Structure Elucidation of Antibiotics.

To date, there are hundreds of characterized natural products with antibacterial activity against pathogenic bacteria, and several have become bonafide antibiotic drugs. The development of antibacterial natural products into antibiotic drugs, both in the past and in the future, hinges upon an accurate description of the exact chemical structure of the compound. Bolstered by some form of mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy is the primary technique for elucidating the chemical structure of organic molecules including natural products. By combining various one-dimensional (1D) and two-dimensional (2D) experiments, the connectivity between atoms is established and a complete "picture" of the molecule is thereby revealed.

Discovery of Thanafactin A, a Linear, Proline-Containing Octalipopeptide from Pseudomonas sp. SH-C52, Motivated by Genome Mining.

Genome mining of the bacterial strains Pseudomonas sp. SH-C52 and Pseudomonas fluorescens DSM 11579 showed that both strains contained a highly similar gene cluster encoding an octamodular nonribosomal peptide synthetase (NRPS) system which was not associated with a known secondary metabolite. Insertional mutagenesis of an NRPS component followed by comparative profiling led to the discovery of the corresponding novel linear octalipopeptide thanafactin A, which was subsequently isolated and its structure determined by two-dimensional NMR and further spectroscopic and chromatographic methods. In bioassays, thanafactin A exhibited weak protease inhibitory activity and was found to modulate swarming motility in a strain-specific manner.

Precursor-Directed Biosynthesis and Fluorescence Labeling of Clickable Microcystins.

Microcystins, cyclic nonribosomal heptapeptides, are the most well-known cyanobacterial toxins. They are exceptionally well studied, but open questions remain concerning their physiological role for the producing microorganism or their suitability as lead compounds for anticancer drug development. One means to study specialized metabolites in more detail is the introduction of functional groups that make a compound amenable for bioorthogonal, so-called click reactions. Although it was reported that microcystins cannot be derivatized by precursor-directed biosynthesis, we successfully used this approach to prepare clickable microcystins. Supplementing different azide- or terminal alkyne containing amino acid analogues into the cultivation medium of microcystin-producing cyanobacteria strains, we found that these strains differ strongly in their substrate acceptance. Exploiting this flexibility, we generated more than 40 different clickable microcystins. We conjugated one of these derivatives with a fluorogenic dye and showed that neither incorporation of the unnatural amino acid analogue nor attachment of the fluorescent label significantly affects the cytotoxicity against cell lines expressing the human organic anion transporting polypeptides 1B1 or 1B3. Using time-lapse microscopy, we observed that the fluorescent microcystin is rapidly taken up into eukaryotic cells expressing these transporters.

Nostotrebin 6 Related Cyclopentenediones and δ-Lactones with Broad Activity Spectrum Isolated from the Cultivation Medium of the Cyanobacterium Nostoc sp. CBT1153.

Cyanobacteria are an interesting source of biologically active natural products, especially chemically diverse and potent protease inhibitors. On our search for inhibitors of the trypanosomal cysteine protease rhodesain, we identified the homodimeric cyclopentenedione (CPD) nostotrebin 6 (1) and new related monomeric, dimeric, and higher oligomeric compounds as the active substances in the medium extract of Nostoc sp. CBT1153. The oligomeric compounds are composed of two core monomeric structures, a trisubstituted CPD or a trisubstituted unsaturated δ-lactone. Nostotrebin 6 thus far has been the only known cyanobacterial CPD. It has been found to be active in a broad variety of assays, indicating that it might be a pan-assay interference compound (PAIN). Thus, we compared the antibacterial and cytotoxic activities as well as the rhodesain inhibition of selected compounds. Because a compound with a δ-lactone instead of a CPD core structure was equally active as nostotrebin 6, the bioactivities of these compounds seem to be based on the phenolic substructures rather than the CPD moiety. While the dimers were roughly equally potent, the monomer displayed slightly weaker activity, suggesting that the compounds show unspecific activity depending upon the number of free phenolic hydroxy groups per molecule.

Scrophularia orientalis extract induces calcium signaling and apoptosis in neuroblastoma cells.

Effective neuroblastoma (NB) treatments are still limited despite treatment options available today. Therefore, this study attempted to identify novel plant extracts that have anticancer effects. Cytotoxicity and increased intracellular calcium levels were determined using the Sulforhodamine B (SRB) assay and Fluo4-AM (acetoxymethyl) staining and fluorescence microscopy in NB cells in order to screen a library of plant extracts. The current study examined the anticancer effects of a dichloromethane extract from Scrophularia orientalis L. (Scrophulariaceae), a plant that has been used in Traditional Chinese Medicine. This extract contained highly potent agents that significantly reduced cell survival and increased calcium levels in NB cells. Further analysis revealed that cell death induced by this extract was associated with intracellular calcium release, opening of the MPTP, caspase 3- and PARP-cleavage suggesting that this extract induced aberrant calcium signaling that resulted in apoptosis via the mitochondrial pathway. Therefore, agents from Scrophularia orientalis may have the potential to lead to new chemo-therapeutic anticancer drugs. Furthermore, targeting intracellular calcium signaling may be a novel strategy to develop more effective treatments for NB.