Isolation, Structure Elucidation, and (Bio)Synthesis of Haprolid, a Cell-Type-Specific Myxobacterial Cytotoxin.

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.

Designation of type strains for seven species of the order Myxococcales and proposal for neotype strains of Cystobacter ferrugineus, Cystobacter minus and Polyangium fumosum.

Ten species of the order Myxococcales with validly published names are devoid of living type strains. Four species of the genus Chondromyces are represented by dead herbarium samples as the type material. For a species of the genus Melittangium and two species of the genus Polyangium, no physical type material was assigned at the time of validation of the names or later on. In accordance with rule 18f of the International Code of Nomenclature of Bacteria the following type strains are designated for these species: strain Cm a14(T) ( = DSM 14605(T) = JCM 12615(T)) as the type strain of Chondromyces apiculatus, strain Cm c5(T) ( = DSM 14714(T) = JCM 12616(T)) as the type strain of Chondromyces crocatus, strain Sy t2(T) ( = DSM 14631(T) = JCM 12617(T)) as the type strain of Chondromyces lanuginosus, strain Cm p51(T) ( = DSM 14607(T) = JCM 12618(T)) as the type strain of Chondromyces pediculatus, strain Me b8(T) ( = DSM 14713(T) = JCM 12633(T)) as the type strain of Melittangium boletus, strain Pl s12(T) ( = DSM 14670(T) = JCM 12637(T)) as the type strain of Polyangium sorediatum and strain Pl sm5(T) ( = DSM 14734(T) = JCM 12638(T)) as the type strain of Polyangium spumosum. Furthermore, the type strains given for three species of the genera Cystobacter and Polyangium had been kept at one university institute and have been lost according to our investigations. In accordance with Rule 18c of the Bacteriological Code, we propose the following neotype strains: strain Cb fe18 ( = DSM 14716  = JCM 12624) as the neotype strain of Cystobacter ferrugineus, strain Cb m2 ( = DSM 14751 = JCM 12627) as the neotype strain of Cystobacter minus and strain Pl fu5 ( = DSM 14668 = JCM 12636) as the neotype strain of Polyangium fumosum. The proposals of the strains are based on the descriptions and strain proposals given in the respective chapters of Bergey's Manual of Systematic Bacteriology (2005).

Isolation, biological activity evaluation, structure elucidation, and total synthesis of eliamid: a novel complex I inhibitor.

Eliamid is a secondary metabolite isolated from two bacterial strains. This molecule features a linear polyketide backbone terminated by a tetramic acid amide moiety. Among other biological activities, eliamid shows a high and specific cytostatic action on human lymphoma and cervix carcinoma cell lines. The 2,4-anti relative configuration of the C-2,C-4-dimethyl substituted amide fragment was assigned by means of Breit's rule. The absolute configuration of all stereocenters was determined by a combination of degradation methods, structural similarity analysis and total synthesis. The stereogenic centers were introduced by vinylogous Mukaiyama aldol reaction and two consecutive Myers alkylations. The use of pentafluorophenyl ester as acylation agent allowed the efficient formation of tetramic acid amide. The longest linear sequence in the synthesis consist of 13 steps and proceeds with 12% overall yield. Differential spectroscopy experiments with beef heart submitochondrial particles established that eliamid is a potent inhibitor of the NADH-ubiquinone oxidoreductase complex. Additionally, biosynthesis of eliamid was investigated by feeding experiments with (13)C-labeled precursors.

Chivosazoles A and F, cytostatic macrolides from myxobacteria, interfere with actin.

Chivosazoles A and F, isolated from Sorangium cellulosum, showed high antiproliferative activity with different mammalian cell lines including human cancer cells. The chivosazoles caused a delay in G2/M phase of the cell cycle, and treated cells often contained two nuclei. By labeling F-actin it was shown that the actin cytoskeleton of the cells starts to break down after a few minutes of treatment. In vitro polymerization assays with purified G-actin revealed that the chivosazoles inhibit actin polymerization and also cause a depolymerization of pyrene-labeled F-actin microfilaments prepared in vitro. Chivosazoles are new tools for the investigation of issues concerning the actin cytoskeleton and they have a different mode of action from the known microfilament-disrupting compounds like rhizopodin and cytochalasin D.

Pedein A and B: production, isolation, structure elucidation and biological properties of new antifungal cyclopeptides from Chondromyces pediculatus (Myxobacteria).

