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1.
Tetrahedron ; 70(27-28): 4156-4164, 2014 Jul 08.
Article in English | MEDLINE | ID: mdl-25045187

ABSTRACT

The lomaiviticins are a family of cytotoxic marine natural products that have captured the attention of both synthetic and biological chemists due to their intricate molecular scaffolds and potent biological activities. Here we describe the identification of the gene cluster responsible for lomaiviticin biosynthesis in Salinispora pacifica strains DPJ-0016 and DPJ-0019 using a combination of molecular approaches and genome sequencing. The link between the lom gene cluster and lomaiviticin production was confirmed using bacterial genetics, and subsequent analysis and annotation of this cluster revealed the biosynthetic basis for the core polyketide scaffold. Additionally, we have used comparative genomics to identify candidate enzymes for several unusual tailoring events, including diazo formation and oxidative dimerization. These findings will allow further elucidation of the biosynthetic logic of lomaiviticin assembly and provide useful molecular tools for application in biocatalysis and synthetic biology.

2.
Antimicrob Agents Chemother ; 58(2): 1092-9, 2014.
Article in English | MEDLINE | ID: mdl-24295976

ABSTRACT

To date, most antibiotics have primarily been developed to target bacteria in the planktonic state. However, biofilm formation allows bacteria to develop tolerance to antibiotics and provides a mechanism to evade innate immune systems. Therefore, there is a significant need to identify small molecules to prevent biofilm formation and, more importantly, to disperse or eradicate preattached biofilms, which are a major source of bacterial persistence in nosocomial infections. We now present a modular high-throughput 384-well image-based screening platform to identify Pseudomonas aeruginosa biofilm inhibitors and dispersal agents. Biofilm coverage measurements were accomplished using non-z-stack epifluorescence microscopy to image a constitutively expressing green fluorescent protein (GFP)-tagged strain of P. aeruginosa and quantified using an automated image analysis script. Using the redox-sensitive dye XTT, bacterial cellular metabolic activity was measured in conjunction with biofilm coverage to differentiate between classical antibiotics and nonantibiotic biofilm inhibitors/dispersers. By measuring biofilm coverage and cellular activity, this screen identifies compounds that eradicate biofilms through mechanisms that are disparate from traditional antibiotic-mediated biofilm clearance. Screening of 312 natural-product prefractions identified the cyclic depsipeptide natural products skyllamycins B and C as nonantibiotic biofilm inhibitors with 50% effective concentrations (EC50s) of 30 and 60 µM, respectively. Codosing experiments of skyllamycin B and azithromycin, an antibiotic unable to clear preattached biofilms, demonstrated that, in combination, these compounds were able to eliminate surface-associated biofilms and depress cellular metabolic activity. The skyllamycins represent the first known class of cyclic depsipeptide biofilm inhibitors/dispersers.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Adhesion/drug effects , Biofilms/drug effects , Depsipeptides/pharmacology , High-Throughput Screening Assays , Peptides, Cyclic/pharmacology , Pseudomonas aeruginosa/drug effects , Azithromycin/pharmacology , Biofilms/growth & development , Dose-Response Relationship, Drug , Drug Combinations , Drug Synergism , Fluorescent Dyes , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Molecular Imaging , Pattern Recognition, Automated , Pseudomonas aeruginosa/growth & development , Pseudomonas aeruginosa/ultrastructure
3.
Bioorg Med Chem ; 17(6): 2154-61, 2009 Mar 15.
Article in English | MEDLINE | ID: mdl-19028101

