naphthoquinones and kalafungin

Topics: Alkylating Agents; Antibiotics, Antineoplastic; Antineoplastic Agents; Chemical Phenomena; Chemistry; Doxorubicin; Mitomycin; Mitomycins; Naphthacenes; Naphthoquinones; Oxidation-Reduction; Quinones; Stereoisomerism

Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; beta-Lactamase Inhibitors; beta-Lactamases; Disease Models, Animal; Humans; Kinetics; Molecular Docking Simulation; Naphthoquinones; Staphylococcal Infections; Staphylococcus aureus; Streptomyces

Medermycin and kalafungin, two antibacterial and antitumor antibiotics isolated from different streptomycetes, share an identical polyketide skeleton core. The present study reported the discovery of kalafungin in a medermycin-producing streptomycete strain for the first time. A mutant strain obtained through UV mutagenesis showed a 3-fold increase in the production of this antibiotic, compared to the wild type strain. Heterologous expression experiments suggested that its production was severely controlled by the gene cluster for medermycin biosynthesis. In all, these findings suggested that kalafungin and medermycin could be accumulated by the same streptomycete and share their biosynthetic pathway to some extent in this strain.

Topics: Anti-Bacterial Agents; Benzoquinones; Chromatography, High Pressure Liquid; Disk Diffusion Antimicrobial Tests; Magnetic Resonance Spectroscopy; Mass Spectrometry; Multigene Family; Mutagenesis; Naphthoquinones; Staphylococcus epidermidis; Streptomyces; Ultraviolet Rays

A general, efficient, and common strategy for the synthesis of (-)-juglomycin A, (+)-kalafungin, (+)-frenolicin B, and (+)-deoxyfrenolicin is reported here. The strategy involves the synthesis of a key building block alkyne from a cheap chiral pool material, D-glucono-δ-lactone, Dötz benzannulation, oxa-Pictet-Spengler reaction, and H(2)SO(4)-mediated epimerization.

Topics: Alkynes; Molecular Structure; Naphthoquinones; Stereoisomerism

The naturally occurring pyranonaphthoquinone (PNQ) antibiotic lactoquinomycin and related aglycones were found to be selective inhibitors of the serine-threonine kinase AKT. A set of synthetic PNQs were prepared and a minimum active feature set and preliminary SAR were determined. PNQ lactones inhibit the proliferation of human tumor cell lines containing constitutively activated AKT and show expected effects on cellular biomarkers. Biochemical data are presented supporting a proposed bioreductive alkylation mechanism of action.

Topics: Alkylation; Antineoplastic Agents; Biomarkers; Cell Line, Tumor; Cell Proliferation; Cysteine; Humans; Lactones; Naphthoquinones; Oncogene Protein v-akt; Pyrans; Stereoisomerism; Structure-Activity Relationship

Many microorganisms produce molecules having antibiotic activity and expel them into the environment, presumably enhancing their ability to compete with their neighbours. Given that these molecules are often toxic to the producer, mechanisms must exist to ensure that the assembly of the export apparatus accompanies or precedes biosynthesis. Streptomyces coelicolor produces the polyketide antibiotic actinorhodin in a multistep pathway involving enzymes encoded by genes that are clustered together. Embedded within the cluster are genes for actinorhodin export, two of which, actR and actA resemble the classic tetR and tetA repressor/efflux pump-encoding gene pairs that confer resistance to tetracycline. Like TetR, which represses tetA, ActR is a repressor of actA. We have identified several molecules that can relieve repression by ActR. Importantly (S)-DNPA (an intermediate in the actinorhodin biosynthetic pathway) and kalafungin (a molecule related to the intermediate dihydrokalafungin), are especially potent ActR ligands. This suggests that along with the mature antibiotic(s), intermediates in the biosynthetic pathway might activate expression of the export genes thereby coupling export to biosynthesis. We suggest that this could be a common feature in the production of many bioactive natural products.

Topics: Anthraquinones; Anti-Bacterial Agents; Bacterial Proteins; Biological Transport; Biosensing Techniques; DNA, Bacterial; Gene Expression Regulation, Bacterial; Ligands; Multigene Family; Mutation; Naphthalenes; Naphthoquinones; Pyrans; Repressor Proteins; Streptomyces coelicolor; Tetracycline

