RESEARCH ARTICLE Novel Inhibitors of Cholesterol Degradation inMycobacterium tuberculosis Reveal How the Bacterium’s Metabolism Is Constrained by the Intracellular Environment

Mycobacterium tuberculosis (Mtb) relies on a specialized set of metabolic pathways to sup-port growth in macrophages. By conducting an extensive, unbiased chemical screen to identify small molecules that inhibit Mtb metabolism within macrophages, we identified a significant number of novel compounds that limit Mtb growth in macrophages and in medi-um containing cholesterol as the principle carbon source. Based on this observation, we de-veloped a chemical-rescue strategy to identify compounds that target metabolic enzymes involved in cholesterol metabolism. This approach identified two compounds that inhibit the HsaAB enzyme complex, which is required for complete degradation of the cholesterol A/B rings. The strategy also identified an inhibitor of PrpC, the 2-methylcitrate synthase, which is required for assimilation of cholesterol-derived propionyl-CoA into the TCA cycle. These chemical probes represent new classes of inhibitors with novel modes of action, and target metabolic pathways required to support growth of Mtb in its host cell. The screen also re-vealed a structurally-diverse set of compounds that target additional stage(s) of cholesterol utilization. Mutants resistant to this class of compounds are defective in the bacterial adenyl