Variations of the Mycobacterium abscessus F-ATP synthase subunit a-c interface alter binding and potency of the anti-TB drug bedaquiline.

The anti-tuberculosis therapeutic bedaquiline (BDQ) is used against Mycobacterium abscessus. In M. abscessus BDQ is only bacteriostatic and less potent compared to M. tuberculosis or M. smegmatis. Here we demonstrate its reduced ATP synthesis inhibition against M. abscessus inside-out vesicles, including the F1FO-ATP synthase. Molecular dynamics simulations and binding free energy calculations highlight the differences in drug-binding of the M. abscessus and M. smegmatis FO-domain at the lagging site, where the drug deploys its mechanistic action, inhibiting ATP synthesis. These data pave the way for improved anti-M. abscessus BDQ analogs.

Impaired ESX-3 Induces Bedaquiline Persistence in Mycobacterium abscessus Growing Under Iron-Limited Conditions.

ESX-3 is a secretion pathway which is essential for mycobactin-mediated iron acquisition under iron-limited conditions. Although present in all Mycobacterium sp., ESX-3 remains to be elucidated in Mycobacterium abscessus. In the study reported here, impaired ESX-3 seriously restricts the growth of M. abscesses under iron-limited conditions; growth is salvaged by functional ESX-3 or iron supplementation. Notably, impaired ESX-3 does not kill M. abscesses when environmental iron is insufficient but induces persistence to bedaquiline, a diarylquinoline class antibiotic used to treat multidrug-resistant mycobacteria. One potential mechanism contributing to persistence is the iron deficiency due to impaired ESX-3 suppressing succinate dehydrogenase activity, which dysregulates the tricarboxylic acid cycle and inactivates bedaquiline. Experiments conducted here also demonstrate that the regulator, MtrA, can bind ESX-3 and promote the survival of M. abscessus. As such, this study suggests that a novel pathway involving MtrA, ESX-3, iron metabolism, and the TCA cycle contributes to bedaquiline persistence in M. abscesses growing under iron-limited conditions.

Structure of mycobacterial ATP synthase bound to the tuberculosis drug bedaquiline.

Tuberculosis-the world's leading cause of death by infectious disease-is increasingly resistant to current first-line antibiotics1. The bacterium Mycobacterium tuberculosis (which causes tuberculosis) can survive low-energy conditions, allowing infections to remain dormant and decreasing their susceptibility to many antibiotics2. Bedaquiline was developed in 2005 from a lead compound identified in a phenotypic screen against Mycobacterium smegmatis3. This drug can sterilize even latent M. tuberculosis infections4 and has become a cornerstone of treatment for multidrug-resistant and extensively drug-resistant tuberculosis1,5,6. Bedaquiline targets the mycobacterial ATP synthase3, which is an essential enzyme in the obligate aerobic Mycobacterium genus3,7, but how it binds the intact enzyme is unknown. Here we determined cryo-electron microscopy structures of M. smegmatis ATP synthase alone and in complex with bedaquiline. The drug-free structure suggests that hook-like extensions from the α-subunits prevent the enzyme from running in reverse, inhibiting ATP hydrolysis and preserving energy in hypoxic conditions. Bedaquiline binding induces large conformational changes in the ATP synthase, creating tight binding pockets at the interface of subunits a and c that explain the potency of this drug as an antibiotic for tuberculosis.

Correlative light electron ion microscopy reveals in vivo localisation of bedaquiline in Mycobacterium tuberculosis-infected lungs.

Correlative light, electron, and ion microscopy (CLEIM) offers huge potential to track the intracellular fate of antibiotics, with organelle-level resolution. However, a correlative approach that enables subcellular antibiotic visualisation in pathogen-infected tissue is lacking. Here, we developed correlative light, electron, and ion microscopy in tissue (CLEIMiT) and used it to identify the cell type-specific accumulation of an antibiotic in lung lesions of mice infected with Mycobacterium tuberculosis. Using CLEIMiT, we found that the anti-tuberculosis (TB) drug bedaquiline (BDQ) is localised not only in foamy macrophages in the lungs during infection but also accumulate in polymorphonuclear (PMN) cells.

Active pulmonary targeting against tuberculosis (TB) via triple-encapsulation of Q203, bedaquiline and superparamagnetic iron oxides (SPIOs) in nanoparticle aggregates.

