Overcoming and preventing bacterial resistance to antibiotics : the development and characterization of novel RNA polymerase and PqsD inhibitors

The treatment of bacterial infections is seriously hampered by the prevalence of resistance to clinically used antibiotics. Thus, there is an urgent need for the development of novel anti-infectives which are able to overcome existing resistances and do not provoke the quick emergence of new ones. In this work, an approach comprising a pharmacophore guided virtual screening is applied to identify novel scaffolds inhibiting the validated bacterial target RNA polymerase (RNAP). Structural modifications of the discovered hits result in potent RNAP inhibitors, which are active against Gram-positive pathogens and exhibit significantly lower resistance frequencies compared to clinically used rifampicin. Subsequent investigations concerning the molecular mechanism of RNAP inhibition reveal the compounds as inhibitors of protein-protein interaction between σ70 and the RNAP core enzyme and suggest the inhibitors’ binding site. In the second part of this work, the discovered compounds are demonstrated to additionally inhibit PqsD, an attractive target to disrupt cell-to-cell communication of Pseudomonas aeruginosa. For this promising anti-virulence concept, which should avoid the occurrence of resistance, bacterial cell death caused by RNAP inhibition is not intended. Thus, the structural requirements needed for PqsD selectivity are elucidated, thereby highlighting the versatility and potential of the discovered benzamidobenzoic acids in the fight against bacterial resistances.Die Behandlung von bakteriellen Infektionen wird durch Resistenzen gegen klinisch verwendete Antibiotika zunehmend gefährdet. Daher besteht großes Interesse an der Entwicklung neuer Antiinfektiva, die in der Lage sind bestehende Resistenzen zu überwinden und die schnelle Entstehung neuer zu vermeiden. In der vorliegenden Arbeit wird ein Pharmakophor-basiertes virtuelles Screening verwendet, um neue Inhibitoren des bakteriellen Targets RNA-Polymerase (RNAP) zu finden. Strukturelle Modifikationen der identifizierten Hits führen zu potenten RNAP-Hemmstoffen, die gegen grampositive Bakterien aktiv sind und weniger häufig zu Resistenzen führen als das klinisch eingesetzte Rifampicin. Nachfolgende Untersuchungen des molekularen Mechanismus der RNAP-Inhibition decken auf, dass die Verbindungen Hemmstoffe der Protein-Protein-Interaktion zwischen σ70 und dem RNAP Core-Enzym sind, und lassen auf die Bindestelle der Inhibitoren schließen. Im zweiten Teil der Arbeit wird gezeigt, dass die entdeckten Verbindungen außerdem PqsD hemmen, ein attraktives Target, das für die Zell-Zell-Kommunikation in Pseudomonas aeruginosa verantwortlich ist. Da in diesem Antivirulenzkonzept, das das Auftreten von Resistenzen verhindern soll, der bakterielle Zelltod durch RNAP-Hemmung unerwünscht ist, werden die strukturellen Voraussetzungen für PqsD-Selektivität aufgeklärt. Dabei werden die Vielseitigkeit und das Potenzial der entdeckten Benzamidobenzoesäuren im Kampf gegen bakterielle Resistenzen beleuchtet

Overcoming and preventing bacterial resistance to antibiotics : the development and characterization of novel RNA polymerase and PqsD inhibitors

