity of Ctn and its fragments (Ctn[1-14]) and (Ctn[15-34]). MIC determination and luminescent cell viability assays were used to evaluate the anti-infective activity of Ctn and its fragments (Ctn[1-14]) and (Ctn[15-34]) as anti-fungal agents against opportunistic yeast and dermatophytes. Cytotoxicity towards healthy eukaryotic cells was assessed in vitro with healthy human kidney-2 (HK-2) cells and erythrocytes. The checkerboard technique was performed to estimate the effects of combining either one of the peptides with amphotericin B. Ctn was the most active peptide against dermatophytes and also the most toxic to healthy eukaryotic cells. Fragments Ctn[1-14] and Ctn[15-35] lost activity against dermatophytes, but became more active against pathogenic yeasts, including several Candida species, both clinical isolates and standard strains, with MICs as low as 5 μm. Interestingly, the two peptide fragments were less cytotoxic to healthy HK-2 cells and less hemolytic to human erythrocytes than the standard-of-care amphotericin B. Also noteworthy was the synergy between Ctn peptides and amphotericin B, with consequent reduction in MICs of both drug and peptides. Altogether, Ctn and its fragments, particularly Ctn[15-34], are promising leads, either alone or in combined regimen with amphotericin B, for the treatment of fungal diseases.

Two new secondary metabolites, named pedein A and B, were isolated from the cell mass of the myxobacterium Chondromyces pediculatus. Their planar structures were elucidated by spectroscopic methods, in particular 2D NMR as 24-membered cyclic hexapeptides composed of a variable tryptophan residue, glycine, sarcosine and three unusual hydroxy beta- and gamma-amino acids. The main component, pedein A, strongly inhibited the growth of yeasts and fungi, induced hemolysis of erythrocytes, and caused changes in membrane permeability of Rhodotorula glutinis. The structures of the pedeins are closely related to the large family of the microsclerodermins, which have been isolated from lithistid sponges of Microscleroderma and Theonella species.

In the course of a screening for cytotoxic compounds from fungi four new terpenoid metabolites, named dasyscyphins A, B, C, and niveulone were isolated from fermentations of Dasyscyphus niveus strain A0101. While dasyscyphin A (1) exhibited no significant biological activities in our test systems dasyscyphin B (2) and dasyscyphin C (3) showed cytotoxic activities against human (HepG2, Hela S3, U937, Colo-320, Jurkat) cell lines with IC50-values of 0.5-3 microg/ml while the activity of niveulone (4) was less pronounced. Only modest or weak antibiotic properties were observed for (2) and (3).

Microbial metabolites were screened for inhibitors of hyphal growth in Candida albicans. Inhibitory activity was found among metabolites of a culture of an actinomycete, which had been isolated from soil. The active substance inhibited hyphal growth and induced growth as a chain of yeast cells under hyphal growth induction conditions. The active substance was purified and analyzed with 1H-NMR, 13C-NMR and mass spectra. The substance was identified as valinomycin, and commercial valinomycin inhibited hyphal growth as effectively as the purified metabolite. The effective concentration was from 0.49 to 62.5 microg/ml. Valinomycin also inhibited hyphal growth in other dimorphic fungi, Candida tropicalis and Aureobasidium pullulans. These results suggest that valinomycin may be a useful tool for understanding the morphological transition of dimorphic fungi.

Two novel antibiotics, watasemycins A and B, were isolated from the fermentation broth of an actinomycete strain. The producing strain TP-A0597 was isolated from the seawater sample collected in Toyama Bay, Japan and identified as Streptomyces sp. based on the taxonomic study. The new antibiotics were obtained by solvent extraction and chromatographic purification and spectroscopic analyses identified that they were new analogs of thiazostatins. Watasemycin possesses a methyl group at 5'-position of thiazostatin instead of a hydrogen atom. Watasemycins showed antibiotic activity against Gram-positive and negative bacteria and yeast.

