The Disorazole Z Family of Highly Potent Anticancer Natural Products from Sorangium cellulosum: Structure, Bioactivity, Biosynthesis, and Heterologous Expression.

Myxobacteria serve as a treasure trove of secondary metabolites. During our ongoing search for bioactive natural products, a novel subclass of disorazoles termed disorazole Z was discovered. Ten disorazole Z family members were purified from a large-scale fermentation of the myxobacterium Sorangium cellulosum So ce1875 and characterized by electrospray ionization-high-resolution mass spectrometry (ESI-HRMS), X-ray, nuclear magnetic resonance (NMR), and Mosher ester analysis. Disorazole Z compounds are characterized by the lack of one polyketide extension cycle, resulting in a shortened monomer in comparison to disorazole A, which finally forms a dimer in the bis-lactone core structure. In addition, an unprecedented modification of a geminal dimethyl group takes place to form a carboxylic acid methyl ester. The main component disorazole Z1 shows comparable activity in effectively killing cancer cells to disorazole A1 via binding to tubulin, which we show induces microtubule depolymerization, endoplasmic reticulum delocalization, and eventually apoptosis. The disorazole Z biosynthetic gene cluster (BGC) was identified and characterized from the alternative producer S. cellulosum So ce427 and compared to the known disorazole A BGC, followed by heterologous expression in the host Myxococcus xanthus DK1622. Pathway engineering by promoter substitution and gene deletion paves the way for detailed biosynthesis studies and efficient heterologous production of disorazole Z congeners. IMPORTANCE Microbial secondary metabolites are a prolific reservoir for the discovery of bioactive compounds, which prove to be privileged scaffolds for the development of new drugs such as antibacterial and small-molecule anticancer drugs. Consequently, the continuous discovery of novel bioactive natural products is of great importance for pharmaceutical research. Myxobacteria, especially Sorangium spp., which are known for their large genomes with yet-underexploited biosynthetic potential, are proficient producers of such secondary metabolites. From the fermentation broth of Sorangium cellulosum strain So ce1875, we isolated and characterized a family of natural products named disorazole Z, which showed potent anticancer activity. Further, we report on the biosynthesis and heterologous production of disorazole Z. These results can be stepping stones toward pharmaceutical development of the disorazole family of anticancer natural products for (pre)clinical studies.

A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansine site of tubulin.

Microtubule-targeting agents (MTAs) like taxol and vinblastine are among the most successful chemotherapeutic drugs against cancer. Here, we describe a fluorescence anisotropy-based assay that specifically probes for ligands targeting the recently discovered maytansine site of tubulin. Using this assay, we have determined the dissociation constants of known maytansine site ligands, including the pharmacologically active degradation product of the clinical antibody-drug conjugate trastuzumab emtansine. In addition, we discovered that the two natural products spongistatin-1 and disorazole Z with established cellular potency bind to the maytansine site on ß-tubulin. The high-resolution crystal structures of spongistatin-1 and disorazole Z in complex with tubulin allowed the definition of an additional sub-site adjacent to the pocket shared by all maytansine-site ligands, which could be exploitable as a distinct, separate target site for small molecules. Our study provides a basis for the discovery and development of next-generation MTAs for the treatment of cancer.

Solving the Puzzle of One-Carbon Loss in Ripostatin Biosynthesis.

Ripostatin is a promising antibiotic that inhibits RNA polymerase by binding to a novel binding site. In this study, the characterization of the biosynthetic gene cluster of ripostatin, which is a peculiar polyketide synthase (PKS) hybrid cluster encoding cis- and trans-acyltransferase PKS genes, is reported. Moreover, an unprecedented mechanism for phenyl acetic acid formation and loading as a starter unit was discovered. This phenyl-C2 unit is derived from phenylpyruvate (phenyl-C3) and the mechanism described herein explains the mysterious loss of one carbon atom in ripostatin biosynthesis from the phenyl-C3 precursor. Through in vitro reconstitution of the whole loading process, a pyruvate dehydrogenase like protein complex was revealed that performs thiamine pyrophosphate dependent decarboxylation of phenylpyruvate to form a phenylacetyl-S-acyl carrier protein species, which is supplied to the subsequent biosynthetic assembly line for chain extension to finally yield ripostatin.

Stereochemical determination of the leupyrrins and total synthesis of leupyrrin A1.

The stereochemical determination of the potent antifungal agents leupyrrin A1 and B1 and the total synthesis of leupyrrin A1 are reported. The relative and absolute configuration was determined by a combination of high field NMR studies, molecular modeling, and chemical derivatization. The expedient total synthesis involves a one-pot sequential Zr-mediated oxidative diyne-cyclization/regioselective opening sequence for preparation of the unique dihydrofuran ring, a highly stereoselective one-pot approach to the butyrolactone, a challenging sp(2)-sp(3) Suzuki coupling and a high-yielding Shiina macrolactonization.

