The glyoxylate shunt as a target for antibacterial intervention in Pseudomonas aeruginosa
Pseudomonas aeruginosa is an opportunistic human pathogen responsible for a large proportion of drug-resistant, hospital-acquired infections worldwide. These infections are notoriously difficult to treat due to P. aeruginosa’s many intrinsic and acquired resistance mechanisms. Despite the critical need for new antibiotics against P. aeruginosa, the current pipeline is unable to meet the demand for treating these infections. P. aeruginosa can thrive in diverse infection scenarios by rewiring its central metabolism, and an example of this is in the lungs of cystic fibrosis patients, where this bacterium metabolises fatty acids into acetyl coenzyme A for biomass production via the glyoxylate shunt. The glyoxylate shunt is comprised of two enzymes: isocitrate lyase and malate synthase G, and allows the synthesis of cellular constituents from C2 nutrient sources, bypassing the decarboxylation steps of the citric acid cycle when glucose is not available. The glyoxylate shunt has potential as a new antibacterial target in P. aeruginosa as there is evidence to support that, besides carbon fixation, the glyoxylate shunt is implicated in virulence and is essential for establishing pulmonary infections. In this dissertation, the glyoxylate shunt was investigated as an antibacterial target in Pseudomonas aeruginosa. The biochemical characterisation of malate synthase G resulted in the first reported crystal structure solved to 1.6 Å resolution. Computational evaluation of the crystal structure revealed two promising binding sites suitable for future in silico drug design, which confirmed malate synthase as an apt drug target. Two 2-aminopyridine derivatives were found that displayed dual inhibition of the glyoxylate shunt enzyme activities as well as P. aeruginosa growth with low micromolar potencies. Insights into the different mechanisms of action on the target enzymes were explored using kinetic analysis and isothermal titration calorimetry. Hit to lead optimisation experiments of the hit compounds provided awareness of their potential for any future development as antibacterial agents. Although the compounds showed promising efficacies, in vitro drug metabolism and safety profiles, I also found that they displayed possible "off-target" effects and had issues in chemical stability. Together, these data indicate that the compounds warrant further chemical modification to improve these characteristics.