Pyrronazols, metabolites from the myxobacteria Nannocystis pusilla and N. exedens, are unusual chlorinated pyrone-oxazole-pyrroles.

The chlorinated pyrrole-oxazole-pyrones pyrronazol A (1), pyrronazol A2 (2), and pyrronazol B (3) were isolated from Nannocystis pusilla strain Ari7, and two chlorinated pyrrole-oxazole isomers, pyrronazols C1 (4) and C2 (5), were isolated from N. pusilla strain Na a174. HRESIMS, NMR, and X-ray crystallographic analysis was used in the structure elucidation including the absolute configuration of pyrronazol A (1). In addition to pyrronazols, 1,6-phenazine-diol (6) and its glycosyl derivative, 1-hydroxyphenazin-6-yl-α-d-arabinofuranoside (7), were isolated and identified from the culture broth of N. pusilla strain Ari7. When tested for biological activity against bacteria, fungi, and yeasts, 1 showed weak antifungal activity against Mucor hiemalis (MIC 33.3 µg/mL) but no antibacterial activity, while 6 showed weak antibacterial and antifungal activity (MIC 33.3 µg/mL) against some of the strains tested. In cell culture experiments 1 showed no significant cytotoxicity, while 6 was active against several cell lines, especially the human ovarian carcinoma cells SK-OV-3 (LD50 2.59 µM).

Nannozinones and sorazinones, unprecedented pyrazinones from myxobacteria.

Nannozinones A (1) and B (2) were discovered as metabolites of the recently isolated Nannocystis pusilla strain MNa10913 belonging to the poorly studied myxobacterial family Nannocystaceae. In contrast, the structurally related sorazinones A (5) and B (6) were isolated from Sorangium cellulosum strain Soce895, which was known as the producer of the antibiotic thuggacin A. The extract also contained methyl indole-3-carboxylate (4). HRESIMS and (1)H, (13)C, and (15)N NMR spectroscopy revealed the structures of nannozinones A (1) and B (2) as unusual dihydropyrrolo- and pyrrolopyrazinone derivatives, while sorazinone A (5) was characterized as an aromatic diketopiperazine and sorazinone B (6) as a dibenzyl 2(1H)-pyrazinone derivative. While the dihydropyrrolo derivative nannozinone A (1) showed weak antibacterial and antifungal activity, nannozinone B (2) inhibited the growth of cell cultures with IC50 values between 2.44 and 16.9 µM. The nannochelin A iron complex (3), which was isolated besides 1 and 2, was even more active, with IC50 values between 0.05 and 1.95 µM. On the other hand, the indole 4 and sorazinones 5 and 6 did not show any significant cytotoxicity and only weak activity against the Gram-positive Nocardia sp.

The binding site of the V-ATPase inhibitor apicularen is in the vicinity of those for bafilomycin and archazolid.

The investigation of V-ATPases as potential therapeutic drug targets and hence of their specific inhibitors is a promising approach in osteoporosis and cancer treatment because the occurrence of these diseases is interrelated to the function of the V-ATPase. Apicularen belongs to the novel inhibitor family of the benzolactone enamides, which are highly potent but feature the unique characteristic of not inhibiting V-ATPases from fungal sources. In this study we specify, for the first time, the binding site of apicularen within the membrane spanning V(O) complex. By photoaffinity labeling using derivatives of apicularen and of the plecomacrolides bafilomycin and concanamycin, each coupled to (14)C-labeled 4-(3-trifluoromethyldiazirin-3-yl)benzoic acid, we verified that apicularen binds at the interface of the V(O) subunits a and c. The binding site is in the vicinity to those of the plecomacrolides and of the archazolids, a third family of V-ATPase inhibitors. Expression of subunit c homologues from Homo sapiens and Manduca sexta, both species sensitive to benzolactone enamides, in a Saccharomyces cerevisiae strain lacking the corresponding intrinsic gene did not transfer this sensitivity to yeast. Therefore, the binding site of benzolactone enamides cannot be formed exclusively by subunit c. Apparently, subunit a substantially contributes to the binding of the benzolactone enamides.