ABSTRACT

Two natural products, diazepinomicin (1) and dioxapyrrolomycin (2), containing stable isotopic labels of (15)N or deuterium, were used to demonstrate the utility of Fourier transform ion cyclotron resonance mass spectrometry for probing natural product biosynthetic pathways. The isotopic fine structures of significant ions were resolved and subsequently assigned elemental compositions on the basis of highly accurate mass measurements. In most instances the mass measurement accuracy is less than one part per million (ppm), which typically makes the identification of stable-isotope labeling unambiguous. In the case of the mono-(15)N-labeled diazepinomicin (1) derived from labeled tryptophan, tandem mass spectrometry located this (15)N label at the non-amide nitrogen. Through the use of exceptionally high mass resolving power of over 125,000, the isotopic fine structure of the molecular ion cluster of 1 was revealed. Separation of the (15)N(2) peak from the isobaric (13)C(15)N peak, both having similar abundances, demonstrated the presence of a minor amount of doubly (15)N-labeled diazepinomicin (1). Tandem mass spectrometry amplified this isotopic fine structure (Deltam=6.32 mDa) from mDa to 1 Da scale thereby allowing more detailed scrutiny of labeling content and location. Tandem mass spectrometry was also used to assign the location of deuterium labeling in two deuterium-labeled diazepinomicin (1) samples. In one case three deuterium atoms were incorporated into the dibenzodiazepine core; while in the other a mono-D label was mainly incorporated into the farnesyl side chain. The specificity of (15)N-labeling in dioxapyrrolomycin (2) and the proportion of the (15)N-label contained in the nitro group were determined from the measurement of the relative abundance of the (14)NO(2)(1-) and (15)NO(2)(1-) fragment ions.


Subject(s)
Biological Products/biosynthesis , Cyclotrons , Dibenzazepines/metabolism , Tandem Mass Spectrometry/methods , Deuterium , Fermentation , Fourier Analysis , Pyrroles/metabolism
4.
J Nat Prod ; 71(11): 1923-6, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18986197

ABSTRACT

Feasible modes of introducing the nitro group into pyrrolomycin antibiotics were investigated based on incorporation of (15)N-labeled arginine and proline into dioxapyrrolomycin, produced by the actinomycete culture LL-F42248. Biosynthesis of nitrated pyrrolomycins was unaffected by the presence of nitric oxide synthase (NOS) inhibitors. The culture was able to grow in nitrogen-free (minimal) media and produce nitrated secondary metabolites. These results indicate that LL-F42248 is capable of fixing nitrogen.


Subject(s)
Actinomyces/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitrogen Fixation/physiology , Arginine/metabolism , Arginine/pharmacology , Atmosphere/chemistry , Molecular Structure , Nitrogen/metabolism , Pyrroles/chemistry , Pyrroles/pharmacology , omega-N-Methylarginine/metabolism , omega-N-Methylarginine/pharmacology
5.
J Nat Prod ; 71(9): 1585-90, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18722414

ABSTRACT

The novel microbial metabolite diazepinomicin/ECO-4601 (1) has a unique tricyclic dibenzodiazepinone core, which was unprecedented among microbial metabolites. Labeled feeding experiments indicated that the carbocyclic ring and the ring nitrogen of tryptophan could be incorporated via degradation to the 3-hydroxyanthranilic acid, forming ring A and the nonamide nitrogen of 1. Genomic analysis of the biosynthetic locus indicated that the farnesyl side chain was mevalonate derived, the 3-hydroxyanthranilic acid moiety could be formed directly from chorismate, and the third ring was constructed via 3-amino-5-hydroxybenzoic acid. Successful incorporation of 4,6-D2-3-hydroxyanthranilic acid into ring A of 1 via feeding experiments supports the genetic analysis and the allocation of the locus to this biosynthesis. These studies highlight the enzymatic complexity needed to produce this structural type, which is rare in nature.


Subject(s)
Alkaloids/chemical synthesis , Dibenzazepines/chemical synthesis , Micromonospora/chemistry , Alkaloids/chemistry , Cyclization , Dibenzazepines/chemistry , Micromonospora/genetics , Molecular Structure
6.
Mol Cancer Ther ; 6(11): 3028-38, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17989320

ABSTRACT

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.


Subject(s)
Cysteine/metabolism , Enzyme Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Adenosine Triphosphate/pharmacology , Allosteric Regulation/drug effects , Animals , Catalysis/drug effects , Cell Line, Tumor , Down-Regulation/drug effects , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Humans , Kinetics , Naphthoquinones/chemistry , Naphthoquinones/pharmacology , Phosphorylation/drug effects , Protein Biosynthesis/drug effects , Protein Kinases/metabolism , RNA Caps/metabolism , Rats , Structure-Activity Relationship , Substrate Specificity/drug effects , TOR Serine-Threonine Kinases , Time Factors
7.
J Nat Prod ; 69(5): 736-41, 2006 May.
Article in English | MEDLINE | ID: mdl-16724832

ABSTRACT

A group of new 10mer linear peptides, designated culicinins A-D (1-4), was isolated from the fermentation broth of the entomopathogenic fungus Culicinomyces clavisporus, strain LL-12I252. The structures of the culicinins were determined by a combination of 2D NMR and MS analysis. The major compound, culicinin D (4), exhibited selective inhibitory activity against PTEN-negative MDA468 tumor cells. Studies on the 3D structure of 4 using NOE data and computer modeling revealed a dominant conformation of the right-handed helix.


