naphthoquinones and medermycin

Medermycin, produced by Streptomyces species, represents a family of antibiotics with significant activity against Gram-positive pathogens. The biosynthesis of this family of natural products has been studied, and new skeletons related to medermycin have rarely been reported until recently. Herein, we report eight chimeric medermycin-type natural products with unusual polycyclic skeletons. The formation of these compounds features some key nonenzymatic steps, which inspired us to construct complex polycyclic skeletons via three efficient one-step reactions under mild conditions. This strategy was further developed to efficiently synthesize analogues for biological activity studies. The synthetic compounds, chimedermycins L and M, and sekgranaticin B, show potent antibacterial activity against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and methicillin-resistant Staphylococcus epidermidis. This work paves the way for understanding the nonenzymatic formation of complex natural products and using it to synthesize natural product derivatives.

Topics: Anti-Bacterial Agents; Biological Products; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Naphthoquinones; Skeleton

Topics: Bacterial Proteins; Biosynthetic Pathways; Genes, Bacterial; Glycosyltransferases; Models, Molecular; Multigene Family; Naphthoquinones; Phylogeny; Streptomyces

Med-ORF10, a single-domain protein with unknown function encoded by a gene located in a gene cluster responsible for the biosynthesis of a novel antitumour antibiotic medermycin, shares high homology to a group of small proteins widely distributed in many aromatic polyketide antibiotic pathways. This group of proteins contain a nuclear transport factor-2 (NTF-2) domain and appear to undergo an evolutionary divergence in their functions. Gene knockout and interspecies complementation suggested that Med-ORF10 plays a regulatory role in medermycin biosynthetic pathway. Overexpression of med-ORF10 in its wild-type strain led to significant increase of medermycin production. It was also shown by qRT-PCR and Western blot that Med-ORF10 controls the expression of genes encoding tailoring enzymes involved in medermycin biosynthesis. Transcriptome analysis and qRT-PCR revealed that Med-ORF10 has pleiotropic effects on more targets. However, there is no similar conserved domain available in Med-ORF10 compared to those of mechanistically known regulatory proteins; meanwhile, no direct interaction between Med-ORF10 and its target promoter DNA was detected via gel shift assay. All these studies suggest that Med-ORF10 regulates medermycin biosynthesis probably via an indirect mode.

Topics: Antineoplastic Agents; Gene Expression Regulation, Bacterial; Multigene Family; Naphthoquinones; Streptomyces

Lactoquinomycin C (

Topics: Cell Line, Tumor; Cytotoxins; Epoxy Compounds; HCT116 Cells; Humans; Inhibitory Concentration 50; Lactones; Naphthoquinones; PC-3 Cells; Streptomyces; Structure-Activity Relationship

A new medermycin derivative, MDN-0171 (1), and two known structurally related compounds, medermycin (2) and antibiotic G15-F (3) were isolated from the acetone extract of culture broths of the marine-derived Streptomyces albolongus strain CA-186053. Their structures were determined using a combination of spectroscopic techniques, including 1D and 2D NMR and electrospray-time of flight mass spectrometry (ESI-TOF MS). Compounds 2 and 3 accounted for the antimicrobial activity (against methicillin-resistant Staphylococcus aureus and Escherichia coli) previously detected in the crude extract of this actinomycete.

Topics: Actinobacteria; Anti-Bacterial Agents; Escherichia coli; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Molecular Structure; Naphthoquinones; Spectrum Analysis; Streptomyces

A new medermycin analog (

Topics: Actinobacteria; Anti-Bacterial Agents; Cell Line, Tumor; Circular Dichroism; Humans; Magnetic Resonance Spectroscopy; Marine Biology; Naphthoquinones

Four new medermycin-type naphthoquinones, strepoxepinmycins A-D (1-4), and one known compound, medermycin (5), were identified from Streptomyces sp. XMA39. Their structures were elucidated by analysis of HRESIMS, 1D and 2D NMR spectroscopic data, and ECD calculations. Among these compounds, strepoxepinmycin A (1) represents a rare 5,10-oxepindione ring system typically formed by a Baeyer-Villiger oxidation, and strepoxepinmycin B (2) is an isolation artifact derived from 1. Bioactivity evaluations of these compounds showed that compounds 3 and 4 exhibited cytotoxicity against HCT-116 and PC-3 cancer cell lines and 4 exhibited moderate inhibition of ROCK 2 protein kinase. In addition, all of the new compounds showed antibacterial activity against Escherichia coli and methicillin-resistant Staphylococcus aureus and antifungal activity against Candida albicans.

