Characterizing novel drugs and chemical probes of biological systems is hindered by difficulties in identifying the mechanism of action (MOA) of biologically active molecules. Here we present a metabolite suppression approach to explore the MOA of antibacterial compounds under nutrient restriction. We assembled an array of metabolites that can be screened for suppressors of inhibitory molecules. Further, we identified inhibitors of E. coli growth under nutrient limitation and charted their interactions with our metabolite array. This strategy led to the discovery and characterization of three novel antibacterial compounds, MAC168425, MAC173979 and MAC13772. We showed that MAC168425 interferes with glycine metabolism, MAC173979 is a time-dependent inhibitor of p-aminobenzoic acid biosynthesis and MAC13772 inhibits biotin biosynthesis. We conclude that metabolite suppression profiling is an effective approach to focus MOA studies on compounds impairing metabolic capabilities. Such bioactives can serve as chemical probes of bacterial physiology and as leads for antibacterial drug development.

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