Two new secondary metabolites, named pedein A and B, were isolated from the cell mass of the myxobacterium Chondromyces pediculatus. Their planar structures were elucidated by spectroscopic methods, in particular 2D NMR as 24-membered cyclic hexapeptides composed of a variable tryptophan residue, glycine, sarcosine and three unusual hydroxy beta- and gamma-amino acids. The main component, pedein A, strongly inhibited the growth of yeasts and fungi, induced hemolysis of erythrocytes, and caused changes in membrane permeability of Rhodotorula glutinis. The structures of the pedeins are closely related to the large family of the microsclerodermins, which have been isolated from lithistid sponges of Microscleroderma and Theonella species.

Discovery and development of the epothilones : a novel class of antineoplastic drugs.

The epothilones are a novel class of antineoplastic agents possessing antitubulin activity. The compounds were originally identified as secondary metabolites produced by the soil-dwelling myxobacterium Sorangium cellulosum. Two major compounds, epothilone A and epothilone B, were purified from the S. cellulosum strain So ce90 and their structures were identified as 16-member macrolides. Initial screening with these compounds revealed a very narrow and selective antifungal activity against the zygomycete, Mucor hiemalis. In addition, strong cytotoxic activity against eukaryotic cells, mouse L929 fibroblasts and human T-24 bladder carcinoma cells was observed. Subsequent studies revealed that epothilones induce tubulin polymerization and enhance microtubule stability. Epothilone-induced stabilisation of microtubules was shown to cause arrest at the G2/M transition of the cell cycle and apoptosis. The compounds are active against cancer cells that have developed resistance to taxanes as a result of acquisition of beta-tubulin overexpression or mutations and against multidrug-resistant cells that overexpress P-glycoprotein or multidrug resistance-associated protein. Thus, epothilones represent a new class of antimicrotubule agents with low susceptibility to key tumour resistance mechanisms. More recently, a range of synthetic and semisynthetic epothilone analogues have been produced to further improve the adverse effect profile (or therapeutic window) and to maximize pharmacokinetic and antitumour properties. Various epothilone analogues have demonstrated activity against many tumour types in preclinical studies and several compounds have been and still are being evaluated in clinical trials. This article reviews the identification and early molecular characterization of the epothilones, which has provided insight into the mode of action of these novel antitumour agents in vivo.

The thuggacins, novel antibacterial macrolides from Sorangium cellulosum acting against selected Gram-positive bacteria.

In our screening program we found an activity against some Gram-positive bacteria, including mycobacteria in the culture supernatant of Sorangium cellulosum strain So ce895. The antibiotic responsible for this activity was isolated and named thuggacin. Initial studies towards the mechanism of action showed that thuggacin A inhibits a late step of the respiratory chain of some bacteria.

Cruentaren, a new antifungal salicylate-type macrolide from Byssovorax cruenta (myxobacteria) with inhibitory effect on mitochondrial ATPase activity. Fermentation and biological properties.

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.

Isolation, Structure Elucidation, Biosynthesis, and Synthesis of Antalid, a Secondary Metabolite from Polyangium species.

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.

Aurafuron A and B, new bioactive polyketides from Stigmatella aurantiaca and Archangium gephyra (Myxobacteria). Fermentation, isolation, physico-chemical properties, structure and biological activity.

New antibiotic polyketides, named aurafuron A (1) and B (2) were isolated from culture extracts of myxobacteria of the species Stigmatella aurantiaca and Archangium gephyra, strain Ar 10844. By multi-step chromatography 1 and 2 were separated from a variety of other non-related co-metabolites, and their structures elucidated by spectroscopic methods as new 5-alkenyl-3 3(2H)-furanones. Aurafurons inhibited the growth of some filamentous fungi and additionally, aurafuron B was weakly active against few Gram-positive bacteria. Both compounds also showed cytotoxic activity against the mouse fibroblast cell line L929.

Disorazol A1, a highly effective antimitotic agent acting on tubulin polymerization and inducing apoptosis in mammalian cells.

