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Argyrins represent a family of cyclic octapeptides exhibiting promising antimicrobial, antitumorigenic and immunosuppressant activities. They derive from a nonribosomal peptide synthetase pathway, which was identified and characterized in this study from the myxobacterial producer strain Cystobacter sp. SBCb004. Using the native biosynthetic gene cluster (BGC) sequence as template synthetic BGC versions were designed and assembled from gene synthesis fragments. A heterologous expression system was established after chromosomal deletion of a well-expressed lipopeptide pathway from the host strain Myxococcus xanthus DK1622. Different approaches were applied to engineer and improve heterologous argyrin production, which was finally increased to 160 mg/L, around 20-fold higher yields compared to the native producer. Heterologous production platform also led to identification of several novel argyrin derivatives (A2, F3, G3, I, J, K, and L). The optimized production system provides a versatile platform for future supply of argyrins and novel derivatives thereof.

Myxobakterien sind gut etablierte Quellen diverser hoch bioaktiver Naturstoffe. Das hier beschriebene Haprolid ist ein neuartiges Makrolacton, das aus vier modifizierten Aminosauren und einem Polyketidfragment aufgebaut ist und aus Byssovorax-cruenta-Har1-Kulturen isoliert wurde. Da sich die vollstandige Bestimmung der stereochemischen Konfiguration als anspruchsvoll erwies, wurde eine bioinformatische Analyse der Biosynthesegene zur Vorhersage der Konfiguration der einzelnen Zentren angewendet. Eine detaillierte Analyse der fur die Haprolidsynthese zustandigen Biosyntheseproteine ergab ein hybrides System aus Polyketidsynthase und Nichtribosomaler Peptidsynthetase und ermoglichte die bioinformatische Konfigurationsanalyse der einzelnen Stereozentren. Durch nachfolgende Totalsynthese von Haprolid konnten alle getroffenen Vorhersagen bestatigt werden. Haprolid zeigte eine zytotoxische Aktivitat gegen bestimmte Zelllinien im nanomolaren Bereich, auf andere Zellen hatte es aber uberraschenderweise kaum Einfluss.

Myxobacteria, a group of antimicrobial producing bacteria, have been successfully cultured and characterized from ten soil samples collected from different parts of Slovakia. A total of 79 myxobacteria belonging to four genera (Myxococcus, Corallococcus, Sorangium, and Polyangium) were isolated based on aspects of their life cycle. Twenty-five of them were purified, fermented, and screened for antimicrobial activities against 11 test microorganisms. Results indicated that crude extracts showed more significant activities against Gram-positive than against Gram-negative bacteria or fungi. Based on a higher degree and broader range of antimicrobial production, the two most potential extracts (K9-5, V3-1) were selected for HPLC fractionation against Micrococcus luteus and Staphylococcus aureus and LC/MS analysis of potential antibiotic metabolites. The analysis resulted in the identification of polyketide-peptide antibiotics, namely corallopyronin A and B (K9-5) and myxalamid B and C (V3-1), which were responsible for important Gram-positive activity in the observed strains. A sequence similarity search through BLAST revealed that these strains showed the highest sequence similarity to Corallococcus coralloides (K9-5, NCBI accession number KX256198) and Myxococcus xanthus (V3-1, NCBI accession number KX256197). Although screening of myxobacteria is laborious, due to difficulties in isolating cultures, this research represented the first report covering the isolation and cultivation of this challenging bacterial group from Slovakian soils as well as the screening of their antimicrobial activity, cultural identification, and secondary metabolite identification.

Die Entwicklung neuer Antibiotika befindet sich in einer ernsthaften Krise, die unter anderem dadurch begrundet ist, dass kaum innovative chemische Grundstrukturen mit Aktivitat gegen Gram-negative und multiresistente Bakterien gefunden werden. Hier berichten wir uber die Entdeckung neuer hochwirksamer Antibiotika aus Myxobakterien, den Cystobactamiden 1–3, die aus Cystobacter sp. isoliert wurden und minimale Hemmkonzentrationen im niedrigen μg mL−1-Bereich aufweisen. Wir beschreiben die Aufreinigung und Strukturaufklarung von drei Derivaten und die Identifizierung und Annotation des dazugehorigen Biosynthesegenclusters. Die molekularen Zielstrukturen der Cystobactamide konnten uber die Analyse des Eigenresistenzmechanismus des Produzentenstammes als bakterielle Typ-IIa-Topoisomerasen identifiziert werden. Da die Optimierungsmoglichkeiten von Chinolonen als Basis fur neue Inhibitoren der Typ-II-Topoisomerasen weitgehend ausgereizt sind, erscheinen die Cystobactamide als hochinteressante Alternativen, die durch Medizinalchemie und biosynthetisches Engineering der Entwicklung neuartiger Antibiotika dienen konnen.

