Antioxidant and Anti-Protease Activities of Diazepinomicin from the Sponge-Associated Micromonospora Strain RV115

Diazepinomicin is a dibenzodiazepine alkaloid with an unusual structure among the known microbial metabolites discovered so far. Diazepinomicin was isolated from the marine sponge-associated strain Micromonospora sp. RV115 and was identified by spectroscopic analysis and by comparison to literature data. In addition to its interesting preclinical broad-spectrum antitumor potential, we report here new antioxidant and anti-protease activities for this compound. Using the ferric reducing antioxidant power (FRAP) assay, a strong antioxidant potential of diazepinomicin was demonstrated. Moreover, diazepinomicin showed a significant antioxidant and protective capacity from genomic damage induced by the reactive oxygen species hydrogen peroxide in human kidney (HK-2) and human promyelocytic (HL-60) cell lines. Additionally, diazepinomicin inhibited the proteases rhodesain and cathepsin L at an IC50 of 70–90 µM. It also showed antiparasitic activity against trypomastigote forms of Trypanosoma brucei with an IC50 of 13.5 µM. These results showed unprecedented antioxidant and anti-protease activities of diazepinomicin, thus further highlighting its potential as a future drug candidate

A Novel Metagenomic Short-Chain Dehydrogenase/Reductase Attenuates Pseudomonas aeruginosa Biofilm Formation and Virulence on Caenorhabditis elegans

In Pseudomonas aeruginosa, the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (QS). N-Acylhomoserine lactones (AHLs) are an important class of signaling molecules involved in bacterial QS and in many pathogenic bacteria infection and host colonization are AHL-dependent. The AHL signaling molecules are subject to inactivation mainly by hydrolases (Enzyme Commission class number EC 3) (i.e. N-acyl-homoserine lactonases and N-acyl-homoserine-lactone acylases). Only little is known on quorum quenching mechanisms of oxidoreductases (EC 1). Here we report on the identification and structural characterization of the first NADP-dependent short-chain dehydrogenase/reductase (SDR) involved in inactivation of N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and derived from a metagenome library. The corresponding gene was isolated from a soil metagenome and designated bpiB09. Heterologous expression and crystallographic studies established BpiB09 as an NADP-dependent reductase. Although AHLs are probably not the native substrate of this metagenome-derived enzyme, its expression in P. aeruginosa PAO1 resulted in significantly reduced pyocyanin production, decreased motility, poor biofilm formation and absent paralysis of Caenorhabditis elegans. Furthermore, a genome-wide transcriptome study suggested that the level of lasI and rhlI transcription together with 36 well known QS regulated genes was significantly (≥10-fold) affected in P. aeruginosa strains expressing the bpiB09 gene in pBBR1MCS-5. Thus AHL oxidoreductases could be considered as potent tools for the development of quorum quenching strategies

New biofilm inhibition strategies, bioactive natural products from marine actinomycetes and semi-synthetic developments on cell wall precursors

Biofilme sind an Oberflächen anhaftende, schleimige Aufwüchse, mit denen sich Mikroorganismen ein eigenes Mikrohabitat geschaffen haben. Innerhalb des Biofilms findet eine chemische Kommunikation mittels sogenannter Autoinducer statt, die als Quorum-Sensing bezeichnet wird. Durch die Ausbildung des Biofilms sind die darin lebenden Mikroorganismen besser gegenüber schädlichen Umwelteinflüssen geschützt, was eine deutlich erhöhte Resistenz gegenüber Antibiotika und Bioziden zur Folge hat. Aufgrund dieser Tatsache ist die Verhinderung der Biofilmbildung an Orten, wo ein Bakterienwachstum unerwünscht ist, von entscheidender Bedeutung. In der vorliegenden Dissertation wurden Verfahren entwickelt, mit deren Hilfe sich die Funktion von Enzymen untersuchen lassen, die zu einer Inaktivierung einer Autoinducer-Klasse führen. Aus diesen Arbeiten konnte das Genprodukt BpiB09 als eine N-Acylhomoserinlacton abbauende Oxidoreduktase charakterisiert werden. Zudem konnte aus einem Screening-Prozess für vier unterschiedliche Naturstoffe eine potentiell biofilminhibierende Wirkung festgestellt werden. Des Weiteren wurden Methoden erarbeitet, um ein Autoinducer-System aus einem Violacin-Produzenten aufzuklären. Die Entdeckung neuer Naturstoffe und das Wissen über ihre biologischen Eigenschaften ist ein wichtiger Bestandteil in der Entwicklung neuer pharmazeutischer Wirkstoffe. In diesem Projekt wurden vier neue Gattungen von marinen Actinomyceten-Stämmen auf neue und biologisch aktive Verbindungen hin untersucht. Aus diesem Screening-Prozess ging das Diazepinomicin hervor. Für diese Verbindung konnten im weiteren Verlauf neue antioxidative und proteaseinhibierende Eigenschaften nachgewiesen werden. Das detaillierte Verständnis der Funktion von Penicillin-Bindeproteinen kann zu neuen Konzepten in der antibiotischen Wirkstoffforschung führen. Für die Aufklärung solcher Funktionalitäten wurden in diesem Projekt drei UDP-MurNAc-Hexapeptid-Glycin-Derivate semisynthetisch dargestellt. Zudem konnte ein massenspektrometrisches Verfahren zur Analytik der Zielverbindungen etabliert werden.Biofilms are mucous overgrowths adhering to surfaces by which microorganisms have created a microhabitat of their own. Inside the biofilm, a chemical interaction called quorum sensing takes place with the help of so called autoinducers. By developing the biofilm, the microorganisms living in it are better protected against harmful environmental influences, which makes them considerably more resistant to antibiotic agents and biocides. For this reason, it is vitally important to prevent the development of biofilms from happening in places where bacterial growth is unwanted. In the dissertation at hand, methods were developed to analyze the function of enzymes causing an inactivation of the autoinducer class. In the course of this work, the gene product BPiB09 could be characterized as an oxireductase that degrades N-Acyl homoserine lactone. Furthermore, a screening process made it possible to detect a potentially biofilm-inhibiting effect of four different natural substances. Over and above that, methods were developed to explain an autoinducer system from a violacein producer. The discovery of new natural products and the knowledge of their biological characteristics is an important part of the development of new pharmaceutical agents. In this project, four new genera of marine Actinomycetes were examined for new and biologically active compounds. From this screening, Diazepinomicin emerged. Later, new antioxidant and protease-inhibiting characteristics could be detected for this compound. A profound comprehension of the function of PBSs may reveal new concepts in antibiotic agent research. To clarify such functionalities, three glycine derivatives were successfully prepared. Moreover, a mass spectrometric method to analyze the target compounds could be established

In situ isobaric lipid mapping by MALDI-ion mobility separation-mass spectrometry imaging

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