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.

GE23077 binds to the RNA polymerase 'i' and 'i+1' sites and prevents the binding of initiating nucleotides.

Using a combination of genetic, biochemical, and structural approaches, we show that the cyclic-peptide antibiotic GE23077 (GE) binds directly to the bacterial RNA polymerase (RNAP) active-center 'i' and 'i+1' nucleotide binding sites, preventing the binding of initiating nucleotides, and thereby preventing transcription initiation. The target-based resistance spectrum for GE is unusually small, reflecting the fact that the GE binding site on RNAP includes residues of the RNAP active center that cannot be substituted without loss of RNAP activity. The GE binding site on RNAP is different from the rifamycin binding site. Accordingly, GE and rifamycins do not exhibit cross-resistance, and GE and a rifamycin can bind simultaneously to RNAP. The GE binding site on RNAP is immediately adjacent to the rifamycin binding site. Accordingly, covalent linkage of GE to a rifamycin provides a bipartite inhibitor having very high potency and very low susceptibility to target-based resistance. DOI: http://dx.doi.org/10.7554/eLife.02450.001.

Modular synthesis of polyene side chain analogues of the potent macrolide antibiotic etnangien by a flexible coupling strategy based on hetero-bis-metallated alkenes.

An efficient procedure for the concise synthesis of hetero-bis-metallated alkenes as useful building blocks for the modular access to highly elaborate polyenes and stabilized analogues is reported. By applying these bifunctional olefins in convergent Stille/Suzuki-Miyaura couplings, novel, carefully selected side chain analogues of the potent RNA polymerase inhibitor etnangien were synthesized by a modular late stage coupling strategy and evaluated for antibacterial and antiproliferative activities.

Opening and closing of the bacterial RNA polymerase clamp.

Using single-molecule fluorescence resonance energy transfer, we have defined bacterial RNA polymerase (RNAP) clamp conformation at each step in transcription initiation and elongation. We find that the clamp predominantly is open in free RNAP and early intermediates in transcription initiation but closes upon formation of a catalytically competent transcription initiation complex and remains closed during initial transcription and transcription elongation. We show that four RNAP inhibitors interfere with clamp opening. We propose that clamp opening allows DNA to be loaded into and unwound in the RNAP active-center cleft, that DNA loading and unwinding trigger clamp closure, and that clamp closure accounts for the high stability of initiation complexes and the high stability and processivity of elongation complexes.

Design, synthesis and biological evaluation of simplified side chains of the macrolide antibiotic etnangien.

Novel simplified side chains of the potent RNA polymerase inhibitor etnangien were designed, synthesized and evaluated for antibacterial activity against Gram-positive bacteria and one Gram-negative bacterium.

Sulfangolids, macrolide sulfate esters from Sorangium cellulosum.

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.

Sandaracinus amylolyticus gen. nov., sp. nov., a starch-degrading soil myxobacterium, and description of Sandaracinaceae fam. nov.

A novel starch-degrading myxobacterium designated NOSO-4(T) (new organism of the Sorangiineae strain 4) was isolated in 1995 from a soil sample containing plant residues, collected in Lucknow, Uttar Pradesh, India. The novel bacterium shows typical myxobacterial characteristics such as gram-negative, rod-shaped vegetative cells, swarming colonies, fruiting body-like aggregates and bacteriolytic activity. The strain is mesophilic, strictly aerobic and chemoheterotrophic. Based on 16S rRNA gene sequences, NOSO-4(T) shows highest similarity (96.2 %) with the unidentified bacterial strain O29 (accession no. FN554397), isolated from leek (Allium porrum) rhizosphere, and to the myxobacteria Jahnella thaxteri (88.9 %) and Chondromyces pediculatus (88.5 %). Major fatty acids are C(17 : 1) 2-OH, C(20 : 4)ω6 (arachidonic acid), and the straight-chain fatty acids C(17 : 0), C(15 : 0) and C(16 : 0). The genomic DNA G+C content of the novel isolate is 66.8 mol%. It is proposed that strain NOSO-4(T) represents a novel species in a new genus, i.e. Sandaracinus amylolyticus gen. nov., sp. nov., but also belongs to a new family, Sandaracinaceae fam. nov. The type strain of the type species, S. amylolyticus sp. nov., is NOSO-4(T) ( = DSM 53668(T) = NCCB 100362(T)).

New target for inhibition of bacterial RNA polymerase: 'switch region'.

