| Summary: | <p>More people are expected to die of antibiotic resistant infections than cancer by 2050. Resistance is seen in virtually all currently available antibiotics, and only two new classes have been developed since the 1960s. The Kef system is a novel prospective antibiotic target for a new antibiotic class. Kef is a H<sup>+</sup>/K<sup>+</sup> antiporter found in Gram negative bacteria including the ESKAPE pathogens, which are difficult to treat. Kef is involved in bacterial homeostasis and protection mechanisms against electrophiles. Glutathione (GSH) is the native ligand of Kef and can react with electrophiles in the cell to form adducts (GSX) which activate the Kef system and protect the cell. The highest affinity GSX is <sup>t</sup>BuSG, a synthetic peptide based ligand, from which was derived the dipeptide ligand 70.</p>
<p>The focus of this research was to design peptide mimetic Kef ligands based on 70, that disrupt the function of the Kef system, and inhibit bacterial growth. The new scaffolds are designed to have improved chemical properties relative to the dipeptides and greater synthetic flexibility for ligand development. Three ligand scaffolds and analogues were designed using amide isosteres to replace the central amide bond: a pyridinone (71, Chapter 2), an oxazole (72, Chapter 3), and an imidazopyridine (73, Chapter 4). </p>
<p>Chapter 2 describes the synthesis of 71, and ten carboxylic acid analogues, using a late stage diversification route. Varying the headgroups allowed exploration of the structure activity relationship (SAR) around the ligand binding site. All the tested analogues were found to bind Kef using water ligand observed via gradient spectroscopy (waterLOGSY) NMR. Differential scanning fluorimetry (DSF) indicated different pyridinone headgroups stabilised and destabilised the Kef system. Chapter 3 describes the synthesis of 72 from an optimised route based on prior literature, however binding was not observed by waterLOGSY NMR or DSF. Chapter 4 describes the synthesis of 73 from a substituted pyridine. The acid showed promising initial results as a peptide mimetic and bound during waterLOGSY NMR analysis. Investigation of the magnitude of stabilisation using DSF was inconclusive, suggesting 73 is weakly binding, and below the DSF detection limit. </p>
<p>Chapter 5 describes the use of the computational technique scaffold hopping to try and identify a new Kef ligand scaffold. ROCS, Spark, Blaze, and MOE programmes were used to search databases for a mimetic of the GSH backbone. A synthetically attractive hit 74 was selected and synthesised with three analogues, but these were not found to bind or activate Kef, from analysis by waterLOGSY NMR and Kirby Bauer disc assay. </p>
<p>The pyridinone and imidazopyridine scaffolds are the first peptide mimetic ligand families for SdKefQCTD. A high affinity Kef ligand could be developed into an antibiotic with a novel mode of action, providing a significant benefit to modern healthcare, with the increasing levels of antibiotic resistance.</p>
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