Investigation of riboswitches as new antibacterial targets : Identification and characterization of novel synthetic and natural riboswitch modulators with effect on bacterial cell growth

The increase in bacterial resistances and the decrease in available antibacterial substances against multi-drug resistant pathogens calls for an intensified search for novel antibiotics. Therefore, in this study riboswitches were investigated as new antibacterial drug targets. Riboswitches, which are RNA elements mostly found in the 5’ UTR of bacterial mRNA, consist of a metabolite-binding aptamer domain and an expression platform. They regulate up to 4% of all bacterial genes, either by inhibiting transcription or translation initiation. As many of the genes regulated by riboswitches are essential for bacterial functioning, manipulation of riboswitch activation may enable control over bacterial growth and viability. In this study, two riboswitches with distinct regulatory mechanisms were investigated: the glmS ribozyme and a thi-box riboswitch. The glmS riboswitch is a catalytically active RNA that regulates gene expression by recognition of glucosamine-6-phosphate (GlcN6P), subsequent self-cleavage, and degradation of glmS mRNA. Thi-box riboswitches recognize thiamine pyrophosphate (TPP) and are involved in the regulation of TPP-biosynthesis genes. While TPP is a fundamental cofactor for the crucial function of many bacterial proteins, GlcN6P is an important precursor of cell wall biosynthesis. Here, it was sought to identify modulators for these two riboswitch classes based on different strategies. Firstly, in silico predicted riboswitch ligands were validated in vitro. Secondly, in vitro and in vivo screenings were performed using rationally designed libraries of compounds with structural analogy to their natural ligand. Finally, also natural products were investigated for riboswitch modulation. The screening of designed compound analogs identified carba-sugars as potent activators of the glmS ribozyme of Staphylococcus aureus. These GlcN6P analogs activate the ribozyme to a level comparable with the natural metabolite in vitro and revealed inhibition of bacterial growth of a vancomycin-intermediate resistant S. aureus strain. Furthermore, compounds suggested by virtual screening, and natural substances such as aminoglycosides were shown to be inhibitors of glmS ribozyme cleavage. Finally, a natural GlcN-nitrosourea-derivative revealed glmS ribozyme activating potential in vitro, albeit at higher concentrations than carba-sugars. The E. coli thi-box riboswitch thiM was shown to be modulated by triazolethiamine (TT) and derivatives in vivo and increasing concentrations of TT led to a decrease in E. coli growth. Additionally, thiamine analogs containing metal-chelating groups that mimic the pyrophosphate moiety of TPP revealed efficient activation the thi-box riboswitch in vivo. This study describes the discovery and characterization of the first glmS ribozyme activating compound with effects on bacterial growth as well as thiM riboswitch activators that act through a distinct mechanism in comparison to the already described thiamine analog pyrithiamine (PT). The different screening assays applied as well as the compounds identified in this study enabled a thorough investigation of riboswitches and their artificial regulation. In the future, some of these promising riboswitch modulators may serve as lead structures for novel antibacterials to combat the increasing number of multi-drug resistant pathogens.Untersuchung von RNA-Schaltern als neue antibakterielle Zielstrukturen : Identifizierung und Charakterisierung neuer synthetischer und natürlicher RNA-Schalter-Modulatoren, die das Bakterienwachstum beeinflussen Durch die Zunahme bakterieller Resistenzen stehen immer weniger antibiotisch wirksame Substanzen gegen multiresistente Bakterienstämme zur Verfügung. Diese Tatsache begründet die fortlaufende, intensive Suche nach neuen Antibiotika. Im Fokus dieser Suche liegen neben Substanzen mit neuen Wirkungsmechanismen auch solche, die neue Zielstrukturen angreifen. In der vorliegenden Arbeit wurden RNA-Schalter (Riboswitches) als neue Zielstrukturen für die Entwicklung antibakterieller Wirkstoffe untersucht. Riboswitches sind RNA-Elemente, die zumeist in der 5’ untranslatierten Region bakterieller mRNA zu finden sind und aus einer Metabolit-bindenden Aptamerdomäne und einer Expressionsdomäne bestehen. Diese RNA-Strukturen regulieren bis zu 4 % aller bakteriellen Gene, entweder durch vorzeitiges Beenden der Transkription oder Hemmung der Translationsinitiation. Da viele Riboswitch-regulierte Gene essentiell für bakterielle Funktionen sind, ermöglicht die Manipulation der Riboswitch-Aktivierung eventuell die Kontrolle von Bakterienwachstum und -überleben. In dieser Arbeit wurden zwei RNA-Schalter mit verschiedenen regulatorischen Mechanismen untersucht: der glmS-RNA-Schalter sowie ein thi-Box Riboswitch. Der glmS Riboswitch ist eine katalytisch aktive RNA (ein Ribozym), welche durch Erkennung von Glucosamin-6-phosphat (GlcN6P), anschließender Selbstspaltung und Abbau der glmS mRNA die Genexpression reguliert. Thi-Box RNA-Schalter erkennen Thiaminpyrophosphat (TPP) und sind an der Regulation von Genen der TPP-Biosynthese beteiligt. Während TPP ein wichtiger Cofaktor für entscheidende Funktionen vieler bakterieller Proteine ist, spielt GlcN6P eine bedeutende Rolle als Vorläufer der Zellwandbiosynthese. In der hier vorliegenden Arbeit wurden verschiedene Strategien zur Identifikation von Modulatoren dieser Riboswitch-Klassen genutzt. Zum einen wurden in silico vorhergesagte Riboswitch-Liganden in vitro validiert. Zum anderen wurden in vitro und in vivo Screenings mit designten Bibliotheken unternommen, die strukturelle Ähnlichkeit zum natürlichen Liganden aufwiesen. Schließlich wurden auch natürliche Substanzen auf ihre Riboswitch-modulierenden Wirkungen hin untersucht. Im Screening der rational designten Substanzanaloga wurden Carba-Zucker als potente Aktivatoren des glmS-Ribozyms von Staphylococcus aureus identifiziert. Diese GlcN6P-Analoga aktivieren das Ribozym in vitro fast genauso stark wie der natürliche Metabolit und hemmen das Wachstum eines intermediär Vancomycin-resistenten S. aureus Stammes. Darüber hinaus konnte gezeigt werden, dass Substanzen, die im virtuellen Screening gefunden worden, sowie bereits bekannte, natürliche Substanzen wie beispielsweise aminoglycoside, die glmS-Ribozym-Selbstspaltung hindern können. Außerdem zeigte eine Glucosamin-Nitrosoharnstoff-Verbindung glmS-Ribozym-Aktivierung in vitro, jedoch bei höheren Konzentrationen als die Carba-Zucker. Desweiteren wurde gezeigt, dass der E. coli thi-Box Riboswitch thiM in vivo durch Triazolthiamin (TT) und Derivate moduliert wird und steigende Konzentrationen von TT zu einer Verminderung des Bakterienwachstums führen. Zusätzlich aktivierten Thiaminanaloga mit Metall-chelatierenden Gruppen, welche möglicherweise das Pyrophosphat von TPP imitieren, den ThiM Riboswitch in vivo. Diese Arbeit beschreibt die Entdeckung und Charakterisierung der ersten synthetischen Substanz, die das glmS Ribozym aktiviert und Bakterienwachstum hemmt, sowie thiM Riboswitch-Aktivatoren, die wahrscheinlich über einen vom bekannten Thiaminanalogon Pyrithiamine (PT) abweichenden Mechanismus auf den Riboswitch wirken. Die genutzen, unterschiedlichen Screening-Systeme sowie die in dieser Studie entdeckten Substanzen ermöglichen eine fundierte Untersuchung von Riboswitches und ihrer künstlichen Regulation. In Zukunft könnten einige dieser vielversprechenden Substanzen als Leitstrukturen für neue Antibiotika genutzt werden, um die steigende Zahl an multiresistenten Pathogenen zu bekämpfen