Glucosylquestiomycin, a novel N-glucopyranoside of questiomycin A, was isolated from the culture broth of Microbispora sp. TP-A0184. The absolute configuration of the sugar was determined as D-configuration by chemical synthesis. The new antibiotic showed antibacterial activity against gram-positive and -negative bacteria and yeasts and cytotoxic activity against U937 cells.

A new secondary metabolite was detected in the culture filtrate and extracts of Streptomyces violaceusniger Tü 4113 by HPLC-diode-array and HPLC-electrospray-mass-spectrometry screening. The compound named spirofungin has a polyketide-spiroketal structure and shows various antifungal activities, particularly against yeasts.

The in vitro activity of a naturally occurring complex carbohydrate, CAN-296, was evaluated by testing 132 clinical and ATCC isolates of yeast and Aspergillus fumigatus, many of which were azole-resistant. The in vitro susceptibility tests were performed by standardized broth micro- and macrodilution methods and results were compared with those obtained for amphotericin B, fluconazole, ketoconazole, flucytosine and the pneumocandin L-733,560. All tested Candida species showed highly uniform susceptibility to CAN-296 at concentrations of 0.078 to 0.312 microgram/ml; non-albicans Candida were as susceptible to CAN-296 as the Candida albicans strains. Multi-azole-resistant Candida species were highly sensitive to CAN-296. Minimum inhibitory concentration measurements did not differ from minimum lethal concentrations by more than two-fold for all tested Candida species. Aspergillus fumigatus, on the other hand, showed only moderate susceptibility to CAN-296. The kinetics of the anti-Candida activity of CAN-296 was investigated by kill-curve experiments using C. albicans and C. glabrata and the results were compared with those obtain for amphotericin B. CAN-296 was found to be rapidly fungicidal in concentrations ranging from 4-16 fold the mean MIC value. The broad spectrum of anti-Candida activity together with the rapid fungicidal effect make this complex carbohydrate a promising agent for clinical use.

An antifungal antibiotic, benanomicin A, binds in the presence of Ca2+ to susceptible fungi and some bacteria, but not to antibiotic-resistant bacteria and mammalian cells. With the susceptible yeast Saccharomyces cerevisiae, benanomicin A binds similarly to whole cells and to protoplasts. Studies using benanomicin A and three structurally related derivatives suggested that a carboxylic acid in the D-alanine moiety and a sugar moiety in the benanomicin A molecule are essential for both binding and antifungal activities against growing S. cerevisiae. An amino substituent on the sugar moiety can be replaced with a hydroxyl group without the loss of activities. Benanomicin A binds to various yeast mannans which differ in glycosidic linkages. These results indicate that binding of benanomicin A to the mannan portion of fungal cells is essential for exertion of the antifungal activity.

The biological activities of karalicin, a new product from the Pseudomonas fluorescens/putida strain SS-3 (CCM 4430) are described. It shows a weak, but specific and irreversible, antiviral activity on Herpes simplex viruses. It also presents some inhibitory activity on different species of yeasts.

A new antibiotic, chivosazol, was isolated from the culture broth of the myxobacterium Sorangium cellulosum strain So ce12. It is a macrocyclic ring with one oxazol ring and a glycosidically bound 6-deoxyglucose (quinovose) at C-11. The antibiotic shows antimicrobial activity against yeasts and filamentous fungi, and is especially potent against mammalian cells. It was not active against bacteria.

A new antibiotic compound, gephyronic acid was isolated from the culture broth of the myxobacterium, Archangium gephyra strain Ar 3895. Up to 3 mg/liter was produced during the logarithmic and stationary growth phase. The compound is an aliphatic acid, which tends to form a hemiacetal. Both forms inhibited growth of yeasts and molds (MIC 1-25 micrograms/ml) and had a cytostatic effect on mammalian cell cultures (IC50 10-60 ng/ml). Gephyronic acid is a specific inhibitor of eukaryotic protein synthesis showing an IC50 of 1-2 x 10(-7) mol/liter in an in vitro translation assay.

