Structure of the spiroketal-macrolide ossamycin.

Ossamycin is a cytotoxic agent of undetermined structure that was originally isolated in 1965 from culture broths of Streptomyces hygroscopicus var. ossamyceticus. Its overall structure and relative stereochemistry have now been determined by single crystal X-ray diffraction studies. Absolute stereochemistry was established according to the previously determined configuration of its aminosaccharide constituent, ossamine. The aglycone of ossamycin possesses a 24-membered macrolide ring system onto which is incorporated both a 6,6-spiroketal and 5-membered hemiketal ring system. The overall three-dimensional structure possesses features in common with the related macrocyclic antibiotics dunaimycin, cytovaricin, and A82548A.

Rapamycin and FK506 differentially inhibit mast cell cytokine production and cytokine-induced proliferation and act as reciprocal antagonists.

We have previously demonstrated that cyclosporine (CSA) and FK506 are able to selectively inhibit cytokine production by murine mast cell lines at concentrations comparable to those observed with thymus-derived lymphocytes (T cells). The selectivity of these effects were demonstrated by the failure of CSA and FK506 to inhibit cytokine-induced mast cell proliferation at equivalent or higher concentrations. In this report, we examined the ability of rapamycin (RAP) to inhibit cytokine production and cytokine-induced proliferation by a factor-dependent murine mast cell line and compared its activity to that of the structurally related macrolide FK506. The mast cell clone, MC/9, was stimulated to produce cytokines with phorbol myristate acetate plus the calcium ionophore A23187, or to proliferate in response to exogenous cytokines such as interleukin-3 and interleukin-4, produced by the helper T cell clone D10.G4. RAP did not inhibit cytokine production by MC/9, even at concentrations greater than 1000 nM. FK506 and CSA inhibited cytokine production with IC50 of 0.8 and 16.2 nM, respectively. In contrast to its lack of effect on cytokine production, RAP potently inhibited cytokine-induced proliferation of MC/9 cells with an IC50 of 1.9 nM. Because RAP and FK506 are structurally related and yet have divergent biological effects, we examined the ability of RAP to antagonize inhibitory effects of FK506 on mast cell cytokine production and the ability of FK506 to antagonize inhibitory effects of RAP on cytokine-induced mast cell proliferation. The addition of RAP in molar excess reversed inhibition of mast cell cytokine production mediated by FK506, but not that of CSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the mechanism of action of A80915A, a semi-naphthoquinone natural product, as an inhibitor of gastric (H(+)-K+)-ATPase.

A semi-naphthoquinone natural product, A80915A, produced by Streptomyces aculeolatus was found to be a potent inhibitor of gastric (H(+)-K+)-ATPase, the enzyme responsible for acid secretion in the stomach. Enzyme activity was measured by potassium-stimulated hydrolysis of ATP or p-nitrophenolphosphate with enzyme prepared from the stomach fundic mucosa of pigs. Concentration-dependent inhibition was observed with an IC50 of about 2-3 microM for both ATPase and p-nitrophenylphosphatase. A Hill plot indicated that the enzyme has two binding sites for A80915A. Inhibition was not affected by the presence of the reducing agent dithiothreitol, indicating a lack of involvement of enzyme sulfhydryl groups. A 30-min incubation of enzyme with increasing drug concentrations followed by a 10-fold dilution did not alter the IC50, indicating that A80915A does not covalently modify the enzyme. Coincubation of enzyme with 3.8 microM A80915A resulted in time-dependent inhibition. The rate of inhibition was slowed significantly by the presence of 20 mM potassium, rubidium and ammonium but not by 20 mM sodium, lithium and choline, or by 40 mM sucrose. The level of inhibition was influenced by the order of addition of potassium and drug to the enzyme. Taken together, these studies indicate that inhibition by A80915A is dependent on the conformation of gastric (H(+)-K+)-ATPase and that potassium slows the rate of inhibition by converting the enzyme to a conformation where the drug binding site is not as accessible. The mode of action of A80915A is distinct from that of two well characterized proton pump inhibitors, omeprazole and SCH 28080.

