Novel Inhibitors of Cholesterol Degradation in Mycobacterium tuberculosis Reveal How the Bacterium’s Metabolism Is Constrained by the Intracellular Environment

Abstract

Mycobacterium tuberculosis (Mtb) relies on a specialized set of metabolic pathways to support 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 medium containing cholesterol as the principle carbon source. Based on this observation, we developed 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 revealed 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 adenylate cyclase Rv1625/Cya. These data implicate cyclic-AMP (cAMP) in regulating cholesterol utilization in Mtb, and are consistent with published reports indicating that propionate metabolism is regulated by cAMP levels. Intriguingly, reversal of the cholesterol-dependent growth inhibition caused by this subset of compounds could be achieved by supplementing the media with acetate, but not with glucose, indicating that Mtb is subject to a unique form of metabolic constraint induced by the presence of cholesterol.

Loving the poison: the methylcitrate cycle and bacterial pathogenesis

“…Building on these observations, an unbiased chemical screen was recently used to identify compounds that inhibit propionate catabolism and thereby diminish M. tuberculosis survival within macrophages. One such compound, V-13-009920, inhibited PrpC and prevented M. tuberculosis growth in cholesterol-containing media [60]. These results highlight the potential of targeting this pathway in prominent human pathogens as an antimicrobial strategy.…”

“…In support of this, PrpC was found to be required for intracellular growth in macrophages, indicating that propionyl-CoA metabolism is critical for maintaining the intracellular lifestyle [34,58,59]. Indeed, deletion of prpC impaired the ability of M. tuberculosis to grow in media containing cholesterol as a primary carbon source, and recent work has established that an M. tuberculosis prpCD double mutant also exhibits a growth defect in murine bone marrow-derived macrophages [60]. These observations are consistent with the notion that M. tuberculosis obtains a significant amount of its carbon requirements from cholesterol [61].…”

Meeting report: Adaptation and communication of bacterial pathogens

“…These chemical probes represent new classes of inhibitors that target metabolic pathways required to support growth of Mtb in its host cell. 30 In addition, studies into the mode of action of existing frontline drugs on intracellular Mtb revealed how hostderived stresses contribute to the rise of a drug tolerant phenotype. This does however represent an opportunity for the identification of synergistic inhibitors that could ameliorate existing drug therapies.…”

Selective Killing of Dormant Mycobacterium tuberculosis by Marine Natural Products

“…Recent studies have shown the value of screening compound libraries under in vivo-like conditions, since M. tuberculosis undergoes profound metabolic shifts during infection, which may alter target vulnerability. Inducing M. tuberculosis dormancy phenotypes in vitro can enrich the discovery of compounds targeting novel and conditionally essential pathways (13)(14)(15)(16)(17)(18). The Wayne model of gradual oxygen depletion is the most well-characterized in vitro dormancy model.…”