A new drug target - the 'switch region' - has been identified within bacterial RNA polymerase (RNAP), the enzyme that mediates bacterial RNA synthesis. The new target serves as the binding site for compounds that inhibit bacterial RNA synthesis and kill bacteria. Since the new target is present in most bacterial species, compounds that bind to the new target are active against a broad spectrum of bacterial species. Since the new target is different from targets of other antibacterial agents, compounds that bind to the new target are not cross-resistant with other antibacterial agents. Four antibiotics that function through the new target have been identified: myxopyronin, corallopyronin, ripostatin, and lipiarmycin. This review summarizes the switch region, switch-region inhibitors, and implications for antibacterial drug discovery.

Insights into the complex biosynthesis of the leupyrrins in Sorangium cellulosum So ce690.

The anti-fungal leupyrrins are secondary metabolites produced by several strains of the myxobacterium Sorangium cellulosum. These intriguing compounds incorporate an atypically substituted γ-butyrolactone ring, as well as pyrrole and oxazolinone functionalities, which are located within an unusual asymmetrical macrodiolide. Previous feeding studies revealed that this novel structure arises from the homologation of four distinct structural units, nonribosomally-derived peptide, polyketide, isoprenoid and a dicarboxylic acid, coupled with modification of the various building blocks. Here we have attempted to reconcile the biosynthetic pathway proposed on the basis of the feeding studies with the underlying enzymatic machinery in the S. cellulosum strain So ce690. Gene products can be assigned to many of the suggested steps, but inspection of the gene set provokes the reconsideration of several key transformations. We support our analysis by the reconstitution in vitro of the biosynthesis of the pyrrole carboxylic starter unit along with gene inactivation. In addition, this study reveals that a significant proportion of the genes for leupyrrin biosynthesis are located outside the core cluster, a 'split' organization which is increasingly characteristic of the myxobacteria. Finally, we report the generation of four novel deshydroxy leupyrrin analogues by genetic engineering of the pathway.

Hepatitis C virus complete life cycle screen for identification of small molecules with pro- or antiviral activity.

Infection with the hepatitis C virus represents a global public health threat given that an estimated 170 million individuals are chronically infected and thus at risk for cirrhosis and hepatocellular carcinoma. A number of direct antiviral molecules are in clinical development. However, side effects, drug resistance and viral genotype-specific differences in efficacy may limit these novel therapeutics. Therefore, a combination of well tolerated drugs with distinct mechanisms of action targeting different steps of the viral replication cycle will likely improve viral response rates and therapy success. To identify small molecules that interfere with different steps of the HCV replication cycle, we developed a novel dual reporter gene assay of the complete HCV life cycle and adapted it to 384-well high-throughput format. The system is based on a highly permissive Huh-7 cell line stably expressing a secreted luciferase. Using these cells and an efficient HCV luciferase reporter virus, perturbations of each step of the viral replication cycle as well as cell viability can be easily and quantitatively determined. The system was validated with a selected set of known HCV entry, replication and assembly inhibitors and then utilized to screen a library of small molecules derived from myxobacteria. Using this approach we identified a number of molecules that specifically inhibit HCV cell entry, or primarily virus assembly and release. Moreover, we also identified molecules that increase viral propagation. These compounds may be useful leads for development of novel HCV inhibitors and could be instrumental for the identification of as yet unknown host-derived viral resistance and dependency factors.

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.

Biosynthesis of thuggacins in myxobacteria: comparative cluster analysis reveals basis for natural product structural diversity.

The thuggacins are macrolide antibiotics that are active against Mycobacterium tuberculosis, the causative agent of tuberculosis. Distinct variants of these structures are produced by the myxobacteria Sorangium cellulosum So ce895 and Chondromyces crocatus Cm c5, which differ in side chain structure and modification by hydroxylation. We report here a comparative analysis of the biosynthetic gene clusters in these strains, which reveals the mechanistic basis for this architectural diversity. Although the polyketide-nonribosomal peptide cores of the molecules are highly similar, the underlying biosynthetic machineries exhibit an unexpected degree of divergence. Furthermore, the S. cellulosum gene cluster contains a crotonyl-CoA reductase (CCR) homolog not present in C. crocatus, which likely participates in assembling the unusual hexyl side chain of the So ce895 thuggacins, whereas the distinct hydroxylation pattern may result from variable action of a conserved FMN-dependent monooxygenase. Indeed, inactivation of the monooxygenase gene in C. crocatus resulted in production of both mono- and di-deshydroxy thuggacin derivatives, providing direct evidence for the role of this enzyme in the pathway. Finally, integration of a Tn5-derived npt promotor upstream of the thuggacin cluster in C. crocatus led to a significant increase in thuggacin production.

Design, synthesis and biological evaluation of simplified analogues of the RNA polymerase inhibitor etnangien.

Novel simplified analogues of the potent RNA polymerase inhibitor etnangien were obtained by total synthesis and evaluated for antibacterial activity against Gram-positive bacteria and one Gram-negative bacterium.