The detection of proline isomerase activity in FK506-binding protein by two-dimensional 1H NMR exchange spectroscopy.

1H NMR assignments of the trans and cis isomers of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were accomplished by two-dimensional NMR techniques. Conformational exchange between the cis and trans isomers was not detected in the two-dimensional exchange spectra (NOESY) until catalytic amounts of FK506-binding protein (FKbp) were added. The addition of FK506 to the enzyme-substrate solution inhibited the enzyme and removed the substrate exchange peaks.

A80915, a new antibiotic complex produced by Streptomyces aculeolatus. Discovery, taxonomy, fermentation, isolation, characterization, and antibacterial evaluation.

New semi-naphthaquinone antibiotics A80915A, B, C, and D were isolated from the fermented broth of Streptomyces aculeolatus A80915 (NRRL 18422). Factors A and C, present in both the broth filtrate and mycelial methanol extract, and factors B and D, found predominantly in the broth filtrate, were recovered by extraction with ethyl acetate. Purification of the individual factors was accomplished by preparative reverse phase high performance liquid chromatograph on C18 bonded silica supports. Factors A through D show antimicrobial activity against Gram-positive aerobic and anaerobic organisms in vitro. Mechanism of action studies demonstrated nearly complete inhibition of macromolecular biosynthesis (protein, RNA, DNA, and cell wall) by A80915 factors A through D. A less highly cyclized semi-naphthaquinone, A80915 factor G, was isolated from the broth of the strain fermented in an alternate medium.

A54145, a new lipopeptide antibiotic complex: discovery, taxonomy, fermentation and HPLC.

A54145 is a complex of new lipopeptide antibiotics that inhibits Gram-positive bacteria and acts as a growth promotant for broiler chicks. Eight factors; A, B, C, D, E, F, A1 and B1; have been isolated and characterized. They contain four similar peptide nuclei, each of which is acylated with either an 2-decanoyl, n-decanoyl, or undecanoyl side chain. Taxonomic studies ascertained that the producing microorganism was a strain of Streptomyces fradiae. Fermentation studies determined that superior antibiotic yields were obtained in stirred bioreactors in a soybean flour-molasses medium employing a continuous glucose feed. These findings, interwoven with the selection of hyper-productive mutants, increased fermentation yields from less than 50 micrograms/ml to more than 1 mg/ml. An analytical HPLC system was developed for the identification and subsequent quantitation of each factor of the A54145 complex.

A54145, a new lipopeptide antibiotic complex: isolation and characterization.

A54145 is a complex of acidic lipopeptide antibiotics which are produced by Streptomyces fradiae and are active against Gram-positive bacteria. The A54145 complex was isolated by adsorption on Diaion HP-20 nonfunctionalized macroreticular resin and/or ion exchange on Amberlite IRA-68 anion exchange resin. Antibacterial factors A, A1, B, B1, C, D, E, and F were obtained in purified form by repeated preparative reverse phase HPLC on C8 and/or C18 bonded-phase supports. The molecular formulae of the factors are C72H109N17O27 (factors A and A1), C73H111N17O27 (factors B, B1, C, and D), C74H113N17O27 (factor E), and C71H107N17O27 (factor F). The identities of the acyl side chains were established as 8-methylnonanoyl (factors F, A, and B1), n-decanoyl (factors A1 and B), and 8-methyldecanoyl (factors C, D, and E).

A54145, a new lipopeptide antibiotic complex: microbial and chemical modification.

A54145 is a complex of acidic lipopeptide antibiotics produced by Streptomyces fradiae NRRL 18158, NRRL 18159, and NRRL 18160. Each antibiotic factor consists of a peptide core bearing an N-terminal acyl substituent. N-Lys-tert-BOC-protected A54145 complex was deacylated by Actinoplanes utahensis; three protected core peptides were isolated. A54145 antibiotic analogs were synthesized by acylation of the tryptophan N-terminus with 2,4,5-trichlorophenyl active esters, followed by deblocking with trifluoroacetic acid.

A54145 a new lipopeptide antibiotic complex: microbiological evaluation.