Natural products have long been a source of useful biological activity for the development of new drugs. Their macromolecular targets are, however, largely unknown, which hampers rational drug design and optimization. Here we present the development and experimental validation of a computational method for the discovery of such targets. The technique does not require three-dimensional target models and may be applied to structurally complex natural products. The algorithm dissects the natural products into fragments and infers potential pharmacological targets by comparing the fragments to synthetic reference drugs with known targets. We demonstrate that this approach results in confident predictions. In a prospective validation, we show that fragments of the potent antitumour agent archazolid A, a macrolide from the myxobacterium Archangium gephyra, contain relevant information regarding its polypharmacology. Biochemical and biophysical evaluation confirmed the predictions. The results obtained corroborate the practical applicability of the computational approach to natural product 'de-orphaning'.

The macrolide archazolid inhibits vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, and potently suppresses cancer cell growth at low nanomolar concentrations. In view of the growing link between inflammation and cancer, we investigated whether inhibition of V-ATPase by archazolid may affect primary human monocytes that can promote cancer by sustaining inflammation through the release of tumor-promoting cytokines. Human primary monocytes express V-ATPase, and archazolid (10-100nM) increases the vesicular pH in these cells. Archazolid (10nM) markedly reduced the release of pro-inflammatory (TNF-α, interleukin-6 and -8) but also of anti-inflammatory (interleukin-10) cytokines in monocytes stimulated with LPS, without affecting cell viability up to 1000nM. Of interest, secretion of interleukin-1β was increased by archazolid. Comparable effects were obtained by the V-ATPase inhibitors bafilomycin and apicularen. The phosphorylation of p38 MAPK and ERK-1/2, Akt, SAPK/JNK or of the inhibitor of NFκB (IκBα) as well as mRNA expression of IL-8 were not altered by archazolid in LPS-stimulated monocytes. Instead, archazolid caused endoplasmic reticulum (ER) stress response visualized by increased BiP expression and accumulation of IL-8 (and TNF-α) at the ER, indicating a perturbation of protein secretion. In conclusion, by interference with V-ATPase, archazolid significantly affects the secretion of cytokines due to accumulation at the ER which might be of relevance when using these agents for cancer therapy.

Resistance of cancer cells towards chemotherapy is the major cause of therapy failure. Hence, the evaluation of cellular defense mechanisms is essential in the establishment of new chemotherapeutics. Archazolid B, a novel vacuolar H(+)-ATPase inhibitor, displayed cytotoxicity in the low nanomolar range on a panel of different tumor cell lines. First, we investigated tumor-specific cytotoxicity of archazolid B by comparing cancer to non-cancer cells. Breast, liver and colon cancer cells displayed higher drug sensitivity than corresponding non-tumorous cells, whereas leukemia cell lines were as sensitive as peripheral mononuclear blood cells. Investigating classical drug resistance mechanisms, archazolid B was identified as a possible substrate of the ABC transporters ABCB1 (P-glycoprotein) and ABCG2 (BCRP), whereas collateral sensitivity was observed in ABCB5-expressing cells. Our results pointed to a possible binding competition of archazolid B with verapamil on P-glycoprotein. However, archazolid B did not reverse resistance towards doxorubicin indicating that it might be a substrate but not an inhibitor of P-glycoprotein mediated transport. Furthermore, the cytotoxicity of archazolid B was independent of the p53 status of the cell. Mechanisms of aquired resistance were investigated establishing an archazolid B-resistant MCF-7 cell line. Interestingly, drug resistance was not conferred by aberrant expression or DNA mutations of the gene encoding vacuolar H(+)-ATPase subunit c, the direct target of archazolids. Instead, long-term treatment with archazolid B led to a slight overexpression of ABCB1 and a significant overexpression of the epidermal growth factor receptor and reduced cell growth, all of which can be assumed to contribute to archazolid B resistance.

