| Summary: | <p>The glutathione-gated potassium efflux system (Kef) is a K<sup>+</sup>/H<sup>+</sup> antiporter found in the majority of Gram negative pathogens, which shows potential as a novel antibiotic target. Kef plays a vital role in the protection of bacteria against toxic electrophiles through the regulation of cytoplasmic pH. Kef is inhibited by glutathione (<b>GSH</b>), ɣ L-glu-L-cys-gly, and activated by glutathione S-conjugates (<b>GSX</b>). Healy <em>et al</em>. have quantified the affinities of <b>GSH</b> and a range of <b>GSX</b> for <em>Shewanella denitrificans</em> Kef (SdKef). <b>GSH</b> was found to have a weak affinity of 900 μM, whereas the strongest GSX, <b><sup><em>t</em></sup></b></p>BuSG, was 400 nM. <p>This dissertation looks to understand the potency shown by <b><sup><em>t</em></sup></b></p>BuSG for <em>Sd</em>Kef by exploring the binding contributions from each group of the tripeptide. Truncated analogues of <b><sup><em>t</em></sup></b>BuSG were synthesised and evaluated using a competition fluorescence assay and <sup>1</sup>H CPMG NMR. In summary, removal of the glycine unit caused a complete loss in affinity for <em>Sd</em>Kef, whereas removing the glutamate unit resulted in a negligible loss. Interactions made by the Glu-Cys amide carbonyl oxygen, the Cys-Gly amide N<em>H</em> and a directional interaction of the conjugated thiol were also found to be important contributors to affinity. <p>The information obtained during this work allowed the development of a membrane permeant, truncated analogue of <b><sup><em>t</em></sup></b></p>BuSG, which lacks the majority of the glutamate, for use in in vivo studies. The truncated analogue is able to activate <em>Sd</em>Kef to elicit K<sup>+</sup> efflux, demonstrating that the majority of the glutamate is not essential for activity. Furthermore, application of this truncated analogue to <em>Escherichia coli</em> cells expressing the <em>sdkef</em> gene in a Kirby Bauer disc diffusion assay has demonstrated for the first time that small molecules activating <em>Sd</em>Kef can elicit inhibition of growth / cell death. Kef thus shows promise as a target for the development of novel antibacterial agents.
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