Mode-of-action profiling reveals glutamine synthetase as a collateral metabolic vulnerability of M. tuberculosis to bedaquiline

Abstract

Combination chemotherapy can increase treatment efficacy and suppress drug resistance. Knowledge of how to engineer rational, mechanism-based drug combinations, however, remains lacking. Although studies of drug activity have historically focused on the primary drug-target interaction, growing evidence has emphasized the importance of the subsequent consequences of this interaction. Bedaquiline (BDQ) is the first new drug for tuberculosis (TB) approved in more than 40 y, and a species-selective inhibitor of the Mycobacterium tuberculosis (Mtb) ATP synthase. Curiously, BDQ-mediated killing of Mtb lags significantly behind its inhibition of ATP synthase, indicating a mode of action more complex than the isolated reduction of ATP pools. Here, we report that BDQ-mediated inhibition of Mtb's ATP synthase triggers a complex metabolic response indicative of a specific hierarchy of ATP-dependent reactions. We identify glutamine synthetase (GS) as an enzyme whose activity is most responsive to changes in ATP levels. Chemical supplementation with exogenous glutamine failed to affect BDQ's antimycobacterial activity. However, further inhibition of Mtb's GS synergized with and accelerated the onset of BDQ-mediated killing, identifying Mtb's glutamine synthetase as a collateral, rather than directly antimycobacterial, metabolic vulnerability of BDQ. These findings reveal a previously unappreciated physiologic specificity of ATP and a facet of mode-of-action biology we term collateral vulnerability, knowledge of which has the potential to inform the development of rational, mechanism-based drug combinations.

Keywords: antibiotics; drug combination; metabolomics; mode-of-action; tuberculosis.

Fig. 1.

Identification of activity-specific metabolic effects of BDQ. (A) Intrabacterial ATP pool sizes, expressed as nanomoles per milligram residual peptide (y axis), after exposure of Mtb to a dose range of BDQ (the active diastereomer) spanning 0–100× MIC or equimolar range of BDQi (the inactive enantiomer) concentrations. (B) Effect of BDQ and BDQi on calculated energy charge (EC) after exposure of Mtb to the same range of BDQ or BDQi concentrations as in A. (C and D) Stereochemical structures of BDQ and BDQi. (E) Schematic of BDQ-mediated inhibition of the Mtb ATP synthase and secondary effect on adenylate (AXP) species (ATP, ADP, and AMP) and EC. (F and G) Growth curves of Mtb strain H37Rv in the absence and presence of varying concentrations of BDQ or BDQi in 7H9 liquid medium. (H) Heat map profile depicting the relative levels of 130 metabolites after 24 h exposure of Mtb to BDQ or BDQi concentrations, as indicated. Columns represent individual treatments as indicated. Rows denote individual metabolites measured. Data were parsed by uncentered Pearson’s correlation with centroid linkage clustering and rendered using the image generation program Java TreeView (http://jtreeview.sourceforge.net/). Data are depicted on a log₂ scale relative to untreated control for each compound. G1, G2, G3, and G4 represent metabolite subgroups exhibiting BDQ-specific, but not BDQi-specific, responses, and are designated activity-specific metabolites. Among them, G3-typed metabolites (starred) show the similar changed kinetics with the EC’s curve. All data points shown in this figure represent the average of 3 technical replicates and are representative of 2 independent experiments.

Fig. 2.

BDQ-specific linkage of ATP and glutamine levels in Mtb. (A) Waterfall plot representation of metabolic correlation network between ATP and BDQ activity-specific metabolites as determined by Pearson pairwise correlation analysis. The blue columns represent the positive correlation pairs, and the orange ones represent the negative correlation pairs. Raw correlation coefficient values are labeled at each column as well listed in SI Appendix, Table S5. (B) Intrabacterial glutamine pool sizes, expressed as nanomoles per microgram residual peptide (y axis), after exposure of Mtb to a dose range of BDQ spanning 0–50× MIC or equimolar range of BDQi concentration. (C) Overlay plot of intrabacterial ATP and glutamine levels after exposure of Mtb to BDQ at 40× MIC over time (0 to 3 d); (D) Overlay plot of BDQ treatment on the EC of viable Mtb (left y axis) and in vitro activity of Mtb GlnA1 (right y axis) in the presence of the in vitro AXP ratio corresponding to the same range of BDQ concentrations. (E) Time course of de novo glutamine synthesis after 3-d preincubation of Mtb with BDQ (20× MIC), BDQi (same range as BDQ), or vehicle control (DMSO) followed by transfer to fresh media containing [15N] glutamate and metabolic profiling at the indicated points (0, 1, 2, and 3 h). (F) The EIC for [15N] glutamine (m/z = 146.0589 [M-H]⁻), as in E. (G) CFU-based assay of Mtb viability after exposure of Mtb to the same range of BDQ or BDQi concentrations, as in E. All data points shown in B–G represent the average of 3 technical replicates and are representative of 2 independent experiments.

Fig. 3.

Synergistic inhibition of Mtb viability and glutamine pools by BDQ and GS inhibitors. (A) In vitro inhibition curves and IC₅₀ values of Mtb GlnA1 by MSO (blue) and 2,4,5-TIM (red). (B, C, and H) Mtb growth inhibition curves of BDQ in the absence (red) and presence (blue) of varying concentrations of MSO (B, 0.25× MIC), Btz043 (C, 0.25× MIC), and TIM (H, 0.25× MIC). Curves depict OD₅₈₀ values after 10 d incubation in 7H9 liquid medium with 0.2% glucose and 0.2% glycerol as carbon sources and drugs, as indicated; the raw values of IC50 and ΔIC50 are listed in SI Appendix, Table S6. (D) CFU-based assay of Mtb viability after exposure to MSO (0.2× MIC), BDQ (1× MIC), or the combination. (E) Total bacterial glutamine pools after exposure of Mtb to BDQ (20× MIC), MSO (1× MIC), or the combination. **P < 0.01 by unpaired Student t test. (F) Schematic depicting mechanism of synergy between BDQ and GS inhibitor, MSO. (G) Partial rescue of BDQ (0.18 µM)-MSO (20 µM) combination with exogenous (5 mM) glutamine. (I) CFU-based assay of Mtb viability after exposure to TIM (0.5× MIC), BDQ (1× MIC), or the combination. For D, E, G, and I, statistical differences were determined by unpaired t test. ns = not significant; **P < 0.01; *0.01 < P < 0.05. All data points shown in this figure represent the average of 3 technical replicates and are representative of 2 independent experiments. Error bars correspond to SEs of measurement.