Processing bodies (P-bodies) are cytoplasmatic mRNP granules containing non-translating mRNAs and proteins from the mRNA decay and silencing machineries. The mechanism of P-body assembly has been typically addressed by depleting P-body components. Here we apply a complementary approach and establish an automated cell-based assay platform to screen for molecules affecting P-body assembly. From a unique library of compounds derived from myxobacteria, 30 specifically inhibited P-body assembly. Gephyronic acid A (GA), a eukaryotic protein synthesis inhibitor, showed the strongest effect. GA also inhibited, under stress conditions, phosphorylation of eIF2α and stress granule formation. Other hits uncovered interesting novel links between P-body assembly, lipid metabolism, and internal organelle physiology. The obtained results provide a chemical toolbox to manipulate P-body assembly and function.

The strain no. VY46 was isolated from agricultural soil of Slovak republic and tested for potential antimicrobial activity against various human pathogens. On the basis of results, strain VY46 significantly inhibited growth of yeast Candida albicans and therefore was used for further characterization. In order to explore the potential bioactivities, extract of the fermented broth culture was prepared with organic solvent extraction method. The ethylacetate extract was subjected to HPLC fractionation against Candida albicans and followed by LC/MS analysis for potential production of anticandidal substances. The analysis resulted in the identification of two antimycins antibiotics, which may be responsible for important anticandidal activity of the strain. On the basis of liquid chromatography and mass spectrometry the antibiotics were identified as Urauchimycin A and Kitamycin A. According tothe results from cultural, morphological, physiological, biochemical and 16S rRNA gene sequence methods, the strain was identified as Streptomyces albidoflavus. In addition, neighbor-joining phylogenetic tree confirmed the relationships of this strain to other members of Streptomyces genera.

The isolation, structure elucidation, and synthesis of antalid (1), a novel secondary metabolite from Polyangium sp., is described herein. The structure elucidation of 1 was performed with the aid of mass spectrometry, high field NMR experiments, and crystal structure analysis. The absolute configuration of antalid was confirmed through the Mosher ester method and ultimately by total synthesis. In addition, the biosynthetic origin of this hybrid PKS-NRPS natural product was unraveled by the in silico analysis of its biosynthetic gene cluster.

Myxobacteria are well-established sources for novel natural products exhibiting intriguing bioactivities. We here report on haprolid (1) isolated from Byssovorax cruenta Har1. The compound exhibits an unprecedented macrolactone comprising four modified amino acids and a polyketide fragment. As configurational assignment proved difficult, a bioinformatic analysis of the biosynthetic gene cluster was chosen to predict the configuration of each stereocenter. In-depth analysis of the corresponding biosynthetic proteins established a hybrid polyketide synthase/nonribosomal peptide synthetase origin of haprolid and allowed for stereochemical assignments. A subsequent total synthesis yielded haprolid and corroborated all predictions made. Intriguingly, haprolid showed cytotoxicity against several cell lines in the nanomolar range whereas other cells were almost unaffected by treatment with the compound.

The antibiotic elansolid C1 (8) was isolated from Chitinophaga sancti strain FxGBF13 after fermentation in the presence of anthranilic acid. Remarkably, 8 was also obtained by addition of anthranilic acid to a crude fermentation extract containing the macrolide elansolid A2 (1*). This Michael-type conjugate addition allowed us to generate 21 new derivatives of elansolid C1 (9-29) by using various nucleophiles. Biological activities of all derivatives were evaluated against Staphylococcus aureus, Micrococcus luteus, and the mouse cell line L929.

Sulfangolids are the first sulfate ester containing secondary metabolites from myxobacteria. The metabolites 1-4 and the structurally related kulkenon (5) were isolated from different strains of the species Sorangium cellulosum. In the course of isolation all metabolites proved to be rather sensitive due to their conjugated double bond systems and the strong acidic nature of the sulfate ester in sulfangolids. The relative configuration of sulfangolid C (3) was assigned by extensive 1D and 2D NMR analysis and molecular modelling. In addition, the biosynthesis of 3 was studied by feeding experiments.

