Azetidines Kill Multidrug-Resistant Mycobacterium tuberculosis without Detectable Resistance by Blocking Mycolate Assembly

Abstract

Tuberculosis (TB) is the leading cause of global morbidity and mortality resulting from infectious disease, with over 10.6 million new cases and 1.4 million deaths in 2021. This global emergency is exacerbated by the emergence of multidrug-resistant MDR-TB and extensively drug-resistant XDR-TB; therefore, new drugs and new drug targets are urgently required. From a whole cell phenotypic screen, a series of azetidines derivatives termed BGAz, which elicit potent bactericidal activity with MIC₉₉ values <10 μM against drug-sensitive Mycobacterium tuberculosis and MDR-TB, were identified. These compounds demonstrate no detectable drug resistance. The mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with cell envelope biogenesis, specifically late-stage mycolic acid biosynthesis. Transcriptomic analysis demonstrates that the BGAz compounds tested display a mode of action distinct from the existing mycobacterial cell wall inhibitors. In addition, the compounds tested exhibit toxicological and PK/PD profiles that pave the way for their development as antitubercular chemotherapies.

Figure 1

BGAz derivatives synthesized.

Figure 2

Assessment of bactericidal activity of BGAz-004 and BGAz-005 against M. tuberculosis H37Rv. Average total viable counts (CFU mL^–1) of M. tuberculosis cultures exposed to either BGAz-004 (Panel A) or BGAz-005 (Panel B) at concentrations: 0 μM (0.1% DMSO) (circle, closed), 3, 6, 12, 24, 48, and 96 μM or isoniazid (Panel C) at concentrations 0 μM (0.1% DMSO), 0.9, 1.8, 3.7, 7.3, 14.6, and 29.2 μM over a 14-day time-course. Samples were taken after 0, 2, 6, 10, and 14 days of antibiotic exposure, serially diluted, and plated by the method of Miles et al. Statistical comparisons were performed at 6, 10, and 14 days of antibiotic exposure at 96 μM BGAz-004 and BGAz-005 using factorial ANOVA and posthoc Tukey’s honestly significant difference test (Panel D). Data represent three biological repeats ± standard deviation.

Figure 3

Assessment of bactericidal activity of BGAz-004 and BGAz-005 against M. tuberculosis H37Rv. Quantitation of Calcien-Violet-AM (CV-AM) and Sytox-green (SG) fluorescence of M. tuberculosis H37Rv, using flow cytometry, after exposure to BGAz-004 (column A) and BGAz-005 (column B) at concentrations: 0 μM (0.1% DMSO), 3, 6, 12, 24, 48, and 96 μM or (column C) isoniazid at concentrations 0 μM (0.1% DMSO), 0.9, 1.8, 3.7, 7.3, 14.6, and 29.2 μM over a 14-day time-course. The percentages of the population that are unstained or stained with each dye (or both dyes) are represented in four gates (rows P1–P4). Row P1: unstained population (CV-AM^–/SG^–); row P2: CV-stained population (CV-AM⁺/SG^–); row P3: dual-stained population (CV-AM⁺/SG⁺); and row P4: SG-stained population (CV-AM^–/SG⁺). Data represent three biological repeats ± standard deviation. Statistical comparisons were made using factorial ANOVA and posthoc Tukey’s honestly significant difference test.

Figure 4

Effect of BGAz-005 on the incorporation of radiolabeled precursors into the major cellular macromolecules of M. smegmatis. The incorporation of (A) [methyl-³H]thymidine (for DNA), (B) [5,6-³H]uridine (for RNA), (C) l-[4,5-³H]leucine (for protein), (D) [³H]meso-diaminopimelic acid (for peptidoglycan), and (E) [¹⁴C]acetic acid (for lipids) was measured over a period of 36 h. The percentage of incorporation measured at 36 h is represented in panel F. Each plot and error bars represent the average of three independent experiments.

Figure 5

Transcriptional response to BGAz-005 exposure demonstrating inhibition of mycobacterial cell envelope biosynthesis. (A) Cluster diagram of all genes showing similarity of biological replicates and separation of drug-treated from carrier control samples. (B) Volcano plot of M. bovis BCG response to BGAz-005, highlighting genes significantly differentially expressed. (C) Heatmap of 286 gene BGAz-005 signature relative to carrier control. Conditions as columns, genes as rows; red coloring highlighting induced genes and blue representing repressed genes. The BGAz-004 signature is clustered alongside, indicating a similar mode of drug action.

Figure 6

BCG cell envelope lipid analysis upon exposure to BGAz-005. BCG were cultured in 7H9 broth and exposed to increasing concentrations of BGAz-005. Lipids were selectively labeled with [¹⁴C]-acetic acid for 12 h, and cell envelope lipids were selectively removed by solvent extraction, separated by TLC (chloroform/methanol/water, 80:20:2, v/v/v), and visualized by autoradiography. (A) Equal volumes of lipids loaded adjusted for BCG growth; (B) equal counts of lipids (25,000 cpm) loaded; (C) mycolic acid methyl ester (MAME) analysis of cell-wall bound mycolates released by 5% TBAH and separated by TLC (petroleum ether/acetone, 95:5, v/v); (D) quantification of BCG lipids from panels A–C by densitometry. M. smegmatis cell envelope lipid analysis upon exposure to BGAz-005. (E) M. smegmatis were cultured in 7H9 broth, exposed to increasing concentrations of BGAz-005 for 6 h and the cell envelope lipids selectively removed by solvent extraction. Equal volumes of lipid adjusted by bacterial growth were separated by TLC (chloroform/methanol/water, 80:20:2, v/v/v) and stained with MPA or (F) alpha-naphthol. (G) Equal volumes of lipid adjusted by bacterial growth were separated by TLC (hexane/diethyl ether/acetic acid), 70:30:1, v/v/v and stained with MPA. (H) Equal volumes of lipid adjusted by bacterial growth were separated by 2D-TLC (direction 1 chloroform/methanol 96:4, v/v, direction 2 toluene/acetone 80:20, v/v) and stained with MPA.

Figure 7

Assessing the MIC shift of BGAz-004 and BGAz-005 against the AG85 complex. MIC values of BGAz-004, BGAz-005, and Ebselen were determined against BCG harboring overexpression vectors and compared to empty vector controls (pTIC6) in order to identify a shift in MIC against fbpA (A), fbpB (B), and fbpC (C). Fold change in MIC shift (D). The MIC₉₉ was calculated using an end point resazurin assay and the Gomperz equation for MIC determination (GraphPad Prism). Data are of triplicate repeats.