Rationale Entwicklung von Antiinfektiva mit neuen Bindestellen und neuen Wirkmechanismen zur Überwindung bakterieller Resistenzen

Bacterial resistances are on the rise which necessitates the development of novel anti-infectives to stay forearmed against bacterial infections. In this work, two different strategies were pursued to accomplish this goal. The “switch region” of bacterial RNA polymerase (RNAP) was chosen as a novel binding site, which was recently found to be targeted by the natural alpha-pyrone antibiotic myxopyronin. It is of particular interest as the alpha-pyrone antibiotics show no cross-resistance to the clinically used rifampicin. Based on a hit compound obtained during a pharmacophore-based virtual screening, a series of small molecule inhibitors was synthesized and their in vitro potency was evaluated. The resulting compounds display good antibacterial activity against Gram positive bacteria and Gram negative Escherichia Coli TolC, coming along with a reduced resistance frequency compared to rifampicin. Furthermore, mutasynthesis was investigated as an approach to generate myxopyronin derivatives. Thereby, the substrate specificity of the involved biosynthetic enzymes was determined and the production of several myxopyronin analogs was analytically proven. A second attempt aimed for a reduction of P. aeruginosa virulence by jamming its quorum sensing enzyme PqsD. The application of molecular docking complemented by biophysical methods enabled the rational design of potent PqsD inhibitors. Moreover the structural features of two distinct inhibitor classes could be successfully combined.Bakterielle Resistenzen sind auf dem Vormarsch. Um weiterhin gegen bakterielle Infektionen gewappnet zu sein, ist die Entwicklung neuer Antiinfektiva erforderlich. In dieser Arbeit wurden zwei Strategien verfolgt um dieses Ziel zu erreichen. Die „Switch Region“ der bakteriellen RNA Polymerase (RNAP), kürzlich als Angriffspunkt des natürlichen alpha-Pyron Antibiotikums Myxopyronin entdeckt, wurde als neue Bindestelle ausgewählt. Da die alpha-Pyron Antibiotika keine Kreuzresistenz mit Rifampicin aufweisen ist sie von besonderem Interesse. Basierend auf einer Hit-Verbindung aus einem Pharmakophor basierten virtuellen Screening, wurde eine Reihe von Inhibitoren synthetisiert und auf ihre in vitro Potenz untersucht. Die resultierenden Verbindungen weisen gute antibakterielle Effekte und eine verringerte Resistenz-Frequenz im Vergleich zu Rifampicin auf. Daneben wurde die Mutasynthese als alternative Herstellungsmethode neuer Myxopyronin-Derivate untersucht. Hierbei konnte die Substratspezifität der beteiligten Biosynthese-Enzyme bestimmt und die Produktion verschiedener Myxopyronin Analoga analytisch nachgewiesen werden. Ein zweiter Versuch hatte eine Reduktion der Virulenz von P. aeruginosa durch Hemmung seines quorum sensing Enzyms PqsD zum Ziel. Der Einsatz von Docking, ergänzt durch biophysikalische Methoden ermöglichte das rationale Design potenter PqsD Inhibitoren. Außerdem konnten die strukturellen Eigenschaften zweier unterschiedlicher Inhibitor-Klassen erfolgreich kombiniert werden

Binding Mode Characterization of Novel RNA Polymerase Inhibitors Using a Combined Biochemical and NMR Approach

Bacterial RNA polymerase (RNAP) represents a validated target for the development of broad-spectrum antibiotics. However, the medical value of RNAP inhibitors in clinical use is limited by the prevalence of resistant strains. To overcome this problem, we focused on the exploration of alternative target sites within the RNAP. Previously, we described the discovery of a novel RNAP inhibitor class containing an ureidothiophene-2-carboxylic acid core structure. Herein, we demonstrate that these compounds are potent against a set of methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) strains (MIC 2–16 μg mL^–1) and rifampicin-resistant <i>Escherichia coli</i> TolC strains (MIC 12.5–50 μg mL^–1). Additionally, an abortive transcription assay revealed that these compounds inhibit the bacterial transcription process during the initiation phase. Furthermore, the binding mode of the ureidothiophene-2-carboxylic acids was characterized by mutagenesis studies and ligand-based NMR spectroscopy. Competition saturation transfer difference (STD) NMR experiments with the described RNAP inhibitor myxopyronin A (<b>Myx</b>) suggest that the ureidothiophene-2-carboxylic acids compete with <b>Myx</b> for the same binding site in the RNAP switch region. INPHARMA (interligand NOE for pharmacophore mapping) experiments and molecular docking simulations provided a binding model in which the ureidothiophene-2-carboxylic acids occupy the region of the <b>Myx</b> western chain binding site and slightly occlude that of the eastern chain. These results demonstrate that the ureidothiophene-2-carboxylic acids are a highly attractive new class of RNAP inhibitors that can avoid the problem of resistance