Numerous loss-of-function mutations in the progranulin (GRN) gene cause frontotemporal lobar degeneration with ubiquitin and TAR–DNA binding protein 43-positive inclusions by reduced production and secretion of GRN. Consistent with the observation that GRN has neurotrophic properties, pharmacological stimulation of GRN production is a promising approach to rescueGRNhaploinsufficiency and prevent disease progression. We therefore searched for compounds capable of selectively increasing GRN levels. Here, we demonstrate that four independent and highly selective inhibitors of vacuolar ATPase (bafilomycin A1, concanamycin A, archazolid B, and apicularen A) significantly elevate intracellular and secreted GRN. Furthermore, clinically used alkalizing drugs, including chloroquine, bepridil, and amiodarone, similarly stimulate GRN production. Elevation of GRN levels occurs via a translational mechanism independent of lysosomal degradation, autophagy, or endocytosis. Importantly, alkalizing reagents rescue GRN deficiency in organotypic cortical slice cultures from a mouse model for GRN deficiency and in primary cells derived from human patients withGRNloss-of-function mutations. Thus, alkalizing reagents, specifically those already used in humans for other applications, and vacuolar ATPase inhibitors may be therapeutically used to prevent GRN-dependent neurodegeneration.

The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.

Im Jahr 1993 wurde das Polyketid Rhizopodin (Abbildung 1) aus dem Myxobakterium Myxococcus stipitatus isoliert. Studien mit der gereinigten Substanz ergaben, dass Rhizopodin bereits bei nanomolaren Konzentrationen einen drastischen Effekt auf das Zytoskelett eukaryotischer Zellen aus bt. Es wurde vermutet, dass diese Eigenschaft auf der F higkeit des Naturstoffs beruht, das eukaryotische Protein Aktin zu binden und dessen Polymerisation zu st ren. Als Struktur f r das Rhizopodin wurde ein Makrolidring bestehend aus 19 Atomen, einem disubstituierten Oxazolring sowie einem konjugierten Diensystem mit insgesamt neun stereogenen Zentren vorgeschlagen (Abbildung 1a). Um den Bindungsmodus des Rhizopodins an G-Aktin und die absolute Konfiguration des Naturstoffs zu ermitteln, haben wir den Komplex aus Rhizopodin und Kaninchenmuskelaktin kristallisiert und seine Kristallstruktur bei 2.4 Aufl sung untersucht. berraschend zeigt die Kristallstruktur, dass Rhizopodin ein C2-symmetrisches Dilacton ist. Dementsprechend besteht Rhizopodin aus einem Makrolidring mit 38 Atomen, zwei disubstituierten Oxazolringen und zwei konjugierten Diensystemen mit insgesamt 18 stereogenen Zentren. Basierend auf der Kristallstruktur beschreiben wir hier die biologisch aktive Konformation dieses Rhizopodin-Dilactons sowie die absolute Konfiguration der 18 stereogenen Zentren. Die Interaktion zwischen Rhizopodin und G-Aktin wurde in vitro anhand von Polymerisationsexperimenten mit Pyrenmodifiziertem G-Aktin untersucht. Es zeigt sich, dass eine steigende Rhizopodin-Konzentration die Aktinpolymerisation zunehmend inhibiert, wobei die Polymerisation ab einem st chiometrischen Verh ltnis von 0.5 Rhizopodin-Dilacton pro G-Aktin vollst ndig unterbunden ist. Rhizopodin f hrt zudem in Gelpermeationsexperimenten zu einer Verringerung des Elutionsvolumens von G-Aktin. Diese Beobachtungen lassen auf eine durch Rhizopodin induzierte Dimerisierung des Aktins schliesen. In vivo w rde vermutlich ein deutlich geringerer st chiometrischer Anteil an Rhizopodin die Dynamik des Zytoskeletts empfindlich st ren, da der Einbau eines einzigen Aktin-Rhizopodin-Dimers in ein Aktinfilament dessen Wachstum unterbinden w rde. Orthorombische Kristalle des gereinigten Aktin-Rhizopodin-Komplexes beugen R ntgenstrahlung bis zu einer Aufl sung von 2.4 . Die Struktur des Komplexes wurde mittels molekularen Ersatzes gel st, wobei als Suchmodell die Struktur des Tetramethylrhodamin(TMR)-gebundenen Aktins aus Kaninchenmuskel (PDB: 1J6Z) eingesetzt wurde. Die asymmetrische Einheit des Aktin-RhizopodinKomplexkristalls umfasst zwei Aktinmonomere (A und B), die ber eine senkrecht zur kristallographischen c-Achse liegende, nicht-kristallographische zweiz hlige Drehachse ineinander berf hrt werden. Die kristallographischen Daten sind in Tabelle 1 zusammengestellt. Abbildung 1. Struktur von Rhizopodin. a) Die urspr nglich vorgeschlagene Monolactonstruktur. b) berarbeitete, C2-symmetrische Dilactonstruktur inklusive der Stereochemie. c) Kugel-Stab-Modell der biologisch aktiven Konformation des Rhizopodins. Die berlagerung des Rhizopodins (gr n) mit einer um 1808 gedrehten Kopie (rot) belegt die C2-Symmetrie des Molek ls. Die Konfigurationszuordnung der stereogenen Zentren (Sternchen) ist konsistent.

