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.

Fig 1. Distribution of hit compound IC₅₀ values in macrophages and in 7H9 OADC.

Dot plot depicting the IC₅₀ values for the most potent 1,359 compounds in 7H9 OADC and in the macrophage infection assays. For both assays, compounds were tested across 8 separate 2-fold dilution series 50–0.4 μM. Universally active compounds with IC₅₀ values < 5.0 μM in macrophages and < 5.0 μM in 7H9 OADC are indicated in green. The conditionally active compounds with IC₅₀ values < 5.0 μM in macrophages and > 50.0 μM 7H9 OADC are depicted in red. Compounds with differential inhibitory activity are indicated in orange, grey, and blue.

Fig 2. Chemical rescue of Mtb ΔIcl1.

(A) Growth of Mtb ΔIcl1 was monitored in 7H9 OADC containing cholesterol (100 μM) or propionate (100 μM) in the presence of V-13–012725 (25 μM) and V-13–011503 (25 μM). Growth rescue by the compounds V-13–012725 and V-13–011503 is specific to cholesterol with no growth is observed in media containing propionate. (B) The compound, V-13–009920 (25 μM) rescues Mtb ΔIcl1 growth in 7H9 OADC media containing cholesterol (100 μM) and propionate (100 μM). Chemical rescue by V-13–009920 is comparable to rescue by vitamin-B12 (10 μg/ml). The data are representative of two independent experiments.

Fig 3. V-13–009920 inhibits the 2-methylcitrate synthase PrpC.

(A) Inhibition of PrpC enzyme activity was monitored by quantifying thiol release from propionyl-CoA leading to the formation of 3-thio-6-nitrobenzoate from DTNB measured at 412 nm. V-13–009920 inhibits pure PrpC enzyme with an IC₅₀ value of 4.0 ± 1.1 uM. (B) Chemical structure of V-13–009920 (5-(4-chlorophenyl)-N-(4-(N-(5-methyl-1,3,4-thiadiazol-2-yl)sulfamoyl)phenyl)-2-(trifluoromethyl)furan-3-carboxamide. Data are representative of two independent experiments.

Fig 4. V-13–012725 and V-13–011503 inhibit cholesterol breakdown.

(A) Growth of wild type Mtb is not inhibited in 7H9 OADC containing cholesterol (100 μM) and experimental compounds. (B) In 7H9 OADC containing cholesterol (100 μM) V-13–012725 and V-13–011503 specifically inhibit cholesterol turnover. (C) V-13–012725 and V-13–011503 directly inhibit the activity of the recombinant HsaAB enzyme complex with IC₅₀ values of 5.0 ± 0.8 and 11.0 ± 2.0 μM, respectively. (D) Chemical structures of V-13–012725, 2-(4-fluorophenyl)-5-methyl-1H-[1, 2, 4]triazolo[1, 5-a]pyrimidin-7-one and V-13–011503, 3,5-dimethyl-N-(5-phenyl-1,3,4-thiadiazol-2-yl)-1,2-oxazole-4-carboxamide. Data are representative of at least two independent experiments and error bars represent s.d.

Fig 5. Orphan cholesterol utilization inhibitors display transcriptional response characteristic of blocked cholesterol utilization.

(A) The methylcitrate genes are repressed in the presence of the Group 1 cholesterol utilization inhibitors. (B) Numerous genes in the KstR1 and KstR2 regulons are repressed in the presence of the orphan cholesterol utilization inhibitors. Data represent the normalized mean of three independent experiments with P-values <0.05. Data represents the ratio of gene expression values compared to DMSO controls.

Fig 6. Acetate rescues inhibition by orphan cholesterol utilization inhibitors.

(A) Wild type Mtb was grown in 7H12 media containing cholesterol (100 μM) and supplemented with the additional carbon substrates glucose (0.1%) and acetate (0.1%) as indicated. Growth inhibition was quantified using an Alamar Blue-based assay. (B) The compounds V-12–003679, V-12–007958, and V-12–007960 inhibit cholesterol utilization in media supplemented with cholesterol and the additional carbon substrates 0.1% glucose (glu) and 0.1% acetate (ace) indicated in parentheses. Data are representative of at least two independent experiments and error bars represent s.d.

Fig 7. Inhibition of cholesterol utilization by the orphan inhibitor V-12–007958 is dependent on cAMP levels.

(A) Transposon mutants with insertions in the gene rv1625/cya are resistant to V-12–007958 in 7H12 media containing cholesterol (100 μM). The transposon insertion sites in the rv1625/cya coding sequence are indicated. (B) The compounds V-12–003679, V-12–007958, and V-12–007960 all stimulate cAMP production in wild type Mtb grown in 7H12 media containing cholesterol (100 μM) and acetate (0.1%). (C) Cholesterol utilization is restored to levels similar to the uninhibited control in a transposon disrupted mutant with an insertion in rv1625/cya. Data are representative of at least two independent experiments, * = P < 0.05, and error bars represent s.d.