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Antibacterial agents targeting aminoacyl-trna synthetases: understanding resistance to known inhibitors, and biological characterisation of novel inhibitors

Gupta, Arya (2017) Antibacterial agents targeting aminoacyl-trna synthetases: understanding resistance to known inhibitors, and biological characterisation of novel inhibitors. PhD thesis, University of Leeds.

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Antibiotics have been the pillars of modern medicine as they have been used for the prevention and treatment of bacterial infectious diseases. However, the rise in antibiotic resistance amongst pathogenic bacteria and the lack of new antibacterial agents reaching the clinic is concerning. The major aim of the consortium, New Antibacterials with Inhibitory Activity on Aminoacyl-tRNA Synthetases (NABARSI), was to discover new antibacterial agents with inhibitory activity against the underexploited drug targets, the aminoacyl-tRNA synthetase (aaRS) enzymes. As a part of NABARSI, this thesis describes the biological characterisation of novel aminoacyl-tRNA synthetase (aaRS) inhibitors and studies to better understand resistance in Staphylococcus aureus to known aaRS inhibitors. From a starting panel of 100,000 compounds screened in silico, 7000 were selected and tested for target binding activity against purified isoleucyl-tRNA synthetase (IleRS) and leucyl-tRNA synthetase (LeuRS) from both Staphylococcus aureus and Escherichia coli. Five of these were found to be inhibitory against E. coli. Conditional mutants of E. coli were used to confirm target specificity of these novel aaRS inhibitors, and it was shown that the antibacterial activity was a direct consequence of inhibition of LeuRS. However, E. coli was found to have a high frequency of spontaneous resistance to the compounds in vitro. Characterisation of spontaneous mutants resistant to these candidate antibacterial agents led to the identification of a novel resistance mechanism to aaRS inhibitors. To aid in assessing anti Gram-positive activity of the compounds developed within NABARSI, conditional mutants of S. aureus for ileS, leuS, serS and thrS were also generated and validated. The strains exhibited an increase in sensitivity (32-64 fold) to their cognate inhibitors compared to the parental strain, thus providing a platform to identify compounds with both whole cell and target specific activity. To better understand the underlying reason(s) of resistance to aaRS inhibitors and provide useful information for the possible development of new derivatives, the molecular basis of resistance to mupirocin and GSK2251052 (GSK’052) in S. aureus was examined. The results presented in this thesis provide evidence that the mupirocin-resistance proteins MupA and MupB, are functional isoleucyl-tRNA synthetase enzymes. Bioinformatic analysis of these proteins suggests the presence of an extra domain, which likely contributes to the observed mupirocin-resistance. Assessment of the resistance liability of GSK’052 in S. aureus indicate that the compound is not a suitable anti-staphylococcal agent and that resistance to the compound pre-exists in the clinic. The findings presented in this thesis highlights the importance of both the thorough biological characterisation of novel candidate antibacterial agents and understanding the molecular basis of resistance to antibacterial agents. Together they are able to provide useful information for developing new antibiotics or potent derivatives of existing ones.

Item Type: Thesis (PhD)
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds)
The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute for Molecular and Cellular Biology (Leeds)
Depositing User: Mr Arya Gupta
Date Deposited: 27 Nov 2017 14:08
Last Modified: 27 Nov 2017 14:08
URI: http://etheses.whiterose.ac.uk/id/eprint/18661

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