Rhizopodin was isolated as cytostatic and weakly antifungal macrolide (1) and later characterized as potent actin-depolymerizing agent. It is produced by the myxobacterium Myxococcus stipitatus, which enables a fermentative supply of the drug for biological studies. We here report a revised structure that characterizes rhizopodin (2) as the first known dimeric bis-lactone exhibiting side chains that terminate in N-methyl-vinylformamide groups, which are otherwise found in smaller marine toxins also targeting the actin cytoskeleton. Compound 2 might function as bivalent inhibitor forming ternary complexes with actin which would explain its high efficacy.

Etnangien (1), a new macrolide antibiotic active against Gram-positive bacteria, was isolated from the culture broth of the myxobacterium Sorangium cellulosum, strains So ce750 and So ce1045. Spectroscopic structure elucidation of 1 revealed a complex macrocyclic lactone bearing a modified C21 carboxylic acid side chain. The latter contains two allylic hydroxyl groups and an all-E hexaene unit, which provides the characteristic UV chromophore of 1. Initial studies toward the mechanism of action showed that bacterial and viral nucleic acid polymerases are inhibited by etnangien (1).

The novel polyketide macrolide archazolid-7-O-β-D-glucopyranoside (3) has been isolated from the myxobacterium Cystobacter violaceus and the structure of this first archazolid-glycoside has been determined by spectroscopic and degradative methods. A synthesis of simplified 7-O analogues, based on regioselective derivatisation of archazolid A, was elaborated. These structurally novel archazolids of natural and synthetic origin were evaluated in detail for V-ATPase inhibition and their biological activities are discussed in terms of their solution conformations, as determined by high-field NMR studies, including J-based conformation analysis and constrained molecular dynamics simulations. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Lysobactin, also known as katanosin B, is a potent antibiotic with in vivo efficacy against Staphylococcus aureus and Streptococcus pneumoniae. It was previously shown to inhibit peptidoglycan (PG) biosynthesis, but its molecular mechanism of action has not been established. Using enzyme inhibition assays, we show that lysobactin forms 1:1 complexes with Lipid I, Lipid II, and Lipid II(A)(WTA), substrates in the PG and wall teichoic acid (WTA) biosynthetic pathways. Therefore, lysobactin, like ramoplanin and teixobactin, recognizes the reducing end of lipid-linked cell wall precursors. We show that despite its ability to bind precursors from different pathways, lysobactin's cellular mechanism of killing is due exclusively to Lipid II binding, which causes septal defects and catastrophic cell envelope damage.

Isolation and structure elucidation of three novel iturin-type lipopeptides, paenilarvins A-C, are described.

The novel macrolide cruentaren A was produced at levels up to 3.2 mg/liter by cultures of the myxobacterium Byssovorax cruenta. The new compound strongly inhibited the growth of yeasts and filamentous fungi and showed high cytotoxicity against L929 mouse fibroblast cells. A minor co-metabolite of cruentaren A, named cruentaren B, and identified as a six-membered lactone isomer of cruentaren A, showed only marginal cytotoxicity and no antifungal activity. Cruentaren A inhibited F0F1 mitochondrial ATP-hydrolysis in submitochondrial particles of yeasts and beef heart.