Topics: Anti-Infective Agents; Antineoplastic Agents; Cell Line, Tumor; Humans; Naphthoquinones; Streptomyces; Water Microbiology

The C-arylglycosides are available in enantiomerically pure form via the Dötz benzannulation reaction between Fischer alkenyl chromium carbene complexes and alkynes; it also could be converted to a precursor of medermycin by O-carbamate directed ipso bromination and nitrile substitution in good overall yields.

Topics: Molecular Structure; Naphthoquinones; Stereoisomerism

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

Medermycin, a polyketide antibiotic, possesses strong bioactivity against a variety of tumors through a novel mechanism and is structurally featured with a pyran ring containing two chiral centers (3S and 15R). By far the biosynthetic origin of such enantiomerical conformations still remains obscure. In the present study, we reported the functional characterization of a proposed ketoreductase Med-ORF12 encoded by medermycin biosynthetic cluster and revealed its involvement in the stereochemical control at C3 center of medermycin. Firstly, bioinformatics analysis of Med-ORF12 suggested that it belongs to a group of stereospecific ketoreductases. Next, a Med-ORF12-deficient mutant was obtained and LC/MS measurements demonstrated that medermycin production was completely abolished in this mutant. Meanwhile, it was found that two shunt products were accumulated at the absence of Med-ORF12. Finally, the reintroduction of Med-ORF12 into this mutant could restore the production of medermycin. In a conclusion, these data supported that Med-ORF12 is essential for the biosynthesis of medermycin and performs its role as a stereospecifc ketoreductase in the tailoring steps of medermycin biosynthetic pathway.

Topics: Alcohol Oxidoreductases; Amino Acid Sequence; Anthraquinones; Antibiotics, Antineoplastic; Bacterial Proteins; Biosynthetic Pathways; Computational Biology; Genes, Bacterial; Genetic Complementation Test; Metabolomics; Molecular Sequence Data; Multigene Family; Mutation; Naphthoquinones; Open Reading Frames; Phylogeny; Pyrans; Sequence Alignment; Stereoisomerism; Streptomyces

Medermycin, as a prominent member of benzoisochromanequinones, possesses strong antitumor activity and is biosynthesized under the control of a 29-ORF-containing biosynthetic gene cluster. Most of ORFs in this gene cluster have not been characterized, including a small protein encoding gene med-ORF10, proposed to play a regulatory role in biosynthesis of medermycin in an unknown mode. In this study, we reported the expression, protein preparation, crystallization and preliminary X-ray diffraction analyses of Med-ORF10 of the wild type Streptomyces strain. Firstly, we cloned and overexpressed med-ORF10 in Escherichia coli and purified the protein with 98% purity and 3 mg/L yield. Then, we crystallized the protein at concentration of 20 mg/mL in condition 22% PEG 3350, 0.2 M magnesium formate and collected the data at 1.78 Å resolution. Finally, we detected the expression of Med-ORF10 in Streptomyces by western blotting. In conclusion, this study confirmed the expression of Med-ORF10 protein in the wild-type strain of Streptomyces AM-7161 and collected the X-ray diffraction data of Med-ORF10 crystal at 1.78 Å resolution. These studies provide evidences for the functional Med-ORF10 protein in Streptomyces strains and facilitate our further investigation.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Cloning, Molecular; Crystallization; Escherichia coli; Metabolic Networks and Pathways; Molecular Sequence Data; Multigene Family; Naphthoquinones; Recombinant Proteins; Sequence Alignment; Streptomyces; X-Ray Diffraction