Cladospirone bisepoxide (1), a novel metabolite, was isolated from cultures of a fungus which was characterized as a coelomycete by the formation of pycnidia. By optimization of media and fermentation conditions, a titer of up to 1.5 g/liter on shake level and 1.16 g/liter on bioreactor scale could be achieved. The isolation of the compound was performed by solvent extraction of the culture broth and subsequent crystallization. Cladospirone bisepoxide displays selective antibiotic activity against several bacteria and fungi and inhibits germinations of Lepidium sativum at low concentrations.

Three novel antibiotics, named chrymutasins A, B and C, were isolated from the fermentation products of a mutant strain obtained by NTG (N-methyl-N'-nitro-N-nitrosoguanidine) treatment. The mutant strain produced the chrymutasins, which differed in the aglycone moiety from chartreusin, and related compounds. The production of these compounds needed a characteristically long fermentation period. The antitumor activity of chrymutasin A is better in vivo than that of chartreusin, the cytotoxic activity against cell lines in vitro is equivalent, and the antimicrobial spectrum is narrower.

Two polyene macrolide, trichomycins A and B, were compared by physico-chemical and microbiological methods. The two antibiotics were found to have the same molecular formula, C58H84N2O18 (MW 1,096), by elemental analysis and FAB-MS. However, 1H and 13C NMR spectrometry studies indicated that the hydroxyl at C-5 of trichomycin A located on C-9 in trichomycin B. Trichomycin B possessed lower activities against fungi and yeasts than those of trichomycin A.

New antifungal antibiotics pradimicins D and E were isolated from the culture filtrates of Actinomadura hibisca P157-2 (ATCC 53557) and its mutant A2660 (ATCC 53762). The structure of pradimicin D is N-[[(5S,6S)-5-O-[4,6-dideoxy-4-(methylamino)-3-O-(beta-D- xylopyranosyl)-beta-D-galactopyranosyl]-5,6,8,13-tetrahydro-1,6,9,14- tetrahydroxy-11-methoxy-3-methyl-8,13-dioxobenzo[a]naphthacen++ +-2-yl] carbonyl]glycine, based on spectral analyses compared to pradimicin A. Pradimicin E is the des-N-methyl analog of pradimicin D. Pradimicins D and E were equal in activity to pradimicin A in vitro against a variety of fungi and in vivo against Candida albicans A9540 in mice.

New acylpeptide antibiotics named cepafungins I, II and III were isolated from the culture broth of a strain identified as Pseudomonas sp. These antibiotics are neutral substances, soluble in aqueous alcohols and dimethyl sulfoxide, and show UV maxima at 260.5 nm. The IR spectra indicated these to be peptides. Molecular formulas C28H46N4O6, C27H44N4O6 and C26H42N4O6 for cepafungins I, II and III were indicated by elemental analysis and SI-MS. Cepafungins exhibited inhibitory activity against yeast and fungi, and antitumor activity against P388 leukemia in mice.

Pholipomycin, a new member of the phosphoglycolipid antibiotics, was primarily active against gram-positive bacteria including clinically isolated resistant bacteria. It differed from other members of the antibiotic family as it also demonstrated activity against gram-negative bacteria. Pholipomycin protected mice from infection with Staphylococcus aureus and was nontoxic to mice having an LD50 (i.v.) of 600 mg/kg. Besides possessing anti-microbiol activity, pholipomycin, when administered orally, appeared to promote growth in chickens and pigs.

A new antibiotic named platenocidin was isolated from the culture broth of Streptomyces H 273 N-SY2 and the producting microbe was classified as Streptomyces platensis. Platenocidin gives 5-hydroxymethyluracil by acid or alkaline hydrolysis and can be considered to belong to one of the nucleoside antibiotics. The antibiotic inhibits the growth of certain species of yeasts, but not that of bacteria.