[structure: see text] The relative and absolute stereochemistry of the structurally unique 24-membered myxobacterial macrolides archazolid A and B, highly potent vacuolar-type ATPase (V-ATPase) inhibitors in vitro and in vivo, was determined on the basis of a combination of extensive high-field NMR studies, including J-based configuration analysis, molecular modeling, and chemical methods.

Secondary metabolism involves a broad diversity of biochemical reactions that result in a wide variety of biologically active compounds. Terminal amide formation during the biosynthesis of the myxobacterial electron-transport inhibitor, myxothiazol, was analyzed by heterologous expression of the unique nonribosomal-peptide synthetase, MtaG, and incubation with a synthesized substrate mimic. These experiments provide evidence that the terminal amide is formed from a carrier protein-bound myxothiazol acid that is thioesterified to MtaF. This intermediate is transformed to an amide by extension with glycine and subsequent oxidative cleavage by MtaG. The final steps of melithiazol assembly involve a highly similar protein-bound intermediate (attached to MelF, a homologue of MtaF), which is transformed to an amide by MelG (homologue of MtaG). In this study, we also show that the amide moiety of myxothiazol A can be hydrolyzed in vivo to the formerly unknown free myxothiazol acid by heterologous expression of melJ in the myxothiazol producer Stigmatella aurantiaca DW4/3-1. The methyltransferase MelK can finally methylate the acid to give rise to the methyl ester, which is produced as the final product in the melithiazol A biosynthetic pathway. These experiments clarify the role of MelJ and MelK during melithiazol assembly.

Two novel highly cytotoxic metabolites, spirangien A (1) and B (2), were isolated from the myxobacterium Sorangium cellulosum (strain So ce90). The structures were elucidated by detailed NMR spectroscopic analysis. The previously unknown molecular framework common to spirangien A and B includes a highly functionalized spiroketal core structure, a side chain bearing a pentaene chromophore and a terminal carboxyl group, and a total of thirteen stereocenters. The absolute configuration at C-3 was determined by degradation and subsequent fragment analysis by GC. The relative stereochemistry of the spiroketal structure was proposed on the basis of vicinal proton couplings and ROESY data for hydroperoxide derivative 4, obtained by ozonolysis of spirangien A, and for 1,3-diene 5, obtained by cross-metathesis with ethylene. X-ray crystal structure analysis of 5 confirmed its structure and unambiguously provided the complete relative stereochemistry of all twelve stereocenters. The 1,3-diene derivative 5 still shows strong cytotoxic activity. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

The structure-activity relationships of epothilones indicate that major modifications are only tolerated in the western ring segment. In particular, C2 methyl of the thiazole ring appears to be most flexible. Its broad modification started from epothilone F, which was obtained from natural epothilone B by hydroxylation via the N-oxide. Some of the prepared derivatives exhibit improved esterase stability in addition to high cytotoxic activity. For these and other favorable properties, amine (BMS-310705) was recently introduced in clinical trials. In an alternative approach, modified side chains were introduced by replacement of the C12,C15 ring segment via ring-opening olefin metathesis (ROM) of epothilone C in the presence of ethylene to 12,13-seco-epothilone C, introduction of a synthetic building block followed by ring-closing olefin metathesis (RCM), and epoxidation to the 16-alkyne analog of epothilone A. The structure of the tetrapeptide tubulysin D was confirmed by total hydrolysis to N-methyl d-pipecolic acid, l-isoleucine, tubuvaline (Tuv), tubuphenylalanine (Tup), formaldehyde, and 3-methylbutyric acid. Mild acidic hydrolysis to cyclo-tubulysin andoxidative degradation to l-valine allowed the assignment of the stereocenters of Tuv, hydrazinolysis, and comparison with synthetic reference samples to that of Tup. The absolute configuration of tubulysin D is: (R)-Mep, (2,3S)-Ile, (1'R ,3'R)-Tuv, and (2S,4R)-Tup.

