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

(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.