Erwinia herbicols (strain A 111) produces two acylated peptide antibiotics herbicolins A and B. Isolation of herbicolins was performed by adsorption on a polystryrol adsorbent followed by elution with methanol. Further purification was achieved by gel filtration on Sephadex LH-20, counter-current distribution or by TLC. Herbicolin A was chemically characterized, containing 2 glycines, 1 L-threonine, 1 D-allo-threonine, 1 D-glutamic acid, 1 D-leucine, 1 L-agrinine and beta-hydroxy myristic acid. Herbicolins A and B are inactive against bacteria, but highly active against yeasts and filamentous fungi.

Two new nikkomycins were isolated from the culture broth of Streptomyces tendae Tü 901/PF 53+-3. The new compounds are the dipeptide nikkomycin pseudo-Z (psi Z) and tripeptide nikkomycin pseudo-J (psi J), which are analogues to nikkomycins Z and J. Nikkomycins pseudo-Z and pseudo-J have a C-glycosidic linkage between uracil and 5-amino-5-deoxy-D-allo-furanuronic acid, which is comparable to the C-glycosidic bond in pseudouridine. The new Cc-nucleoside nikkomycins exhibit a lower biological activity than the CN-nucleoside nikkomycins.

Whole shaken cultures of 20 random, unidentified actinomycetes were extracted with n-butanol at pH 4.5, 7.0 and 8.5, respectively. Residues of butanol-extractable materials (BXM) were reconstituted (100X) in buffers and freeze-dried. BXM were surprisingly well tolerated in animals and were screened against influenza A viral pneumonia in mice. One culture yielded BXM-80 which suppressed both chemical (LPS) and viral (NDV) pneumonia in mice as well as inhibited rat foot pad edema induced by carrageenin. Aspirin, Butazolidin, hydrocortisone, indomethacin, and prednisolone, which are known to inhibit carrageenin-induced rat foot pad edema were tested against chemical (LPS) and viral (NDV) pneumonia in mice. Only hydrocortisone and prednisolone suppressed LPS pneumonia. All of these 5 compounds failed to inhibit NDV pneumonia. Microbial products are suggested as a source for new and unique anti-inflammatory agents.

A new antifungal antibiotic, named neo-enactin, was isolated from the cultured mycelia of Strain H 829-MY 10, identified as a new subspecies of Streptoverticillium and named Streptoverticillium olivoreticuli subsp. neoenacticus. The antibiotic was produced with a tetraene antifungal antibiotic, found mainly in the cultured mycelia of Strain H 829-MY 10 and it was extracted with methanol. The antibiotic is of a basic nature and it can be extracted with ethyl acetate at alkaline pH. Purification of neo-enactin was carried out by partition chromatography on cellulose and elution with ethyl acetate bufferized with phosphate buffer (pH 8.0). Neo-enactin shows strong antifungal activity and potentiates the antifungal activity of polyene antifungal antibiotics.

The new antibiotic gardimycin has an interesting in vitro antibacterial activity against Gram-positive bacteria and Neisseria gonorrhoeae. Parenteral administration gives a high degree of protection against experimental infections in mice. It also shows some chemotherapeutic activity when given rectally.

Six new antibiotics were isolated as the minor components related to aculeacin A from the culture broth of Aspergillus aculeatus M-4214 and named as aculeacins B, C, D, E, F and G. Their physico-chemical properties were analogous to those of aculeacin A and they showed significant activity against fungi. All of the minor components liberated palmitic acid on alkaline hydrolysis. Amino acid analysis showed that threonine and hydroxyproline are common constituents of aculeacins.

Aculeacin A, a new antifungal antibiotic was isolated from the mycelial cake of Aspergillus aculeatus M-4214. The antibiotic is a white amorphous powder soluble in lower alcohols and hardly soluble in other organic solvents or water. Aculeacin A gave palmitic acid and five ninhydrin-positive products including theonine, hydroxyproline upon acid hydrolysis. The antibiotic showed a potent activity against molds and yeasts, but exhibited no antibacterial activity. Aculeacin A has relatively low toxicity in mice.