The ActVA-ActVB system from Streptomyces coelicolor isatwo-component flavin-dependent monooxygenase that belongs to an emerging class of enzymes involved in various oxidation reactions in microorganisms. The ActVB component is a NADH:flavin oxidoreductase that provides a reduced FMN to the second component, ActVA the proper monooxygenase. In this work, we demonstrate that the ActVA-ActVB system catalyzes the aromatic monohydroxylation of dihydrokalafungin by molecular oxygen. In the presence of reduced FMN and molecular oxygen, the ActVA active site accommodates and stabilizes an electrophilic flavin FMN-OOH hydroperoxide intermediate species as the oxidant. Surprisingly, we demonstrate that the quinone form of dihydrokalafungin is not oxidized by the ActVA-ActVB system, whereas the corresponding hydroquinone is an excellent substrate. The enantiomer of dihydrokalafungin, nanaomycin A, as well as the enantiomer of kalafungin, nanaomycin D, are also substrates in their hydroquinone forms. The previously postulated product of the ActVA-ActVB system, the antibiotic actinorhodin, was not found to be formed during the oxidation reaction.

Topics: Anthraquinones; Flavins; FMN Reductase; Hydrogen Peroxide; Hydroquinones; Hydroxylation; Mixed Function Oxygenases; Naphthoquinones; Oxidants; Quinones; Streptomyces coelicolor; Substrate Specificity

The new antibiotic tetrahydrokalafungin was produced by the transformants of kalafungin producing S. tanashiensis and kalafungin-nonproducing mutants carrying the recombinant plasmid pKU523. This plasmid consists of pKU501 (J. Antibiotics 44: 995 approximately 1005, 1991) which contains the gene cluster for kalafungin biosynthesis, and additional 5 kb stability region of SCP2*.

Topics: Anti-Bacterial Agents; Molecular Structure; Naphthoquinones; Plasmids; Streptomyces; Structure-Activity Relationship

We have developed a high capacity screen for compounds that inhibit the 3C protease of human rhinovirus-1b. The assay uses a recombinant strain of Escherichia coli expressing both the protease and a tetracycline resistance-conferring protein modified to contain the minimal protease cleavage site. Cultures growing in microtiter plates containing tetracycline are treated with potential inhibitors and simultaneously monitored for change in growth over time using an oxygen probe. Most of the cultures, not containing an inhibitor of the 3C protease, show reduced growth due to cleavage of the essential gene product; normal growth is seen only in the infrequent culture that contains an inhibitor. In the present example, we have used the tetA gene of plasmid pACYC184 as the modified gene. The system has been validated using inhibitors of protease 3C, and has been used to identify three new inhibitors of the enzyme, active in the micromolar range.

Topics: 3C Viral Proteases; Amino Acid Sequence; Antiporters; Arabinose; Autoanalysis; Bacterial Proteins; Citrinin; Cysteine Endopeptidases; Escherichia coli; Gene Expression; Molecular Sequence Data; Naphthoquinones; Oligopeptides; Plasmids; Protease Inhibitors; Pyrones; Recombinant Fusion Proteins; Tetracycline; Tetracycline Resistance; Viral Proteins

Large actI, III-homologous DNA fragments were isolated from genomic libraries of the strains that produce the benzoisochromanequinone antibiotics kalafungin and nanaomycin A methyl ester, Streptomyces tanashiensis strain Kala and Streptomyces sp. OM-173, respectively. These libraries were prepared in Escherichia coli JM108 by using a novel Streptomyces-E. coli bifunctional cosmid, pKU205, and screened with polyketide synthase genes (actI and III) for actinorhodin biosynthesis from Streptomyces coelicolor A3(2) as probes. The cloned DNA fragments (28 and 42 kb) were analyzed by hybridization with DNA containing actinorhodin biosynthetic genes (actI, II, III, IV, VA, VB, VI and VII). Both fragments hybridized with the actI, III, VA and VI regions, but not with the actII, IV, VB and VII regions. The cloned fragment of S. tanashiensis DNA was analyzed by complementation tests with kalafungin-nonproducing mutants. Seven genes (kalI approximately VII), which correspond to seven steps in kalafungin biosynthesis, were found to be located on a 14 kb continuous DNA fragment. Five of the genes were located on the regions homologous to the genes for actinorhodin biosynthesis, but the other two genes were not. Although kalafungin is an intermediate or shunt product in actinorhodin biosynthesis in S. coelicolor A3(2), the genes for kalafungin biosynthesis in S. tanashiensis are not identical with those in S. coelicolor A3(2).