Subject(s)
Antineoplastic Agents/isolation & purification , Hypocreales/chemistry , Oligopeptides/isolation & purification , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Drug Screening Assays, Antitumor , Humans , Models, Chemical , Molecular Structure , New York , Nuclear Magnetic Resonance, Biomolecular , Oligopeptides/chemistry , Oligopeptides/pharmacology , Peptaibols , Tumor Cells, Cultured
8.
J Nat Prod ; 69(1): 29-33, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16441063

ABSTRACT

Streptomyces fumanus, intramurally coded as culture LL-F42248, produces a series of pyrrolomycins including dioxapyrrolomycin (1) as the principal component. Our biosynthetic studies revealed that feeding labeled acetate to growing cultures of S. fumanus yielded pyrrolomycins labeled in the phenyl ring only. When l-[methyl-13C]methionine was fed, the labeled carbon atom was found in the methoxy group of pyrrolomycins H-J and in the methylenedioxy bridge of dioxapyrrolomycin. A Na15NO3-enriched medium was employed to produce 15N-labeled pyrrolomycins in which both nitrogen atoms were highly enriched, whereas feeding of 15N-labeled l-proline furnished pyrrolomycins labeled in the pyrrole moiety. Thus, S. fumanus elaborates the pyrrolomycin skeleton from proline and a polyketide precursor. Since the organism readily converted 13C- or 15N-labeled pyrrolomycin C, G, or H into the correspondingly labeled dioxapyrrolomycin, these minor pyrrolomycins are actually precursors of the ultimate product, dioxapyrrolomycin.


Subject(s)
Streptomyces/chemistry , Molecular Structure , Nitrates/metabolism , Nuclear Magnetic Resonance, Biomolecular , Pyrroles/chemical synthesis , Pyrroles/chemistry , Pyrroles/isolation & purification , Pyrroles/pharmacology , Streptomyces/metabolism
9.
J Nat Prod ; 68(8): 1262-5, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16124774

ABSTRACT

Five new polyketide metabolites, phaeochromycins A-E (1-5), were isolated from an actinomycete designated Streptomyces phaeochromogenes LL-P018, cultured from a soil sample collected from a riverbank in Westevenger, Germany. Phaeochromycins A and C were found to be weak inhibitors of MAPKAP kinase-2 (IC50 = 39 and 130 microM, respectively). The structures of the compounds were determined by spectroscopic analysis, primarily two-dimensional NMR, and revealed that phaeochromycins A, B, C, and E were octaketides, elaborated from a C4 starter unit, related to shunt products of the actinorhodin pathway, namely, mutactin, dehydromutactin, SEK34b, and BSM1. Phaeochromycin D (4) is an unusual partially cyclized degraded octaketide intermediate.


Subject(s)
Anti-Inflammatory Agents/isolation & purification , Enzyme Inhibitors/isolation & purification , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrones/isolation & purification , Streptomyces/chemistry , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Arthritis, Rheumatoid/drug therapy , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Germany , Humans , Inhibitory Concentration 50 , Intracellular Signaling Peptides and Proteins , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Pyrones/chemistry , Pyrones/pharmacology , Soil Microbiology
10.
J Antibiot (Tokyo) ; 58(4): 271-4, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15981414

ABSTRACT

A new prenylated naphthoquinone antibiotic, fumaquinone (5,7-dihydroxy-2-methoxy-3-methyl-6-(3-methyl-but-2-enyl)[1,4]naphthoquinone) was isolated from cultures of Streptomyces fumanus (LL-F42248). Its chemical structure was determined primarily by NMR spectroscopy. Preliminary feeding experiments indicated the naphthoquinone is of polyketide origin, while the O-methyl and aromatic C-methyl groups are derived from methionine.


