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Title: Bacterial kinases as potential targets for broad-spectrum antibiotics
Author: Batten, Laura Elizabeth
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2013
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Polyphosphate biosynthesis and the stringent response play an important role in the virulence of pathogenic bacteria. Our objective is to validate these pathways as an antimicrobial target and to identify inhibitors of the key enzymes polyphosphate kinase (PPK), (p)ppGpp synthetase I (RelA) and (p)ppGpp synthetase/hydrolase II (SpoT). The role of polyphosphate and (p)ppGpp metabolism in Francisella virulence has been explored with deletion mutants. These exhibited defects for intracellular growth in macrophages and were attenuated in mice, indicating a key role for the FtPPK, FtRelA and FtSpoT in the virulence of Francisella. The development of three in vitro activity assays will enable the discovery of PPK inhibitors. Ion-pairing HPLC analysis has been used to measure substrate kinetics, providing evidence that FtPPK belongs to the PPK2 superfamily with little preference between substrates (KM: ADP - 369 μM; GDP - 624 μM) as displayed by other PPK2 enzymes. 31P NMR spectroscopy has been used to monitor the overall time course of the PPK reaction. To facilitate high-throughput screening, a coupled luminescence based activity assay has been developed in a 96-well plate format. These assays can also be applied to the discovery of inhibitors for FtRelA and FtSpoT. Understanding the structural basis of inhibitor action requires a crystal structure of the target enzyme. For FtPPK, a crystallisation screen has identified conditions for obtaining suitable crystals and data has been collected to 2.1 Å resolution. Future studies will use the high throughput assay to identify PPK inhibitors; NMR and HPLC assays to characterise the mode of action and crystal structures of PPK:inhibitor complexes will identify the precise molecular interactions.
Supervisor: Roach, Peter Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry ; QR Microbiology ; RS Pharmacy and materia medica