Rapidly spreading antibiotic resistance and the low discovery rate of new antimicrobial compounds demand more effective strategies for early drug discovery. One bottleneck in the drug discovery pipeline is the identification of the modes of action (MoAs) of new compounds. We have developed a rapid systematic metabolome profiling strategy to classify the MoAs of bioactive compounds. The method predicted MoA-specific metabolic responses in the nonpathogenic bacterium after treatment with 62 reference compounds with known MoAs and different metabolic and nonmetabolic targets. We then analyzed a library of 212 new antimycobacterial compounds with unknown MoAs from a drug discovery effort by the pharmaceutical company GlaxoSmithKline (GSK). More than 70% of these new compounds induced metabolic responses in indicative of known MoAs, seven of which were experimentally validated. Only 8% (16) of the compounds appeared to target unconventional cellular processes, illustrating the difficulty in discovering new antibiotics with different MoAs among compounds used as monotherapies. For six of the GSK compounds with potentially new MoAs, the metabolome profiles suggested their ability to interfere with trehalose and lipid metabolism. This was supported by whole-genome sequencing of spontaneous drug-resistant mutants of the pathogen and in vitro compound-proteome interaction analysis for one of these compounds. Our compendium of drug-metabolome profiles can be used to rapidly query the MoAs of uncharacterized antimicrobial compounds and should be a useful resource for the drug discovery community.

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Choline favors the pathogenesis of Pseudomonas aeruginosa because hemolytic phospholipase C and phosphorylcholine phosphatase (PchP) are synthesized as a consequence of its catabolism. The experiments performed here resulted in the identification of the factors that regulate both the catabolism of choline and the gene coding for PchP. We have also identified and characterized the promoter of the pchP gene, its transcriptional organization and the factors that affect its expression. Deletion analyses reveal that the region between -188 and -68 contains all controlling elements necessary for pchP expression: a hypothetical -12/-24 promoter element, a consensus sequence for the integration host factor (-141/-133), and a palindromic sequence resembling a binding site for a potential enhancer binding protein (-190/-174). Our data also demonstrate that choline catabolism and NtrC (nitrogen regulatory protein) are necessary for the full expression of pchP and is partially dependent on σ(54) factor.

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