The treatment of bacterial infections is seriously hampered by the prevalence of resistance to clinically used antibiotics. Thus, there is an urgent need for the development of novel anti-infectives which are able to overcome existing resistances and do not provoke the quick emergence of new ones. In this work, an approach comprising a pharmacophore guided virtual screening is applied to identify novel scaffolds inhibiting the validated bacterial target RNA polymerase (RNAP). Structural modifications of the discovered hits result in potent RNAP inhibitors, which are active against Gram-positive pathogens and exhibit significantly lower resistance frequencies compared to clinically used rifampicin. Subsequent investigations concerning the molecular mechanism of RNAP inhibition reveal the compounds as inhibitors of protein-protein interaction between σ70 and the RNAP core enzyme and suggest the inhibitors’ binding site. In the second part of this work, the discovered compounds are demonstrated to additionally inhibit PqsD, an attractive target to disrupt cell-to-cell communication of Pseudomonas aeruginosa. For this promising anti-virulence concept, which should avoid the occurrence of resistance, bacterial cell death caused by RNAP inhibition is not intended. Thus, the structural requirements needed for PqsD selectivity are elucidated, thereby highlighting the versatility and potential of the discovered benzamidobenzoic acids in the fight against bacterial resistances.Die Behandlung von bakteriellen Infektionen wird durch Resistenzen gegen klinisch verwendete Antibiotika zunehmend gefährdet. Daher besteht großes Interesse an der Entwicklung neuer Antiinfektiva, die in der Lage sind bestehende Resistenzen zu überwinden und die schnelle Entstehung neuer zu vermeiden. In der vorliegenden Arbeit wird ein Pharmakophor-basiertes virtuelles Screening verwendet, um neue Inhibitoren des bakteriellen Targets RNA-Polymerase (RNAP) zu finden. Strukturelle Modifikationen der identifizierten Hits führen zu potenten RNAP-Hemmstoffen, die gegen grampositive Bakterien aktiv sind und weniger häufig zu Resistenzen führen als das klinisch eingesetzte Rifampicin. Nachfolgende Untersuchungen des molekularen Mechanismus der RNAP-Inhibition decken auf, dass die Verbindungen Hemmstoffe der Protein-Protein-Interaktion zwischen σ70 und dem RNAP Core-Enzym sind, und lassen auf die Bindestelle der Inhibitoren schließen. Im zweiten Teil der Arbeit wird gezeigt, dass die entdeckten Verbindungen außerdem PqsD hemmen, ein attraktives Target, das für die Zell-Zell-Kommunikation in Pseudomonas aeruginosa verantwortlich ist. Da in diesem Antivirulenzkonzept, das das Auftreten von Resistenzen verhindern soll, der bakterielle Zelltod durch RNAP-Hemmung unerwünscht ist, werden die strukturellen Voraussetzungen für PqsD-Selektivität aufgeklärt. Dabei werden die Vielseitigkeit und das Potenzial der entdeckten Benzamidobenzoesäuren im Kampf gegen bakterielle Resistenzen beleuchtet

Composing compound libraries for hit discovery--rationality-driven preselection or random choice by structural diversity?

In order to identify new scaffolds for drug discovery, surface plasmon resonance is frequently used to screen structurally diverse libraries. Usually, hit rates are low and identification processes are time consuming. Hence, approaches which improve hit rates and, thus, reduce the library size are required

Benzamidobenzoic acids as potent PqsD inhibitors for the treatment of Pseudomonas aeruginosa infections.

Targeting PqsD is a promising novel approach to disrupt bacterial cell-to-cell-communication in Pseudomonas aeruginosa. In search of selective PqsD inhibitors, two series of benzamidobenzoic acids - one published as RNAP inhibitors and the other as PqsD inhibitors - were investigated for inhibitory activity toward the respective other enzyme. Additionally, novel derivatives were synthesized and biologically evaluated. By this means, the structural features needed for benzamidobenzoic acids to be potent and, most notably, selective PqsD inhibitors were identified. The most interesting compound of this study was the 3-Cl substituted compound 5 which strongly inhibits PqsD (IC₅₀ 6.2 μM) while exhibiting no inhibition of RNAP

Discovery of Novel Bacterial RNA Polymerase Inhibitors: Pharmacophore-Based Virtual Screening and Hit Optimization

The bacterial RNA polymerase (RNAP) is a validated target for broad spectrum antibiotics. However, the efficiency of drugs is reduced by resistance. To discover novel RNAP inhibitors, a pharmacophore based on the alignment of described inhibitors was used for virtual screening. In an optimization process of hit compounds, novel derivatives with improved in vitro potency were discovered. Investigations concerning the molecular mechanism of RNAP inhibition reveal that they prevent the protein–protein interaction (PPI) between σ⁷⁰ and the RNAP core enzyme. Besides of reducing RNA formation, the inhibitors were shown to interfere with bacterial lipid biosynthesis. The compounds were active against Gram-positive pathogens and revealed significantly lower resistance frequencies compared to clinically used rifampicin

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