A54145 complex is made up of eight factors; A, A1, B, B1, C, D, E, and F which were active in vitro (MIC 0.25 approximately greater than 32 micrograms/ml) against Gram-positive aerobic organisms. The complex, factor B and B1 were found to be active against two strains of Clostridium perfringens. A calcium dependence study on some of the factors showed that their in vitro antibacterial activity was greatly enhanced by the presence of calcium (50 mg/liter) in the media. Resistance build-up was seen when Staphylococcus sp. and Streptococcus sp. were passed seven times in the presence of sublethal concentrations of A54145 antibiotics. This resistance disappeared immediately when the resistant organisms were passed in the absence of the antibiotics. Factor A was very effective against Staphylococcus aureus and Streptococcus pyogenes infections in mice (sc ED50s of 3.3 approximately 2.4 mg/kg x 2, respectively). Factor B was more active against S. pyogenes in vivo (sc ED50, 0.9 mg/kg x 2). Acute mouse toxicities were determined with these antibiotics. Semisynthetic derivatives were evaluated.

Structure elucidation of A58365A and A58365B, angiotensin converting enzyme inhibitors produced by Streptomyces chromofuscus.

A58365A and A58365B, angiotensin converting enzyme inhibitors isolated from the culture filtrate of Streptomyces chromofuscus NRRL 15098, are homologous compounds of molecular formulas C12H13NO6 and C13H15NO6. The molecular similarities of the two inhibitors were established by comparison of their 1H NMR, 13C NMR, and UV spectra. Catalytic hydrogenation of A58365A led to a tetrahydro-deoxy derivative, C12H17NO5; extensive 1H NMR decoupling studies at 360 MHz allowed all the non-exchangeable protons of the derivative to be connected in a continuous substructure. This fragment was combined with information from other spectroscopic methods to suggest the structures for A58365A (1) and A58365B (2); the conclusions were confirmed by an X-ray crystallographic analysis of A58365A-dimethyl ester.

Method for the detection and quantitation of chitinase inhibitors in fermentation broths; isolation and insect life cycle effect of A82516.

A new method of screening for chitinase inhibitors in crude fermentation broths as a means of discovering new insecticidal leads has been developed. In this procedure soluble Remazol brilliant violet 5R dye-coupled chitin degradation products released from insoluble chitin azure substrate by hydrolysis with Streptomyces griseus chitinase are filtered in 0.45 micron Millititer HA 96 well filtration plates and collected in 96 well microtiter plates. Inhibitors of this reaction are detected by a decrease in absorbance (570 nm) of the filtrate. A chitinase inhibitor, designated A82516, produced by culture A82516 was discovered using this screen. Purified A82516 was found to have an IC50 of 3.7 X 10(-6) M for S. griseus chitinase. At a test concentration of 0.27 mg/ml, A82516 was 100% effective in preventing development of house fly larvae to pupae. Allosamidin, a recently reported chitinase inhibitor in vitro, has spectral properties identical to A82516.

Isolation of A58365A and A58365B, angiotensin converting enzyme inhibitors produced by Streptomyces chromofuscus.

A58365A and A58365B, angiotensin converting enzyme inhibitors, were isolated from the culture filtrate of Streptomyces chromofuscus NRRL 15098. A58365A and A58365B are homologous nitrogen-containing bicyclic structures of molecular formulae C12H13NO6 and C13H15NO6.

Antineoplastic evaluation of Pacific basin marine algae.

Extracts of 107 marine alga specimens from Pacific islands were tested for P-388 lymphocytic leukemia and Ehrlich ascites tumor in mice. Several specimens showed high antitumor activity in both systems, with some featuring a notable lack of toxicity.

Antileukemia activity in the Osillatoriaceae: isolation of Debromoaplysiatoxin from Lyngbya.

Chloroform extracts of several seaweeds, of the family Oscillatoriaceae, from Enewetak Atoll, Marshall Islands, display activity against P-388 lymphocytic mouse leukemia. A P-388 active compound, debromoaplysiatoxin, has been isolated from Lyngbya gracilis and characterized. This compound also has dermonecrotic activity and may be the dermatitis-producing substance in L. majuscula, the causative agent of "swimmers' itch" outbreaks in Hawaiian waters.