Structure Elucidation, Total Synthesis, Antibacterial In Vivo Efficacy and Biosynthesis Proposal of Myxobacterial Corramycin.

Herein, we describe the myxobacterial natural product Corramycin isolated from Corallococcus coralloides. The linear peptide structure contains an unprecedented (2R,3S)-γ-N-methyl-ß-hydroxy-histidine moiety. Corramycin exhibits anti-Gram-negative activity against Escherichia coli (E. coli) and is taken up via two transporter systems, SbmA and YejABEF. Furthermore, the Corramycin biosynthetic gene cluster (BGC) was identified and a biosynthesis model was proposed involving a 12-modular non-ribosomal peptide synthetase/polyketide synthase. Bioinformatic analysis of the BGC combined with the development of a total synthesis route allowed for the elucidation of the molecule's absolute configuration. Importantly, intravenous administration of 20 mg kg-1 of Corramycin in an E. coli mouse infection model resulted in 100 % survival of animals without toxic side effects. Corramycin is thus a promising starting point to develop a potent antibacterial drug against hospital-acquired infections.

Production of the Bengamide Class of Marine Natural Products in Myxobacteria: Biosynthesis and Structure-Activity Relationships.

The bengamides, sponge-derived natural products that have been characterized as inhibitors of methionine aminopeptidases (MetAPs), have been intensively investigated as anticancer compounds. We embarked on a multidisciplinary project to supply bengamides by fermentation of the terrestrial myxobacterium M. virescens, decipher their biosynthesis, and optimize their properties as drug leads. The characterization of the biosynthetic pathway revealed that bacterial resistance to bengamides is conferred by Leu 154 of the myxobacterial MetAP protein, and enabled transfer of the entire gene cluster into the more suitable production host M. xanthus DK1622. A combination of semisynthesis of microbially derived bengamides and total synthesis resulted in an optimized derivative that combined high cellular potency in the nanomolar range with high metabolic stability, which translated to an improved half-life in mice and antitumor efficacy in a melanoma mouse model.

Biosynthetic Studies of Telomycin Reveal New Lipopeptides with Enhanced Activity.

Telomycin (TEM) is a cyclic depsipeptide antibiotic active against Gram-positive bacteria. In this study, five new natural telomycin analogues produced by Streptomyces canus ATCC 12646 were identified. To understand the biosynthetic machinery of telomycin and to generate more analogues by pathway engineering, the TEM biosynthesis gene cluster has been characterized from S. canus ATCC 12646: it spans approximately 80.5 kb and consists of 34 genes encoding fatty acid ligase, nonribosomal peptide synthetases (NRPSs), regulators, transporters, and tailoring enzymes. The gene cluster was heterologously expressed in Streptomyces albus J1074 setting the stage for convenient biosynthetic engineering, mutasynthesis, and production optimization. Moreover, in-frame deletions of one hydroxylase and two P450 monooxygenase genes resulted in the production of novel telomycin derivatives, revealing these genes to be responsible for the specific modification by hydroxylation of three amino acids found in the TEM backbone. Surprisingly, natural lipopeptide telomycin precursors were identified when characterizing an unusual precursor deacylation mechanism during telomycin maturation. By in vivo gene inactivation and in vitro biochemical characterization of the recombinant enzyme Tem25, the maturation process was shown to involve the cleavage of previously unknown telomycin precursor-lipopeptides, to yield 6-methylheptanoic acid and telomycins. These lipopeptides were isolated from an inactivation mutant of tem25 encoding a (de)acylase, structurally elucidated, and then shown to be deacylated by recombinant Tem25. The TEM precursor and several semisynthetic lipopeptide TEM derivatives showed rapid bactericidal killing and were active against several multidrug-resistant (MDR) Gram-positive pathogens, opening the path to future chemical optimization of telomycin for pharmaceutical application.

