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>.