Concerted action of P450 plus helper protein to form the amino-hydroxy-piperidone moiety of the potent protease inhibitor crocapeptin.

The crocapeptins are described here as cyclic depsipeptides, isolated from cultures of the myxobacterium Chondromyces crocatus . Structure elucidation of the compounds revealed a cyanopeptolin-like skeleton, containing the characteristic amino-hydroxy-piperidone (Ahp)-heterocycle. Like the cyanopeptolins, the myxobacterial crocapeptins proved to be serine protease inhibitors. The nonribosomal origin of the peptide was confirmed by mutagenesis experiments, and the biosynthesis gene cluster was sequenced. It could be shown that the Ahp-heterocycle originates from a proline residue in the precursor molecule precrocapeptin, which is converted to crocapeptin by the tailoring enzymes CpnE and CpnF. Conversion of precrocapeptin isolated from a cpnF mutant into crocapeptin was achieved using recombinant CpnF, a cytochrome P450 enzyme responsible for hydroxylation of the proline residue in precrocapeptin. Addition of protein CpnE resulted in strongly increased conversion rates toward Ahp containing product. A mutant with 10-fold increased production of crocapeptin A was created through insertion of the Pnpt-promotor in front of the NRPS gene.

Rescue of progranulin deficiency associated with frontotemporal lobar degeneration by alkalizing reagents and inhibition of vacuolar ATPase.

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 rescue GRN haploinsufficiency 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 with GRN loss-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.

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.

The biofilm inhibitor carolacton disturbs membrane integrity and cell division of Streptococcus mutans through the serine/threonine protein kinase PknB.

Carolacton, a secondary metabolite isolated from the myxobacterium Sorangium cellulosum, disturbs Streptococcus mutans biofilm viability at nanomolar concentrations. Here we show that carolacton causes leakage of cytoplasmic content (DNA and proteins) in growing cells at low pH and provide quantitative data on the membrane damage. Furthermore, we demonstrate that the biofilm-specific activity of carolacton is due to the strong acidification occurring during biofilm growth. The chemical conversion of the ketocarbonic function of the molecule to a carolacton methylester did not impact its activity, indicating that carolacton is not functionally activated at low pH by a change of its net charge. A comparative time series microarray analysis identified the VicKRX and ComDE two-component signal transduction systems and genes involved in cell wall metabolism as playing essential roles in the response to carolacton treatment. A sensitivity testing of mutants with deletions of all 13 viable histidine kinases and the serine/threonine protein kinase PknB of S. mutans identified only the ΔpknB deletion mutant as being insensitive to carolacton treatment. A strong overlap between the regulon of PknB in S. mutans and the genes affected by carolacton treatment was found. The data suggest that carolacton acts by interfering with PknB-mediated signaling in growing cells. The resulting altered cell wall morphology causes membrane damage and cell death at low pH.

Damage of Streptococcus mutans biofilms by carolacton, a secondary metabolite from the myxobacterium Sorangium cellulosum.

BACKGROUND: Streptococcus mutans is a major pathogen in human dental caries. One of its important virulence properties is the ability to form biofilms (dental plaque) on tooth surfaces. Eradication of such biofilms is extremely difficult. We therefore screened a library of secondary metabolites from myxobacteria for their ability to damage biofilms of S. mutans. RESULTS: Here we show that carolacton, a secondary metabolite isolated from Sorangium cellulosum, has high antibacterial activity against biofilms of S. mutans. Planktonic growth of bacteria was only slightly impaired and no acute cytotoxicity against mouse fibroblasts could be observed. Carolacton caused death of S. mutans biofilm cells, elongation of cell chains, and changes in cell morphology. At a concentration of 10 nM carolacton, biofilm damage was already at 35% under anaerobic conditions. A knock-out mutant for comD, encoding a histidine kinase specific for the competence stimulating peptide (CSP), was slightly less sensitive to carolacton than the wildtype. Expression of the competence related alternate sigma factor ComX was strongly reduced by carolacton, as determined by a pcomX luciferase reporter strain. CONCLUSIONS: Carolacton possibly interferes with the density dependent signalling systems in S. mutans and may represent a novel approach for the prevention of dental caries.