Medermycin, an aromatic polyketide antibiotic produced by streptomyces, possesses a stereochemical-pyran-ring lactone critical for its strong anticancer activity. The med-ORF12 located in the biosynthetic gene cluster of medermycin encodes a stereochemical ketoreductase. Based on many indirect data, we proposed it to be involved in the enantioselective reduction at C-3 in the formation of the pyran ring of medermycin. The direct genetic evidence about the function of med-ORF12 in the medermycin-producing strain has yet to be obtained. Enzymatic features, expression and regulation pattern of Med-ORF12 in the medermycin-producer still remain obscure.. The present study aimed to investigate the expression profiles of med-ORF12 and relationship between Med-ORF12 and medermycin accumulation in medermycin-producers using prokaryotic expression, protein purification, polyclonal antiserum preparation, western blot.. First, we established a prokaryotic expression system of med-ORF12 using a pET vector and optimized the induction conditions to accumulate the soluble Med-ORF12. Subsequently, we acquired the polyclonal antiserum against Med-ORF12 by immunizing the New Zealand rabbit with the purified protein. Finally, we detected the expression pattern of med-ORF12 in the medermycin producers with the obtained polyclonal antiserum, and found that med-ORF12 could express with a fairly high amount during the late stationary phase of the medermycin-producers, consistent with the accumulation of medermycin as a secondary metabolite.. These data indicated that Med-ORF12 expressing efficiently in the secondary metabolism could be involved in the biosynthesis of medermycin in the medermycin-producers.

Topics: Animals; Antibiotics, Antineoplastic; Genes, Bacterial; Immune Sera; Multigene Family; Naphthoquinones; Open Reading Frames; Plasmids; Rabbits

Streptomyces sp. AM-7161 is a producer of an aromatic polyketide medermycin with strong antibacterial and antitumor activity. It has been inefficient to perform genetic manipulations in this strain, which heavily hinders the genetic analysis of some interesting problems concerning tailoring modifications in medermycin biosynthesis. Here, condition optimizations of sporulation and mycelium growth of this strain as well as protoplast preparation and regeneration were conducted systematically. Based on these results, protoplast transformation system was established, and two types of foreign plasmids (integrative and auto-replicating) were efficiently introduced into this strain. Additionally, a conjugation system to mediate plasmid transfer between Escherichia coli and Streptomyces sp. AM-7161 was also optimized and established. These results provide the practical procedures to perform more convenient genetic analysis of medermycin biosynthesis in this strain.

Topics: Anti-Bacterial Agents; Conjugation, Genetic; Escherichia coli; Gene Transfer, Horizontal; Genetic Engineering; Genetic Vectors; Genetics, Microbial; Molecular Biology; Naphthoquinones; Plasmids; Protoplasts; Streptomyces; Transformation, Bacterial

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

The serine/threonine kinase AKT/PKB plays a critical role in cancer and represents a rational target for therapy. Although efforts in targeting AKT pathway have accelerated in recent years, relatively few small molecule inhibitors of AKT have been reported. The development of selective AKT inhibitors is further challenged by the extensive conservation of the ATP-binding sites of the AGC kinase family. In this report, we have conducted a high-throughput screen for inhibitors of activated AKT1. We have identified lactoquinomycin as a potent inhibitor of AKT kinases (AKT1 IC(50), 0.149 +/- 0.045 micromol/L). Biochemical studies implicated a novel irreversible interaction of the inhibitor and AKT involving a critical cysteine residue(s). To examine the role of conserved cysteines in the activation loop (T-loop), we studied mutant AKT1 harboring C296A, C310A, and C296A/C310A. Whereas the ATP-pocket inhibitor, staurosporine, indiscriminately targeted the wild-type and all three mutant-enzymes, the inhibition by lactoquinomycin was drastically diminished in the single mutants C296A and C310A, and completely abolished in the double mutant C296A/C310A. These data strongly implicate the binding of lactoquinomycin to the T-loop cysteines as critical for abrogation of catalysis, and define an unprecedented mechanism of AKT inhibition by a small molecule. Lactoquinomycin inhibited cellular AKT substrate phosphorylation induced by growth factor, loss of PTEN, and myristoylated AKT. The inhibition was substantially attenuated by coexpression of C296A/C310A. Moreover, lactoquinomycin reduced cellular mammalian target of rapamycin signaling and cap-dependent mRNA translation initiation. Our results highlight T-loop targeting as a new strategy for the generation of selective AKT inhibitors.