Discovery, Structure Elucidation, and Biological Characterization of Nannocystin†A, a Macrocyclic Myxobacterial Metabolite with Potent Antiproliferative Properties.

Microbial natural products are a rich source of bioactive molecules to serve as drug leads and/or biological tools. We investigated a little-explored myxobacterial genus, Nannocystis sp., and discovered a novel 21-membered macrocyclic scaffold that is composed of a tripeptide and a polyketide part with an epoxyamide moiety. The relative and absolute configurations of the nine stereocenters was determined by NMR spectroscopy, molecular dynamics calculations, chemical degradation, and X-ray crystallography. The compound, named nannocystin A (1), was found to inhibit cell proliferation at low nanomolar concentrations through the early induction of apoptosis. The mode of action of 1 could not be matched to that of standard drugs by transcriptional profiling and biochemical experiments. An initial investigation of the structure-activity relationship based on seven analogues demonstrated the importance of the epoxide moiety for high activity.

Pau d'arco activates Nrf2-dependent gene expression via the MEK/ERK-pathway.

Pau d'arco is a plant-derived traditional medicine that acts by poorly understood molecular mechanisms. Here, we studied the effect of pau d'arco on the cytoprotective transcription factor Nrf2. An aqueous extract of pau d'arco stimulated Nrf2-dependent gene expression and led to nuclear localization of Nrf2 in vitro. Chromatographic separation and mass spectrometry of the extract identified benzene trioles or benzene tetraoles within the active fractions. The extract stimulated the mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase (MEK)/extracellular-signal-regulated kinase (ERK1/2) pathway. The pharmacological inhibition of MEK, but not of p38 mitogen-activated protein kinase, glycogen synthase kinase-3 or phosphoinositide 3-kinase was required for the activation of Nrf2-dependent gene expression by pau d'arco, but not for the nuclear translocation of Nrf2. In vivo pau d'arco increased the expression of Nrf2-target genes in the intestine. The results suggest that the activation of Nrf2 could mediate beneficial effects of pau d'arco, in particular in the intestine.

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.

Etnangien, a macrolide-polyene antibiotic from Sorangium cellulosum that inhibits nucleic acid polymerases.

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).

Coniosetin, a novel tetramic acid antibiotic from Coniochaeta ellipsoidea DSM 13856.

The coprophilic ascomycete Coniochaeta ellipsoidea DSM 13856 forms the new antibiotic coniosetin (1) in surface cultures grown on a medium containing malt extract and oatmeal. The structure of the compound C25H35NO4, MW 413, was determined by 2D-NMR and mass spectrometric studies. Coniosetin belongs to the class of tetramic acids; it consists of a substituted aliphatic bicyclic ring system linked to a tetramic acid subunit through a carbonyl center. The absolute configuration was determined by measuring its circular dichroism spectrum and comparing the data with those of equisetin. Coniosetin has a pronounced antibacterial and antifungal action, inhibiting even multi drug-resistant strains of Staphylococcus aureus at a concentration of 0.3 microg/ml, though it is inactive against Gram-negative bacteria.

Cyclipostins, novel hormone-sensitive lipase inhibitors from Streptomyces sp. DSM 13381. II. Isolation, structure elucidation and biological properties.

Hormone-sensitive lipase (HSL) is a key enzyme of lipid metabolism and its control is therefore a target in the treatment of diabetes mellitus. Cultures of the Streptomyces species DSM 13381 have been shown to potently inhibit HSL. Ten inhibitors of HSL, termed cyclipostins, have been isolated from the mycelium of this microorganism and a further nine related compounds detected. Their structures were characterized by 2-D NMR experiments and by mass spectrometry and were found to comprise neutral cyclic enol phosphate esters with an additional y-lactone ring. On account of their ester-bound fatty alcohol side chain, the cyclipostins have physicochemical properties similar to those of triglycerides. The outstanding characteristic of the cyclipostins is their strong anti-HSL activity, with IC50 values in the nanomolar range.