This potentiation was biochemically accompanied by a sophisticated depletion of glutamine pools (strain made resistant to at least one 1 antibiotic exhibits hypersensitivity to another mechanistically unrelated antibiotic weighed against its drug-susceptible parent (36). compared to the isolated reduced amount of ATP swimming pools. Right here, we record that BDQ-mediated inhibition of Mtbs ATP synthase causes a complicated metabolic response indicative of a particular hierarchy of ATP-dependent reactions. We determine glutamine synthetase (GS) as an enzyme whose activity can be most attentive to adjustments in ATP amounts. Chemical substance supplementation with exogenous glutamine didn’t influence BDQs antimycobacterial activity. Nevertheless, additional inhibition of Mtbs GS synergized with and accelerated the starting point of BDQ-mediated eliminating, determining Mtbs 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. Growing concern IGFIR about antibiotic resistance and the faltering pipeline of new antibiotics have fueled renewed interest in combination chemotherapies for their ability to increase treatment efficacy and reduce rates of drug toxicity and resistance. However, the development of such drug combinations remains largely empiric (1). Traditional drug development has focused on exploiting the primary drugCtarget interaction (2, 3). Yet, recent work has begun to emphasize the functional importance of specific secondary or downstream consequences that follow this interaction and mediate drug activity, sometimes referred to as drug HI TOPK 032 mode of action (MoA) (4C8). Among modern infectious diseases, the importance of combination chemotherapies is established perhaps nowhere HI TOPK 032 more clearly than for tuberculosis (TB). Once associated with mortality rates of as high as 50%, TB was transformed into a predictably curable disease with the advent of antibiotics. This success was achieved only after the introduction of drug combinations that were prompted by early recognition of treatment relapse and secondary drug resistance after monotherapy. Unfortunately, while therapeutically effective, TB chemotherapies remain longer and more complex than for virtually any other bacterial infection, giving rise to unwanted rates of treatment failure, continued transmission, and the emergence of drug resistance (2, 3). Bedaquiline (BDQ) is a species-selective inhibitor of the c and subunits of the (Mtb) ATP synthase and first new TB drug approved in more than 40 y (9C11). BDQ exhibits bactericidal activity against Mtb in vitro, in mouse models of pulmonary TB, and in patients when given for either 2 or 6 mo in combination with a background regimen with multidrug-resistant TB (9, 12). On this basis, BDQ was approved by the US Food and Drug Administration for the treatment of pulmonary multidrug-resistant TB as part of combination therapy in adults (13). Unfortunately, this approval was accompanied by a black box warning because of concerns about potential HI TOPK 032 cardiac toxicities, while reports of clinical resistance to BDQ followed soon after (14). Here, we characterize the downstream secondary consequences of BDQ-mediated inhibition of the Mtb ATP synthase. We show that despite the hundreds of annotated ATP-dependent metabolic reactions in the cell, treatment of Mtb with BDQ has a preferential indirect secondary effect on glutamine biosynthesis, and that this effect can be exploited to increase the potency of BDQ when combined with an inhibitor of Mtbs glutamine synthetases. These studies thus reveal a previously underrecognized facet of MoA biology with the potential to inform the development of rational, drug-specific combination therapies. Results Identification of Activity-Specific Metabolic Effects of BDQ. Although potent, BDQ is widely recognized for a characteristic lag in onset of bactericidal activity, with little to no effect on bacterial viability during the first 1 to 2 2 d at concentrations up to 300 minimum inhibitory concentration (MIC) (15). This lag has been ascribed to the activity of adaptive mechanisms that enable Mtb to persist in slowed or arrested states of replication, where its ATP levels can decrease as much as 90% (16C18). We made use.