Topics: Adenosine Triphosphate; Allosteric Regulation; Animals; Catalysis; Cell Line, Tumor; Cysteine; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Humans; Kinetics; Naphthoquinones; Phosphorylation; Protein Biosynthesis; Protein Kinases; Proto-Oncogene Proteins c-akt; Rats; RNA Caps; Structure-Activity Relationship; Substrate Specificity; Time Factors; TOR Serine-Threonine Kinases

Medermycin shows the same trans (3S,15R) configuration as actinorhodin in the pyran ring crucial for its bioactivity. One medermycin biosynthetic gene, med-ORF12, is assumed to be involved in the stereochemical control at C-3. Functional complementation suggested that it plays a similar role as actVI-ORF1 previously proved to determine the stereospecificity at C-3 in actinorhodin biosynthesis. Co-expression of med-ORF12 with actinorhodin early biosynthetic genes further demonstrated that med-ORF12 encodes a ketoreductase responsible for the enantioselective reduction at C-3 in the formation of the pyran ring.

Topics: Alcohol Oxidoreductases; Molecular Structure; Multigene Family; Naphthoquinones; Open Reading Frames; Stereoisomerism; Streptomyces

Medermycin is a Streptomyces aromatic C-glycoside antibiotic classified in the benzoisochromanequinones (BIQs), which presents several interesting biosynthetic problems concerning polyketide synthase (PKS), post-PKS tailoring and deoxysugar pathways. The biosynthetic gene cluster for medermycin (the med cluster) was cloned from Streptomyces sp. AM-7161. Completeness of the clone was proved by the heterologous expression of a cosmid carrying the entire med cluster in Streptomyces coelicolor CH999 to produce medermycin. The DNA sequence of the cosmid (36 202 bp) revealed 34 complete ORFs, with an incomplete ORF at either end. Functional assignment of the deduced products was made for PKS and biosynthetically related enzymes, tailoring steps including strereochemical control, oxidation, angolosamine pathway, C-glycosylation, and regulation. The med cluster was estimated to be about 30 kb long, covering 29 ORFs. An unusual characteristic of the cluster is the disconnected organization of the minimal PKS genes: med-ORF23 encoding the acyl carrier protein is 20 kb apart from med-ORF1 and med-ORF2 for the two ketosynthase components. Secondly, the six genes (med-ORF14, 15, 16, 17, 18 and 20) for the biosynthesis of the deoxysugar, angolosamine, are all contiguous. Finally, the finding of a glycosyltransferase gene, med-ORF8, suggests a possible involvement of conventional C-glycosylation in medermycin biosynthesis. Comparison among the three complete BIQ gene clusters - med and those for actinorhodin (act) and granaticin (gra) - revealed some common genes whose deduced functions are unavailable from database searches (the 'unknowns'). An example is med-ORF5, a homologue of actVI-ORF3 and gra-ORF18, which was highlighted by a recent proteomic analysis of S. coelicolor A3(2).

Topics: Amino Acid Sequence; Anthraquinones; Anti-Bacterial Agents; Base Sequence; Biological Transport, Active; Cloning, Molecular; DNA, Bacterial; Genes, Bacterial; Genes, Regulator; Glycosylation; Hydroxylation; Molecular Sequence Data; Multienzyme Complexes; Multigene Family; Naphthoquinones; Open Reading Frames; Oxidation-Reduction; Phylogeny; Quinones; Sequence Homology, Amino Acid; Stereoisomerism; Streptomyces

