Mce3R Stress-Resistance Pathway Is Vulnerable to Small-Molecule Targeting That Improves Tuberculosis Drug Activities

Abstract

One-third of the world's population carries Mycobacterium tuberculosis (Mtb), the infectious agent that causes tuberculosis (TB), and every 17 s someone dies of TB. After infection, Mtb can live dormant for decades in a granuloma structure arising from the host immune response, and cholesterol is important for this persistence of Mtb. Current treatments require long-duration drug regimens with many associated toxicities, which are compounded by the high doses required. We phenotypically screened 35 6-azasteroid analogues against Mtb and found that, at low micromolar concentrations, a subset of the analogues sensitized Mtb to multiple TB drugs. Two analogues were selected for further study to characterize the bactericidal activity of bedaquiline and isoniazid under normoxic and low-oxygen conditions. These two 6-azasteroids showed strong synergy with bedaquiline (fractional inhibitory concentration index = 0.21, bedaquiline minimal inhibitory concentration = 16 nM at 1 μM 6-azasteroid). The rate at which spontaneous resistance to one of the 6-azasteroids arose in the presence of bedaquiline was approximately 10^-9, and the 6-azasteroid-resistant mutants retained their isoniazid and bedaquiline sensitivity. Genes in the cholesterol-regulated Mce3R regulon were required for 6-azasteroid activity, whereas genes in the cholesterol catabolism pathway were not. Expression of a subset of Mce3R genes was down-regulated upon 6-azasteroid treatment. The Mce3R regulon is implicated in stress resistance and is absent in saprophytic mycobacteria. This regulon encodes a cholesterol-regulated stress-resistance pathway that we conclude is important for pathogenesis and contributes to drug tolerance, and this pathway is vulnerable to small-molecule targeting in live mycobacteria.

Keywords: cholesterol; codrug; low oxygen.

Figure 1

Structures and synthesis of 6-azasteroids. Key intermediate 1 was prepared as described in the literature⁻ and subjected to further elaboration as shown.

Figure 2

6-Azasteroids improve the efficacy of INH. CDC1551 Mtb in 7H9 medium with cholesterol as a carbon source was incubated with 20 μM 6-azasteroid, finasteride, dutasteride, or DMSO control and varying concentrations of INH (2-fold dilutions) for 14 days at 37 °C. The INH concentration at which no growth occurred was recorded as the MIC. Results are shown as the ratio of the INH MIC in the presence of azasteroid or DMSO to the MIC of INH alone (0.2–0.4 μM). A ratio of 0.5–1 is considered to indicate no change in the INH MIC. The data shown are representative of at least two experiments.

Figure 3

Azasteroids 4a and 6a inhibit Mtb growth and are bactericidal in combination with INH or BDQ. (A) CDC1551 Mtb was cultured in 7H9 medium with glycerol as a carbon source and was diluted to an OD (600 nm) of 0.2. These cultures were incubated with 10 μM 4a or 6a or with DMSO as a control (two replicates), and the growth of Mtb was monitored by OD (600 nm) for 15 days. Data are representative of at least two experiments. (B) H37Rv(mlux) Mtb was cultured in 7H9/glycerol medium and diluted to approximately 5–10 RLU per well. These cultures were incubated with INH, BDQ, and/or 4a at the indicated concentrations or with DMSO as a control, and the growth of Mtb was monitored by autoluminescence for 9 days. (C) H37Rv(mlux) Mtb was cultured in 7H9/glycerol medium and diluted to approximately 5–10 RLU per well. These cultures were incubated with INH, BDQ, and/or 6a at the indicated concentrations or with DMSO as a control, and the growth of Mtb was monitored by autoluminescence for 9 days. (D) CDC1551 Mtb was cultured in 7H9/glycerol medium and diluted to an OD (600 nm) of approximately 0.002. These cultures were incubated with BDQ and/or 6a at the indicated concentrations or with DMSO as a control. After 2 or 7 days, the cultures were diluted, and the CFUs were determined by serial dilution onto agar plates. Error bars in panels B–D indicate SDs (n = 2). The limit of detection is 10 CFU/mL.

Figure 4

6-Azasteroids and INH or BDQ interact under low-oxygen conditions. Isobolographic analysis of codrug LORA MIC in H37Rv(mlux) Mtb: (A) INH and 4a or 6a and (B) BDQ and 4a or 6a. The lines connecting 400 μM INH or 0.21 μM BDQ and 13 μM 4a or 6a denote lines of additivity. H37Rv(mlux) Mtb was cultured and diluted to approximately 5–10 RLU per well. These cultures were incubated with INH or BDQ and 4a or 6a at a checkerboard of concentrations (3-fold dilutions) or with DMSO as a control under hypoxic conditions for 10 days. The RLU values for H37Rv(mlux) Mtb after 28 h of normoxic recovery were recorded. The drug concentrations in wells with at least 90% inhibition of growth relative to that in the no-drug control are plotted. Plotted values are averages (n = 3). (C) H37Rv(mlux) Mtb was cultured at approximately 1 × 10⁵ CFU/mL. These cultures were incubated with BDQ, 4a (10 μM), or 6a (10 μM), alone or in combination, at the indicated concentrations under hypoxic conditions for 10 days. The cultures were diluted, and CFUs were determined by serial dilution onto agar plates. Plotted values are averages with SDs (n = 3); *p < 0.05. The limit of detection is 10 CFU/mL.

Figure 5

Differentially expressed transcripts as analyzed by RNA-seq are visualized in heatmaps. The transcriptional profiles of Mtb strains CDC1551 (WT), ASR1, and ASR2 exposed for 6 h to 4a (30 μM) or 6a (30 μM) were compared with the profile of the corresponding untreated group. Gene names are indicated to the right of the heatmap, and strains and treatment conditions are shown on the bottom. The color scales represent log₂-fold changes in gene expression in treated groups relative to expression in untreated groups; each colored cell represents the mean of biological triplicates. Red, blue, and black lines denote the echA13-fadE17-fadE18, mel2, and mce3 operons, respectively.

Figure 6

Efficacy of 6-azasteroids requires Mce3R-regulated genes. Genes in the Mce3R regulon are required for 6-azasteroid antimycobacterial activity. Cholesterol transcriptionally regulates the Mce3R genes, and this regulon is repressed by Mce3R, a tetR-like repressor protein.