Lünse, Christina E.: Investigation of riboswitches as new antibacterial targets : Identification and characterization of novel synthetic and natural riboswitch modulators with effect on bacterial cell growth. - Bonn, 2013. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-29907
@phdthesis{handle:20.500.11811/5597,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-29907,
author = {{Christina E. Lünse}},
title = {Investigation of riboswitches as new antibacterial targets : Identification and characterization of novel synthetic and natural riboswitch modulators with effect on bacterial cell growth},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2013,
month = jan,

note = {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.},

url = {http://hdl.handle.net/20.500.11811/5597}
}

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