A bioassay-guided fractionation of the crude methanol extract of the myxobacterium Hyalangium minutum, strain NOCB-2(T) (DSM 14724(T)), led to the isolation of hyaladione (1), a novel S-methyl cyclohexadiene-dione. The structure of 1 was established by HRESIMS, NMR, and IR spectroscopy as well as X-ray crystallography. Compound 1 was active against growing mammalian cell lines, with IC(50) values ranging from 1.23 to 3.93 μM, in addition to a broad spectrum of antibacterial and antifungal activities, including inhibition of pathogenic methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa with an MIC of 0.83 and 8.5 μg mL(-1), respectively.

The structures of argyrins A-H were elucidated by NMR spectroscopy, chemical degradation and X-ray analysis as cyclic octapeptides. Argyrins A and B, in addition to the common amino acids tryptophan, glycine, dehydroalanine and alanine or alpha-aminobutyric acid, sarcosine, contain 2-(1-aminoethyl)thiazol-4-caboxylic acid and the novel amino acid 4'-methoxytryptophan. In argyrins C and D the latter is replaced by 4'-methoxy 2'-methyltryptophan. According to NMR analysis the solution and crystal conformations of argyrins A and B are identical in CDCl3 and slightly different in acetone-d6. Argyrins A and B are identical with the antibiotics A21459 A and B, whose structures are revised with respect to 4'-methoxytryptophan.

The development of new antibiotics faces a severe crisis inter alia owing to a lack of innovative chemical scaffolds with activities against Gram-negative and multiresistant pathogens. Herein, we report highly potent novel antibacterial compounds, the myxobacteria-derived cystobactamids 1-3, which were isolated from Cystobacter sp. and show minimum inhibitory concentrations in the low μg mL(-1) range. We describe the isolation and structure elucidation of three congeners as well as the identification and annotation of their biosynthetic gene cluster. By studying the self-resistance mechanism in the natural producer organism, the molecular targets were identified as bacterial type IIa topoisomerases. As quinolones are largely exhausted as a template for new type II topoisomerase inhibitors, the cystobactamids offer exciting alternatives to generate novel antibiotics using medicinal chemistry and biosynthetic engineering.

Among myxobacteria only strains of Myxococcus fulvus are able to produce in addition to the well known myxothiazol A (1) an analog, myxothiazol Z (2), with an ester-type β-methoxyacrylate pharmacophore. Feeding experiments with labeled precursors established its biosynthesis from the amide (1) presumably via an O-methyl imidate (3).

New cytostatic compounds, tubulysins, were isolated from the culture broth of strains of the myxobacteria Archangium gephyra and Angiococcus disciformis. The compounds are peptides partly consisting of unusual amino acids and are distantly related to the dolastatins. The tubulysins were not active against bacteria and only little against fungi, but showed high cytostatic activity against mammalian cell lines with IC50 values in the picomolar range. An incubation with 50 ng/ml tubulysin A led to a complete disappearance of the microtubuli network of the cells within 24 hours. The more active tubulysin D induced multipolar spindles: At 0.5 ng/ml all mitotic cells showed more than four spindle poles.

A new antibiotic compound, gephyronic acid was isolated from the culture broth of the myxobacterium, Archangium gephyra strain Ar 3895. Up to Smg/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 1-25μg/ml) and had a cytostatic effect on mammalian cell cultures (IC50 10-60ng/ml). Gephyronic acid is a specific inhibitor of eukaryotic protein sythesis showing an IC50 of 1-2 × 10-7mol/liter in an in vitro translation assay.

The gliding bacterium Sandaracinus amylolyticus, strain NOSO-4T, was recently characterized as the first representative of a new myxobacterial genus. A screening of the culture broth for antibiotically active metabolites followed by isolation and characterization revealed two unique 3-formylindol derivatives, indiacen A (1) and its chloro derivative indiacen B (2). Both are active against Gram-positive and Gram-negative bacteria as well as the fungus Mucor hiemalis. The biosynthetic origin of the isoprene-like side chain in 1 and 2 was studied by in vivo feeding experiments with 13C-labeled precursors.

The structure of the title compound, a constituent of the myxobacterium Archangium gephyra (Ar 3895) which exists as a separable mixture of keto and hemiketal isomers, is unambiguously confirmed as (I).