tRNA Modifications: Impact on Structure and Thermal Adaptation

Transfer RNAs (tRNAs) are central players in translation, functioning as adapter molecules between the informational level of nucleic acids and the functional level of proteins. They show a highly conserved secondary and tertiary structure and the highest density of post-transcriptional modifications among all RNAs. These modifications concentrate in two hotspots—the anticodon loop and the tRNA core region, where the D- and T-loop interact with each other, stabilizing the overall structure of the molecule. These modifications can cause large rearrangements as well as local fine-tuning in the 3D structure of a tRNA. The highly conserved tRNA shape is crucial for the interaction with a variety of proteins and other RNA molecules, but also needs a certain flexibility for a correct interplay. In this context, it was shown that tRNA modifications are important for temperature adaptation in thermophilic as well as psychrophilic organisms, as they modulate rigidity and flexibility of the transcripts, respectively. Here, we give an overview on the impact of modifications on tRNA structure and their importance in thermal adaptation

Carba-sugars Activate the glmS-Riboswitch of Staphylococcus aureus

The glmS-riboswitch is unique among riboswitch families as it represents a metabolite-dependent ribozyme that undergoes self-cleavage upon recognition of glucosamin-6-phosphate. The glmS-riboswitch is located in the 5′-untranslated region of bacterial genes involved in cell wall biosynthesis. Therefore, this riboswitch represents a promising target for developing new antibiotics. We describe the metabolite-dependent glmS-riboswitch of pathologically relevant and vancomycin-resistant Staphylococcus aureus and the discovery and synthesis of a carba-sugar with potency similar to that of the native metabolite glucosamine-6-phosphate in modulating riboswitch activity. This compound represents a valuable lead structure for the development of antibiotics with a novel mode of action

Numerous small hammerhead ribozyme variants associated with Penelope-like retrotransposons cleave RNA as dimers

<p>Hammerhead ribozymes represent the most common of the 9 natural classes of self-cleaving RNAs. The hammerhead catalytic core includes 11 highly-conserved nucleotides located largely within the unpaired regions of a junction formed by stems I, II and III. The vast majority of previously reported examples carry an additional pseudoknot or other tertiary interactions between nucleotides that precede stem I and nucleotides in the loop of stem II. These extra contacts are critical for high-speed RNA catalysis. Herein, we report the discovery of ∼150,000 additional variant hammerhead representatives that exhibit diminished stem III substructures. These variants are frequently associated with <i>Penelope</i>-like retrotransposons, which are a type of mobile genetic element. Kinetic analyses indicate that these RNAs form dimers to cleave RNA.</p