Optimization of CoaD Inhibitors against Gram-Negative Organisms through Targeted Metabolomics

Drug-resistant Gram-negative bacteria are of increasing concern worldwide. Novel antibiotics are needed, but their development is complicated by the requirement to simultaneously optimize molecules for target affinity and cellular potency, which can result in divergent structure–activity relationships (SARs). These challenges were exemplified during our attempts to optimize inhibitors of the bacterial enzyme CoaD originally identified through a biochemical screen. To facilitate lead optimization, we developed mass spectroscopy assays based on the hypothesis that levels of CoA metabolites would reflect the cellular enzymatic activity of CoaD. Using these methods, we were able to monitor the effects of cellular enzyme inhibition at compound concentrations up to 100-fold below the minimum inhibitory concentration (MIC), a common metric of growth inhibition. Furthermore, we generated a panel of efflux pump mutants to dissect the susceptibility of a representative CoaD inhibitor to efflux. These approaches allowed for a nuanced understanding of the permeability and efflux liabilities of the series and helped guide optimization efforts to achieve measurable MICs against wild-type <i>E. coli</i>

Fragment-Based Drug Discovery of Inhibitors of Phosphopantetheine Adenylyltransferase from Gram-Negative Bacteria

The discovery and development of new antibiotics capable of curing infections due to multidrug-resistant and pandrug-resistant Gram-negative bacteria are a major challenge with fundamental importance to our global healthcare system. Part of our broad program at Novartis to address this urgent, unmet need includes the search for new agents that inhibit novel bacterial targets. Here we report the discovery and hit-to-lead optimization of new inhibitors of phosphopantetheine adenylyltransferase (PPAT) from Gram-negative bacteria. Utilizing a fragment-based screening approach, we discovered a number of unique scaffolds capable of interacting with the pantetheine site of <i>E. coli</i> PPAT and inhibiting enzymatic activity, including triazolopyrimidinone <b>6</b>. Structure-based optimization resulted in the identification of two lead compounds as selective, small molecule inhibitors of bacterial PPAT: triazolopyrimidinone <b>53</b> and azabenzimidazole <b>54</b> efficiently inhibited <i>E. coli</i> and <i>P. aeruginosa</i> PPAT and displayed modest cellular potency against the efflux-deficient <i>E. coli</i> Δ<i>tolC</i> mutant strain

Fragment-Based Drug Discovery of Inhibitors of Phosphopantetheine Adenylyltransferase from Gram-Negative Bacteria

The discovery and development of new antibiotics capable of curing infections due to multidrug-resistant and pandrug-resistant Gram-negative bacteria are a major challenge with fundamental importance to our global healthcare system. Part of our broad program at Novartis to address this urgent, unmet need includes the search for new agents that inhibit novel bacterial targets. Here we report the discovery and hit-to-lead optimization of new inhibitors of phosphopantetheine adenylyltransferase (PPAT) from Gram-negative bacteria. Utilizing a fragment-based screening approach, we discovered a number of unique scaffolds capable of interacting with the pantetheine site of <i>E. coli</i> PPAT and inhibiting enzymatic activity, including triazolopyrimidinone <b>6</b>. Structure-based optimization resulted in the identification of two lead compounds as selective, small molecule inhibitors of bacterial PPAT: triazolopyrimidinone <b>53</b> and azabenzimidazole <b>54</b> efficiently inhibited <i>E. coli</i> and <i>P. aeruginosa</i> PPAT and displayed modest cellular potency against the efflux-deficient <i>E. coli</i> Δ<i>tolC</i> mutant strain