Biological activity of volatiles from marine and terrestrial bacteria.

The antiproliferative activity of 52 volatile compounds released from bacteria was investigated in agar diffusion assays against medically important microorganisms and mouse fibroblasts. Furthermore, the activity of these compounds to interfere with the quorum-sensing-systems was tested with two different reporter strains. While some of the compounds specific to certain bacteria showed some activity in the antiproliferative assay, the compounds common to many bacteria were mostly inactive. In contrast, some of these compounds were active in the quorum-sensing-tests. γ-Lactones showed a broad reactivity, while pyrazines seem to have only low intrinsic activity. A general discussion on the ecological importance of these findings is given.

The biosynthesis of branched dialkylpyrazines in myxobacteria.

The biosynthesis of the volatiles 2,5- and 2,6-diisopropylpyrazine (2 and 3, resp.) released by the myxobacteria Nannocystis exedens subsp. cinnabarina (Na c29) and Chondromyces crocatus (strains Cm c2 and Cm c5) was studied. Isotopically labeled precursors and proposed pathway intermediates were fed to agar plate cultures of the myxobacteria. Subsequently, the volatiles were collected by use of a closed loop stripping apparatus (CLSA), and incorporation into the pyrazines was followed by GC/MS analysis. [(2)H(8)]Valine was smoothly incorporated into both pyrazines clearly establishing their origin from the amino acid pool. The cyclic dipeptide valine anhydride (16)--a potential intermediate on the biosynthetic pathway to branched dialkylpyrazines--was synthesized containing (2)H(1) labels in specific positions. Feeding of [(2)H(16)]-16 and [(2)H(12)]-16 in both valine subunits mainly resulted in the formation of pyrazines derived from only one labeled amino acid, whereas only traces of the expected pyrazines with two labeled subunits were found. To investigate the origin of nitrogen in the pyrazines, a feeding experiment with [(15)N]valine was performed, resulting in the incorporation of the (15)N label. The results contradict a biosynthetic pathway via cyclic dipeptides, but rather point to a pathway on which valine is reduced to valine aldehyde. Its dimerization to 2,5-diisopropyldihydropyrazine 36 and subsequent oxidation results in 2. The proposed biosynthetic pathway neatly fits the results of earlier labeling studies and also explains the formation of the regioisomer 2,6-diisopropylpyrazine 3 by isomerization during the first condensation step of two molecules valine aldehyde. A general biosynthetic pathway to different classes of pyrazines is presented.

New structural variants of homoserine lactones in bacteria.

N-Acylhomoserine lactones (AHLs) are used by a wide variety of bacteria for cell-cell communication in "quorum-sensing". These compounds are derived from L-homoserine lactone and a fatty acid, which varies in chain-length, degree of saturation, and the presence or absence of an oxygen atom at C-3. In this study we describe for the first time the occurrence of acyl chains carrying a methyl branch, and present a GC-MS-based method that can be used to distinguish these compounds from unbranched isomers. The bacterium Aeromonas culicicola produces several methyl branched AHLs. In Jannaschia helgolandensis--a marine bacterium of the Roseobacter clade--a doubly unsaturated AHL, (2E,9Z)-N-(2,9-hexadecadienoyl)-L-homoserine lactone, occurs. The location and configuration of the double bonds was proven by spectrometric investigation and synthesis. Finally, a method was developed to establish the absolute configuration of 3-hydroxyalkanoyl-HSLs by mild cleavage and chiral gas chromatography. The AHLs synthesized during this study were tested in sensor systems specific for certain AHL types. The results show that these compounds display varying responses to the respective sensors; this underlines the importance of determining the whole bouquet of AHLs and its function to fully understand their importance for regulatory functions in bacteria.