Topics: Anthraquinones; Antifungal Agents; Cloning, Molecular; DNA; Genes, Bacterial; Genomic Library; Hybridization, Genetic; Naphthoquinones; Streptomyces

An alkalophilic actinomycete, strain OPC-553 regarded as Nocardiopsis dassonvillei subsp. prasina, produced the cytotoxic substance, TS-1, which showed a marked inhibitory activity against L5178Y mouse leukemic cell in vitro. The cytotoxicity of TS-1 on this cell was very strong and its ID50 was 0.018 micrograms/ml. Through direct comparison of its spectral data with those of an authentic sample, TS-1 was identified as the antifungal antibiotic, kalafungin, already isolated from the culture broth of Streptomyces tanashiensis. However, the isolation of kalafungin from an alkalophilic actinomycete and its cytotoxicity are reported for the first time in this paper.

Topics: Actinomycetales; Animals; Antibiotics, Antineoplastic; Antifungal Agents; Cell Survival; Drug Screening Assays, Antitumor; Leukemia L5178; Mice; Naphthoquinones; Tumor Cells, Cultured

The site of regulation of nanaomycin biosynthesis by inorganic phosphate was studied with washed cells previously grown in a chemically defined medium containing a high- or low-phosphate concentration. The former mycelia produced only about one-tenth the amount of nanaomycin A from acetate as did the latter mycelia. On the other hand, the bioconversions of nanaomycin D to A and nanaomycin A to E were only slightly affected. It is suggested that the site of regulation of nanaomycin biosynthesis by inorganic phosphate lies within steps between acetate and nanaomycin D.

Topics: Anti-Bacterial Agents; Chemical Phenomena; Chemistry; Culture Media; Hydrogen-Ion Concentration; Naphthoquinones; Phosphates; Streptomyces

The mutants of Streptomyces tanashiensis strain Kala, which were specifically blocked in the synthesis of the benzoisochromanequinone antibiotic kalafungin, were isolated and classified into seven phenotypic classes on the basis of the antibiotic activity and cosynthetic properties. The polarity of cosynthetic reactions and the production of kalafungin by a converter strain showed that the seven mutant classes could be arranged in the most probable linear sequence of biosynthetic blocks. Since kalafungin, which closely resembles the undimerized form of actinorhodin, was accumulated in one of the biosynthetically blocked mutants of the actinorhodin-producing Streptomyces coelicolor A3(2), the cosynthesis between kalafungin-nonproducing mutants of S. tanashiensis and actinorhodin-nonproducing mutants of S. coelicolor was performed. The results of these experiments showed that the early steps in kalafungin biosynthesis in S. tanashiensis and actinorhodin biosynthesis in S. coelicolor were similar, but the entire biosynthetic pathway of kalafungin in these two streptomycetes was not identical.

Topics: Antifungal Agents; Chromatography, Thin Layer; Culture Media; Molecular Structure; Mutation; Naphthoquinones; Streptomyces

Topics: Antifungal Agents; Chromatography, Thin Layer; Fermentation; Molecular Structure; Naphthoquinones; Nocardia; Stereoisomerism

From two types of class V act mutants of Streptomyces coelicolor two monomeric precursors of actinorhodin have been isolated and their structures determined. One is the known antibiotic kalafungin and the other a new compound. Their relationship to actinorhodin biosynthesis is discussed.

Topics: Anthraquinones; Anti-Bacterial Agents; Chemical Phenomena; Chemistry; Circular Dichroism; Fermentation; Magnetic Resonance Spectroscopy; Naphthoquinones; Streptomyces

A new complex of anti-Gram-positive antibiotics was produced by the fermentation of Actinoplanes arizonaensis sp. nov. The antibiotics were recovered from the fermentation broth with Amberlite XAD-7 resin and from the mycelium by acetone lysis. UV, IR, MS and NMR spectral studies characterized these compounds as kalafungin-type antibiotics. They differ from other known members by an unusual oxidation pattern on the aromatic ring. They vary from one another by the degree and position of O-methylation on the aromatic ring and in the aliphatic portion of the molecules. The structure of one component was confirmed by X-ray diffraction analysis.

Topics: Actinomycetales; Anti-Bacterial Agents; Magnetic Resonance Spectroscopy; Naphthoquinones; Optical Rotation; Structure-Activity Relationship

Topics: Animals; Anti-Bacterial Agents; Fermentation; Male; Naphthoquinones; Platelet Aggregation; Rats; Rats, Inbred Strains; Structure-Activity Relationship

Topics: Animals; Anthelmintics; Ascaris; Carbohydrates; Fructose; Glucose; Naphthoquinones; Sucrose

Topics: Antifungal Agents; Naphthoquinones; Stereoisomerism

Nanaomycin (NNM) D had a higher growth inhibitory activity than NNM-A against a Gram-negative marine bacterium, Vibrio alginolyticus. These quinone antibiotics were reduced by the respiratory chain-linked flavin dehydrogenase of the organism and the reduced forms of NNMs were quickly autoxidized by molecular oxygen to produce superoxide radicals (O2-). NNM-D was more effective than NNM-A both in the induction of KCN-insensitive oxygen consumption with the intact cells and in the production of O2- by the redox cycling. The growth inhibitory activities of NNM-D and A were partly reduced by raising the superoxide dismutase level of the cells. Thus, the ability to produce O2- at the cell membrane was correlated to the antibacterial activities of NNM-D and A.

