Characterization and targeting of the 2-methylcitrate cycle in Pseudomonas aeruginosa
Pseudomonas aeruginosa is an opportunistic human pathogen responsible for a significant proportion of hospital-acquired infections worldwide. P. aeruginosa infections are especially difficult to treat due to its numerous intrinsic and acquired resistance mechanisms. Even though P. aeruginosa was consequently recognised by the World Health Organization as a critical pathogen requiring urgent novel therapeutic agents, the current pipeline for drug discovery has been unable to meet this pressing demand. By rewiring its central metabolism, P. aeruginosa can thrive in diverse infection scenarios. For example, P. aeruginosa can colonize the cystic fibrosis lung by metabolizing long- and short-chain fatty acids, cholesterol and amino acids generated by degradation and fermentation of lung mucin. The metabolism of the above-mentioned nutrients requires a functional 2-methylcitrate cycle (2-MCC) to metabolize propionyl coenzyme A (PrCoA), a metabolic by-product. In addition, the 2-MCC also allows the utilization of propionate as a carbon source. As propionate and its derived catabolites, including PrCoA, are lethally toxic to cells, the 2-MCC is often viewed as a propionate detoxifying pathway. Generally, the 2-MCC is comprised of three core enzymes: 2-methylcitrate synthase (PrpC), 2-methylcitrate dehydratase (PrpD) and 2-methylisocitrate lyase (PrpB). The 2-MCC presents itself as a potential target for therapeutic intervention in P. aeruginosa; in addition to its role in carbon fixation, it is also involved in virulence and establishing infection. In this dissertation, core enzymes in the 2-MCC were characterized: PrpC, PrpD, and PrpB. The biochemical characterization entailed kinetic analysis of enzymes for the subsequent drug-design pipeline. Structural characterization of these three enzymes resulted in multiple crystal structures solved at near-atomic resolution. PrpC and PrpB were targeted for the development of novel antipseudomonal agents using high throughput fragment-based screening (HT-FBS) and X-ray crystallography. This was followed by fragment optimization, leading to one of the initial hits inhibiting PrpC activity in vitro, and preventing growth of P. aeruginosa in the presence of propionate. Putative mechanisms of inhibition are proposed based on the structural data. PrpD, on the other hand, was not included in the HT-FBS due to its non-essential nature in P. aeruginosa. However, NMR spectroscopy identified an unexpected PrpD substrate and product stereochemistry. The crystal structure of P. aeruginosa PrpD shed light on the likely active site and revealed the residues that are critical for activity. Together, these data provide an excellent foundation for targeting this class of enzymes and offers strategies aimed at further improving the inhibitory activity of the fragment hits.