Total synthesis and biological activity of neopeltolide and analogues.

Combining the core structure of neopeltolide, lactone 16 a, with the oxazole-containing side chain 23 via a Mitsunobu reaction provided the cytotoxic natural product neopeltolide (2). The side chain 23 was prepared from oxazolone 24 via the corresponding triflate. Key steps in the preparation of 23 were a Sonogashira coupling, an enamine alkylation, and a Still-Gennari Horner-Emmons reaction. By changing the Leighton reagent in the allylation step, the 11-epimer of lactone 16 a, compound 50 was prepared. This led to 11-epi-neopeltolide 51. The 5-epimer of neopeltolide, compound 52, could be obtained from the minor isomer of the Prins cyclization. Furthermore, a range of analogues with modifications in the side chain were prepared. All derivatives were checked for toxicity effects on mammalian cell cultures and inhibitory effects on NADH oxidation in submitochondrial particles of bovine heart. Modifications in the lactone part are tolerated to some degree. On the other hand, shortening the distance between the oxazole and the lactone causes a significant drop in activity. Analogue 65 with an additional double bond is equally or even more active than neopeltolide itself.

Iromycins from Streptomyces sp. and from synthesis: new inhibitors of the mitochondrial electron transport chain.

Two new alpha-pyridone metabolites, iromycins E and F, were isolated from cultures of strain Streptomyces sp. Dra 17, thus expanding the recently discovered iromycin family. The inhibitory potential on the mitochondrial respiratory chain was examined and revealed that iromycin metabolites block NADH oxidation in beef heart submitochondrial particles with different efficacy, yet remarkably show only very low cytotoxicity. Difference spectroscopic studies indicated that iromycins inhibit the electron transport at the site of complex I (NADH-ubiquinone oxidoreductase). Derivatives of the natural products were semisynthetically prepared and provided detailed insights into structure-activity relationships. Drawn from these results, there are strong similarities with the piericidins, which are among the most potent complex I inhibitors of the mitochondrial electron transport chain. Furthermore, total synthesis afforded new analogues, and the non-natural iromycin S (IC50 = 58 ng/mL) emerged as the most active compound, thus opening avenues of future studies with the iromycins as new valuable biochemical tools.

Biosynthesis of aurachins A-L in Stigmatella aurantiaca: a feeding study.

The isolation of aurachins A-L (1-11) from Stigmatella aurantiaca strain Sg a15 is described. Their structures and relative configurations were deduced from spectroscopic data, in particular NMR. Three structural types were identified: A-type aurachins (1, 2, 6) are C-3 oxygen-substituted quinolines carrying a farnesyl residue on C-4, C-type aurachins (3, 4, 7-11) are C-4 oxygen-substituted quinolines carrying a farnesyl residue on C-3, and C-type aurachin E (5) has a [1,1a,8,d]imidazoloquinoline structure. Feeding of (13)C-labeled precursors showed that the quinoline ring is constructed from anthranilic acid and acetate, and the farnesyl residue from acetate by both the mevalonate and nonmevalonate pathways. Further, feeding of labeled aurachin C (3) indicated the A-type aurachins are derived by a novel intramolecular 3,4-migration of the farnesyl residue that is induced by a 2,3-epoxidation and terminated by a reduction step. (18)O-Labeling experiments indicated the new oxygen substituents originate from atomospheric oxygen. On the basis of these results a biosynthetic scheme covering all aurachins is proposed. It is further proposed that quinolones with an unorthodox substitution pattern, such as the 2-geranylquinolones from Pseudonocardia sp. and the 3-heptylquinolones from Pseudomonas sp., are formed by related rearrangement mechanisms.

Semisynthesis and antiplasmodial activity of the quinoline alkaloid aurachin E.