Topics: Anti-Bacterial Agents; Electron Transport; NADH Dehydrogenase; Naphthoquinones; Oxygen Consumption; Superoxide Dismutase; Superoxides; Vibrio; Water Microbiology

Nanaomycin D reductase which is involved in the biosynthesis of the antifungal antibiotic nanaomycin catalyzes the formation of nanaomycin A from nanaomycin D in the presence of NADH under anaerobic conditions. On the other hand, under aerobic conditions NADH is consumed and nanaomycin A formation is markedly reduced. These findings suggest that nanaomycin A synthesis is not due to the direct reduction of the 5-membered lactone ring of nanaomycin D. Reduction of various quinones by the enzyme was examined. It was found that nanaomycin A is converted to its hydroquinone derivative in the presence of NADH under anaerobic conditions, whereas NADH consumption alone is observed under aerobic conditions. When p-benzoquinone, 1,4-naphthoquinone or menadione is used instead of nanaomycin D, NADH is also consumed. These results indicate that: (1) these compounds act as electron acceptors, (2) O2 functions as final electron acceptor under aerobic conditions, and (3) nanaomycin D reductase is, in fact, an NADH dehydrogenase (quinone). Changes in the UV-absorption spectrum of a reaction mixture containing nanaomycin D and NADH indicate that a hydroquinone derivative is formed as an intermediate during nanaomycin A formation. Similar results were obtained when nanaomycin D is reduced chemically with NaBH4 or Zn powder. It was concluded that nanaomycin D is converted to a hydroquinone derivative and that nanaomycin A is then formed nonenzymatically through intramolecular electron transfer.

Topics: Aerobiosis; Anaerobiosis; Antifungal Agents; Biotransformation; Chemical Phenomena; Chemistry; Hydroquinones; NADH Dehydrogenase; Naphthoquinones; Oxidation-Reduction; Oxidoreductases

Nanaomycin D reductase, catalyzing the conversion of nanaomycin D to nanaomycin A, which is the first step in the biosynthetic sequence (D leads to A leads to E leads to B) in Streptomyces rosa var. notoensis, was purified from the crude extract of the strain by ammonium sulfate fractionation and column chromatography on DEAE-cellulose, Sephadex G-100 and hydroxyapatite to give an electrophoretically homogeneous preparation. The enzyme was found to be a flavoprotein which contains FAD as a prosthetic group and has a molecular weight of 68,000 daltons. It catalyzed the reductive transformation of nanaomycin D to nanaomycin A in the presence of NADH under anaerobic conditions. The Km values were 250 microM for nanaomycin D and 62 microM for NADH. The enzyme was inhibited by 1 mM Cu2+ ion and by NADH at concentrations over 50 microM. The optimal pH was 5.0 and the optimal temperature was 37 degrees C. Several benzoisochromane-quinone antibiotics other than nanaomycin D, kalafungin (enantiomer of nanaomycin D), griseucin A and frenolicin B were converted to the corresponding reduced products by the enzyme. However, granaticin and 4 alpha, 10 alpha-epoxynanaomycin D were not converted.

Topics: Anti-Bacterial Agents; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; NAD; Naphthoquinones; Oxidoreductases; Spectrum Analysis; Streptomyces; Substrate Specificity; Temperature

The biosynthetic relationship of the nanaomycins produced by Streptomyces rosa var. notoensis OS-3966 was studied by means of a bioconversion method using the antibiotic cerulenin, a specific inhibitor of fatty acid and polyketide biosyntheses. Nanaomycin D was considered to be the first component produced from the hypothetical intermediate "polyketide". It is proposed that the biosynthesis sequence for the nanaomycin is: nanaomycin D leads to nanaomycin A leads to nanaomycin E leads to nanaomycin B. Nanaomycin B can be converted to nanaomycin A by non-enzymatic dehydration; however, nanaomycin A is rapidly bioconverted to nanaomycin E, which is the major component synthesized by the nanaomycin-producing strain.

Topics: Antifungal Agents; Biotransformation; Cerulenin; Fatty Acids; Naphthoquinones; Streptomyces; Time Factors