Subject(s)
Naphthoquinones/chemistry , Streptomyces/chemistry , Acetates/metabolism , Cyclotrons , Magnetic Resonance Spectroscopy , Mass Spectrometry , Naphthoquinones/metabolism , Protein Prenylation , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform Infrared , Streptomyces/metabolism
11.
Appl Environ Microbiol ; 71(4): 1971-6, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15812028

ABSTRACT

The natural product rapamycin, produced during fermentation by Streptomyces hygroscopicus, is known for its potent antifungal, immunosuppressive, and anticancer activities. During rapamycin biosynthesis, the amino acid l-pipecolate is incorporated into the rapamycin molecule. We investigated the use of precursor-directed biosynthesis to create new rapamycin analogs by substitution of unusual l-pipecolate analogs in place of the normal amino acid. Our results suggest that the l-pipecolate analog (+/-)-nipecotic acid inhibits the biosynthesis of l-pipecolate, thereby limiting the availability of this molecule for rapamycin biosynthesis. We used (+/-)-nipecotic acid in our precursor-directed biosynthesis studies to reduce l-pipecolate availability and thereby enhance the incorporation of other pipecolate analogs into the rapamycin molecule. We describe here the use of this method for production of two new sulfur-containing rapamycin analogs, 20-thiarapamycin and 15-deoxo-19-sulfoxylrapamycin, and report measurement of their binding to FKBP12.


Subject(s)
Gene Expression Regulation, Bacterial , Protein Precursors/metabolism , Sirolimus/analogs & derivatives , Sirolimus/metabolism , Streptomyces/metabolism , Biotechnology/methods , Nipecotic Acids/metabolism , Pipecolic Acids/metabolism , Streptomyces/genetics , Streptomyces/growth & development , Tacrolimus Binding Protein 1A/metabolism
12.
J Nat Prod ; 68(2): 277-9, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15730262

ABSTRACT

Along with dioxapyrrolomycin (1), four new pyrrolomycin antibiotics, namely, pyrrolomycin G (3), pyrrolomycin H (4), pyrrolomycin I (5), and pyrrolomycin J (6), were produced in cultures of Streptomyces fumanus. Apart from dioxapyrrolomycin, pyrrolomycin G and pyrrolomycin H are the only other chiral members of the pyrrolomycin family of antibiotics, and their absolute stereochemistry was deduced to be 13S. Here, we report the isolation, structure elucidation, and antimicrobial activity of these new pyrrolomycins.


Subject(s)
Anti-Bacterial Agents , Pyrroles/isolation & purification , Streptomyces/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/classification , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Microbial Sensitivity Tests , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Pyrroles/chemistry , Pyrroles/pharmacology
13.
Mycologia ; 97(2): 444-53, 2005.
Article in English | MEDLINE | ID: mdl-16396352

ABSTRACT

Penicillium dravuni is a new monoverticillate, sclerotium-forming species that was isolated from the alga Dictyosphaeria versluyii collected in Dravuni, Fiji. This species morphologically is similar to P. turbatum in the P. turbatum subseries of the P. thomii series of the Monoverticillata. The nuclear ribosomal internal transcribed spacer region exhibited 97% sequence similarity to known Penicillium spp. in the GenBank database. Phylogenetic analyses revealed that P. dravuni is related most closely to Eupenicillium brefeldianum, E. levitum, E. reticulosporum, E. javanicum, E. ehrlichii and PF simplicissimum. However this new species shares only a distant ancestor with this clade because it branches by itself early in the lineage. P. dravuni also is known to produce the secondary metabolites dictyosphaeric acids A and B and carviolin.


Subject(s)
Chlorophyta/microbiology , Penicillium/classification , Penicillium/isolation & purification , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Ribosomal Spacer/chemistry , DNA, Ribosomal Spacer/genetics , Fiji , Microscopy , Microscopy, Electron, Scanning , Molecular Sequence Data , Penicillium/cytology , Penicillium/physiology , Phylogeny , Sequence Analysis, DNA , Spores, Fungal/cytology , Water Microbiology
16.
J Nat Prod ; 67(8): 1396-9, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15332862

ABSTRACT

Fungal isolate F01V25 was obtained from the alga Dictyosphaeria versluyii collected near Dravuni, Fiji, in 2001 and represented a previously undescribed Penicillium sp. Fermentation of isolate F01V25 resulted in the production of two new polyketides, dictyosphaeric acids A and B, along with the known anthraquinone carviolin. The relative stereochemistry of dictyosphaeric acids A and B was determined using the J-based configuration analysis method in conjunction with ROE and NOE correlations.