A one-step synthesis of the rare aurachin E (1) from the easily accessible aurachin C (2) and cyanogen bromide is described. 3-Bromocarbamoylquinoline (5) is formed in a side reaction with concomitant loss of the 3-farnesyl residue. In an alternative approach, aurachin D (3) was reacted with phosgene and sodium azide to form the imidazolone ring of 1 via N-acylation. Unexpectedly, the initial reaction occurred at the carbonyl group of 3 to give 1H-pyrrolo[3,2-c]quinoline 4. The reaction sequence represents a novel route to this type of compound. Aurachin E, contrary to other aurachins, combines a high in vitro antiplasmodial activity with low cytotoxicity and absence of mitochondrial respiratory inhibition.

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.

Cruentaren A, a highly cytotoxic benzolactone from Myxobacteria is a novel selective inhibitor of mitochondrial F1-ATPases.

Cruentaren A, a new antifungal benzolactone produced by the myxobacterium Byssovorax cruenta, proved to be highly cytotoxic against various human cell lines. It inhibited the proliferation of different cancer cell lines including a multidrug-resistant KB line at low nanomolar levels. It arrested human histocytic lymphoma cells (U-937) in G(0/1) phase, but did not trigger an apoptotic process. Studies to uncover the molecular target of cruentaren A showed that the novel compound, despite its structural similarity to the benzolactone enamides apicularen and salicylihalamide, was no V-ATPase inhibitor. In contrast, cruentaren specifically inhibited mitochondrial F(O)F(1)-ATPases with IC50 values of 15-30 nM. Although the exact binding site of cruentaren remains undefined, inhibition was shown to occur by interaction with the catalytic F(1) domain. Since mitochondrial ATPases play a crucial role in the pathophysiology of several human disorders including cancer, cruentaren or synthetic derivatives thereof could form the basis of future therapeutic strategies.

Molecular and biochemical studies of chondramide formation-highly cytotoxic natural products from Chondromyces crocatus Cm c5.

The jaspamide/chondramide family of depsipeptides are mixed PKS/NRPS natural products isolated from marine sponges and a terrestrial myxobacterium that potently affect the function of the actin cytoskeleton. As a first step to improve production in heterologous host cells and permit genetic approaches to novel analogs, we have cloned and characterized the chondramide biosynthetic genes from the myxobacterium Chondromyces crocatus Cm c5. In addition to the expected PKS and NRPS genes, the cluster encodes a rare tyrosine aminomutase for beta-tyrosine formation and a previously unknown tryptophan-2-halogenase. Conditions for gene transfer into C. crocatus Cm c5 were developed, and inactivation of several genes corroborated their proposed function and served to define the boundaries of the cluster. Biochemical characterization of the final NRPS adenylation domain confirmed the direct activation of beta-tyrosine, and fluorinated chondramides were produced through precursor-directed biosynthesis.

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.

Screening of Compounds against Gardnerella vaginalis Biofilms.

Bacterial vaginosis (BV) is a common infection in reproductive age woman and is characterized by dysbiosis of the healthy vaginal flora which is dominated by Lactobacilli, followed by growth of bacteria like Gardnerella vaginalis. The ability of G. vaginalis to form biofilms contributes to the high rates of recurrence that are typical for BV and which unfortunately make repeated antibiotic therapy inevitable. Here we developed a biofilm model for G. vaginalis and screened a large spectrum of compounds for their ability to prevent biofilm formation and to resolve an existing G. vaginalis biofilm. The antibiotics metronidazole and tobramycin were highly effective in preventing biofilm formation, but had no effect on an established biofilm. The application of the amphoteric tenside sodium cocoamphoacetate (SCAA) led to disintegration of existing biofilms, reducing biomass by 51% and viability by 61% and it was able to increase the effect of metronidazole by 40% (biomass) and 61% (viability). Our data show that attacking the biofilm and the bacterial cells by the combination of an amphoteric tenside with the antibiotic metronidazole might be a useful strategy against BV.