The synthesis of an isomeric mixture of 4-O-acetyl-3-azido-2,3,6-trideoxy-beta-D-arabino-hexopyranosyl analogues 6 of the C-glycosylpyranonaphthoquinone antibiotic medermycin is described. The key 3-acetyl-6-(4-O-acetyl-3-azido-2,3,6-trideoxy-beta-D-arabino- hexopyranosyl)-5-methoxy-1,4-naphthoquinone 8 was prepared via Stille coupling of 6-(3-azido-2,3,6-trideoxy-beta-D-arabino-hexopyranosyl)-3-bromo-1,4- naphthoquinone 17 with (alpha-ethoxyvinyl)tributyl-stannane followed by hydrolysis and oxidation of the resultant hydroquinone 18. Bromonaphthoquinone 17 in turn was afforded by oxidative demethylation of 6-(4-O-acetyl-3-azido-2,3,6-trideoxy-beta-D-arabino-hexopyranosyl)-3- bromo-1,4,5-trimethoxynaphthalene 16 formed by regioselective bromination of 6-(4-acetyl-3-azido-2,3,6-trideoxy- beta-D-arabino-hexopyranosyl)-1,4,5-trimethoxynaphthalene 10. This latter naphthalene 10 was prepared via direct C-glycosylation of naphthol 12 with glycosyl donor 11 using BF3.Et2O in acetonitrile. The regioselectivity of the bromination of naphthalene 10 was independently determined by reductive monomethylation of the 6-(4-O-acetyl-3-azido-2,3,6-trideoxy-beta-D-arabino- hexopyranosyl)-5-methoxy-1,4-naphthoquinone 22 to naphthol 23 followed by selective ortho bromination to bromide 24 and methylation to 16. Attempts to effect acetylation of 6-(4-O-acetyl-3-azido-2,3,6-trideoxy-beta-D-arabino- hexopyranosyl)-3-bromo-1,4,5-trimethoxynaphthalene 16 and 3-bromo-6-(3-dimethylamino-2,3,6-trideoxy-beta-D-arabino- hexopyranosyl)-1,4,5-trimethoxynaphthalene 26 via Stille coupling with (alpha-ethoxyvinyl)tributylstannane were low yielding thereby establishing the necessity to use an azido group as a latent dimethylamino group and a more electrophilic bromonaphthoquinone as the coupling partner for the Stille reaction. Addition of 2-trimethylsilyloxyfuran 9 to 3-acetyl-6-(4-O-acetyl-3-azido-2,3,6-trideoxy-beta-D-arabino-hexopyranosyl)- 5-methoxy-1,4-naphthoquinone 8 afforded the furofuran adducts 7 and 19 as an inseparable mixture of diastereomers. Oxidative rearrangement of this diastereomeric mixture using ceric ammonium nitrate afforded the inseparable diastereomeric furonaphthopyrans 6 and 20.

Topics: Anti-Bacterial Agents; Arabinose; Azides; Furans; Naphthoquinones; Stereoisomerism

[reaction: see text] On the basis of chemical and spectral data, the structure of the medermycin/lactoquinomycin A has been revised, which has also led to the revision of related C-glycosylated naphthoquinone antibiotics such as lactoquinomycin B, menoxymycins A and B, G15-F, and G15-G.

Topics: Anti-Bacterial Agents; Glycosylation; Magnetic Resonance Spectroscopy; Molecular Structure; Naphthoquinones

[structure: see text] Herein we report a significant body of spectroscopic data that supports the originally proposed structure of medermycin/lactoquinomycin A. In addition, we demonstrate that these data are inconsistent with the revised structure reported recently in the literature.

Topics: Anti-Bacterial Agents; Molecular Structure; Naphthoquinones; Nuclear Magnetic Resonance, Biomolecular

Topics: Anti-Bacterial Agents; Antibiotics, Antineoplastic; Microbial Sensitivity Tests; Naphthoquinones; Stereoisomerism; Structure-Activity Relationship

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

Topics: Animals; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Bacteria; Chemical Phenomena; Chemistry; Lethal Dose 50; Mice; Naphthoquinones; Sarcoma, Yoshida; Solubility; Streptomyces