Disorazol A1, a macrocyclic polyketide compound that is produced by the myxobacterium Sorangium cellulosum showed a remarkably high cytostatic activity. It inhibited the proliferation of different cancer cell lines including a multidrug-resistant KB line at low picomolar levels. In presence of disorazol A1, the nuclei of the cells increased in size and the cells often became multinucleate. Low concentrations of disorazol (<100 pM) induced an apoptotic process, characterized by enhanced capase-3 activity and DNA laddering, and abnormal, multipolar mitotic spindles. Low concentrations also induced an accumulation of p53 protein in the nucleus. At higher concentrations, we observed an accumulation of the cells in the G2/M-phase of the cell cycle, and a depletion of microtubules. In vitro, disorazol A1 inhibited the polymerization of tubulin in a concentration-dependent manner and independently of microtubule-associated proteins. Correspondingly it induced a complete depolymerization of microtubules prepared in vitro. Formation of defined degradation structures was not observed. Disorazol is a novel, highly effective antimitotic agent. Efforts are going on to develop it as an anticancer drug.

Archazolids, new cytotoxic macrolactones from Archangium gephyra (Myxobacteria). Production, isolation, physico-chemical and biological properties.

Novel cytotoxic compounds, archazolid A and B, were isolated from the culture broth of strains of the myxobacteria Archangium gephyra and Cystobacter sp. Archazolids consist of a macrocyclic lactone ring with a thiazole side chain and showed high activity against mammalian cells. The IC50 values with different cell lines ranged from 0.1 to 1 ng/ml. An incubation of PtK2 potoroo cells with archazolid A (5 ng/ml) led to the formation of vacuoles in the ER, a phenomenon that is typical for inhibitors of V-ATPases, like concanamycin and bafilomycin. We therefore assume that archazolid is an inhibitor of the V-ATPase of higher cells.

Studies on the biosynthesis of epothilones: hydroxylation of Epo A and B to epothilones E and F.

When Sorangium cellulosum So ce90 is grown without XAD adsorber resin there is a steady state of epothilone A and B biosynthesis and hydroxylation of these products to epothilones E and F. This biotransformation at position C-21 of the thiazole ring is not restricted to producers of epothilones. It is carried out by a substrate induced monooxygenase. Epothilones E and F are further degraded by opening of the lactone ring by an esterase. Steps of degradation of different strains of S. cellulosum are compared.

Cyrmenins, new beta-methoxyacrylate inhibitors of the electron transport. Production, isolation, physico-chemical and biological properties.

New antibiotic compounds, named cyrmenins, were isolated from the culture broth of strains of the myxobacteria Cystobacter armeniaca and Archangium gephyra. The compounds belong to the group of beta-methoxyacrylate (MOA) inhibitors and are the first naturally occuring nitrogen-linked MOAs. The cyrmenins show nearly the same antifungal activity as strobilurin A, but are less toxic in a growth inhibition assay with L929 mouse cells. Cyrmenins inhibit NADH oxidation by submitochondrial particles from beef heart. Investigations by difference spectroscopy showed that cyrmenin B1 blocks the electron transport within the cytochrome bc1-segment (complex III) of the respiratory chain.

Argyrins, immunosuppressive cyclic peptides from myxobacteria. I. Production, isolation, physico-chemical and biological properties.

A group of cyclic peptides consisting of 8 amino acid residues, named argyrins A to H, were isolated from the culture broth of strains of the myxobacterium Archangium gephyra. Argyrin B was found to be a potent inhibitor of T cell independent antibody formation by murine B cells and strongly inhibited the two way murine mixed lymphocyte reaction. All argyrins had slight antibiotic activity, especially against Pseudomonas sp., and inhibited growth of mammalian cell cultures. The growth inhibition was often incomplete and varied highly with different cell lines.

Maracin and Maracen: New Types of Ethynyl Vinyl Ether and α-Chloro Divinyl Ether Antibiotics from Sorangium cellulosum with Specific Activity Against Mycobacteria.

Like something originating from the chemistry laboratory rather than from Nature: However, the title compounds 1 and 2 are, in fact, synthesized by myxobacteria of the species Sorangium cellulosum according to the same assembly pattern from acetate and are excreted into the culture medium. Both compounds have remarkably good in vitro activity against the pathogen responsible for tuberculosis, Mycobacterium tuberculosis.

N-Oxidation of Epothilone A-C and O-Acyl Rearrangement to C-19- and C-21-Substituted Epothilones.

A bothersome side reaction in the last step of a total synthesis of epothilone led to the formation of the thiazol-N-oxide 1. Obtained from epothilones prepared by fermentation, these biologically active N-oxides allow the extremely short synthesis of the highly active epothilones 2 with modified side chains by an O-acyl rearrangement.