Subject(s)
Lactones/isolation & purification , Penicillium/chemistry , Eukaryota , Fiji , Lactones/chemistry , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular
17.
Org Lett ; 5(14): 2385-8, 2003 Jul 10.
Article in English | MEDLINE | ID: mdl-12841736

ABSTRACT

[reaction: see text] Two novel sulfur-containing analogs of the immunosuppressive natural product rapamycin (1) were obtained by feeding cultures of Streptomyces hygroscopicus with l-nipecotic acid (4) and either (S)-1,3-thiazane-4-carboxylic acid (5) or (S)-1,4-thiazane-3-carboxylic acid (6). The structures of the two new compounds, 20-thiarapamycin (2) and 15-deoxo-19-sulfoxylrapamycin (3), were determined by spectroscopic methods.


Subject(s)
Immunosuppressive Agents/chemistry , Sirolimus/analogs & derivatives , Sulfur/chemistry , Magnetic Resonance Spectroscopy , Sirolimus/chemistry
18.
J Org Chem ; 68(5): 2014-7, 2003 Mar 07.
Article in English | MEDLINE | ID: mdl-12608826

ABSTRACT

Chemical investigation of a Penicillium brocae, obtained from a tissue sample of a Fijian Zyzyya sp. sponge, yielded two known diketopiperazines and three novel cytotoxic polyketides, brocaenols A-C. The brocaenols contain an unusual enolized oxepine lactone ring system that to the best of our knowledge is unprecedented in the literature. The structures were elucidated by using 2D-NMR methods including an INADEQUATE experiment. The absolute stereochemistry of brocaenol A was established by using a modified Mosher method. The taxonomy of the producing fungus was elucidated by using both morphological and rDNA sequence analysis.


Subject(s)
Heterocyclic Compounds, 3-Ring/isolation & purification , Penicillium/chemistry , Porifera/chemistry , Animals , Fermentation , Fiji , Heterocyclic Compounds, 3-Ring/chemistry , Inhibitory Concentration 50 , Models, Molecular , Molecular Conformation , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Stereoisomerism
19.
J Antibiot (Tokyo) ; 56(12): 1033-44, 2003 Dec.
Article in English | MEDLINE | ID: mdl-15015731

ABSTRACT

Inhibitors of the enzymes involved in fatty acid biosynthesis (FAB) have been reported as antibacterial agents. These include thiolactomycin, cerulenin, triclosan, diazoborine, naphthyridinones, aminopyridines and pyridoindoles. Our search for new FAB inhibitors, using a lacZ reporter cell-based screen, led to several confirmed hits. Culture F92S91, later identified as a Pseudomonas sp. based on 16S profiling, was found to produce two alpha-pyrones (I and II) and three high molecular weight peptides. The pyrones were unstable under acidic conditions, and they were rearranged into a furanone derivative (III). Of these compounds, pyrone I was the most active with MICs (microg/ml) against B. subtilis (1 to approximately 2), MRSA (2 to approximately 4), M. catarrhalis (4) and VRE (2 to approximately 64). Effects on macromolecular synthesis and membrane functions were tested in B. subtilis. Pyrone I nonspecifically inhibited incorporation of radiolabeled precursors into DNA, RNA and protein within 5 minutes of drug exposure, similar to that of triclosan. Both compounds also inhibited the cellular uptake of these precursors. Cerulenin did not have an effect until 30 minutes of drug treatment. Pyrone I and triclosan were membrane-active (BacLight test); however, pyrone I (at < or = 128 microg/ml concentration) was not hemolytic to human RBCs in contrast to triclosan, which was hemolytic at 16 microg/ml. These data suggest that pyrone-I, unlike triclosan, selectively affects bacterial membrane function.


Subject(s)
Pseudomonas/metabolism , Pyrones/metabolism , Bacillus subtilis/drug effects , Fermentation , Hemolysis , Marine Biology , Microbial Sensitivity Tests , Phylogeny , Pseudomonas/genetics , Pyrones/classification , Pyrones/pharmacology , RNA, Ribosomal, 16S/genetics
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