Tuberculosis (TB) has gained attention over the past few decades by becoming one of the top ten leading causes of death worldwide. This infectious disease of the lungs is orally treated with a medicinal armamentarium. However, this route of administration passes through the body's first-pass metabolism which reduces the drugs' bioavailability and toxicates the liver and kidneys. Inhalation therapy represents an alternative to the oral route, but low deposition efficiencies of delivery devices such as nebulizers and dry powder inhalers render it challenging as a favorable therapy. It was hypothesized that by encapsulating two potent TB-agents, i.e. Q203 and bedaquiline, that inhibit the oxidative phosphorylation of the bacteria together with a magnetic targeting component, superparamagnetic iron oxides, into a poly (D, L-lactide-co-glycolide) (PDLG) carrier using a single emulsion technique, the treatment of TB can be a better therapeutic alternative. This simple fabrication method achieved a homogenous distribution of 500 nm particles with a magnetic saturation of 28 emu/g. Such particles were shown to be magnetically susceptible in an in-vitro assessment, viable against A549 epithelial cells, and were able to reduce two log bacteria counts of the Bacillus Calmette-Guerin (BCG) organism. Furthermore, through the use of an external magnet, our in-silico Computational Fluid Dynamics (CFD) simulations support the notion of yielding 100% deposition in the deep lungs. Our proposed inhalation therapy circumvents challenges related to oral and respiratory treatments and embodies a highly favorable new treatment regime.

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

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.

Molecular mechanistic insights into uncoupling of ion transport from ATP synthesis.

A procedure is evolved to assess the maximum uncoupling activity of the classical unsubstituted phenolic uncouplers of mitochondrial oxidative phosphorylation (OX PHOS) 2,4-dinitrophenol and 2,6-dinitrophenol. The uncoupler concentrations, C, required for maximum uncoupling efficacy are found to be a strong function of the pH, and a linear relationship of pC with pH is obtained between pH 5 to pH 9. The slopes of the uncoupler concentrations in the aqueous and lipid phases as a function of pH have been estimated. It is shown that the experimental results can be derived from first principles by an enzyme kinetic model for uncoupling that is based on the same equations as formulated for the coupling of ion transport to ATP synthesis in a companion paper after imposition of the special conditions arising from the uncoupling process. The results reveal the catalysis of a reaction that involves both the anionic and protonated forms of the phenolic uncouplers in the vicinity of their binding sites in a non-aqueous region of the cristae membranes of mitochondria. The rate-limiting step in the overall process of uncoupling has been identified based on the uncoupling data. The data cannot be explained by a simple conduction of protons by uncouplers from one bulk aqueous phase to another as postulated by Mitchell's chemiosmotic theory. It is shown that Nath's two-ion theory of energy coupling/uncoupling in ATP synthase is consistent with the results. A molecular mechanism for uncoupling of ATP synthesis by the dinitrophenols is presented and the chief differences between coupling and uncoupling in ATP catalysis are summarized. The pharmacological consequences of our analysis of uncoupling are discussed, with particular reference to the mode of action of the anti-tuberculosis drug bedaquiline that specifically targets the c-subunit of the F1FO-ATP synthase and uncouples respiration from ATP synthesis in Mycobacterium tuberculosis. Hence the work is shown to be important both from the point of view of fundamental biology and is also pregnant with possibilities for practical pharmaceutical applications.

The NMR solution structure of Mycobacterium tuberculosis F-ATP synthase subunit ε provides new insight into energy coupling inside the rotary engine.

Mycobacterium tuberculosis (Mt) F1 F0 ATP synthase (α3 :ß3 :γ:δ:ε:a:b:b':c9 ) is essential for the viability of growing and nongrowing persister cells of the pathogen. Here, we present the first NMR solution structure of Mtε, revealing an N-terminal ß-barrel domain (NTD) and a C-terminal domain (CTD) composed of a helix-loop-helix with helix 1 and -2 being shorter compared to their counterparts in other bacteria. The C-terminal amino acids are oriented toward the NTD, forming a domain-domain interface between the NTD and CTD. The Mtε structure provides a novel mechanistic model of coupling c-ring- and ε rotation via a patch of hydrophobic residues in the NTD and residues of the CTD to the bottom of the catalytic α3 ß3 -headpiece. To test our model, genome site-directed mutagenesis was employed to introduce amino acid changes in these two parts of the epsilon subunit. Inverted vesicle assays show that these mutations caused an increase in ATP hydrolysis activity and a reduction in ATP synthesis. The structural and enzymatic data are discussed in light of the transition mechanism of a compact and extended state of Mtε, which provides the inhibitory effects of this coupling subunit inside the rotary engine. Finally, the employment of these data with molecular docking shed light into the second binding site of the drug Bedaquiline. DATABASE: Structural data are available in the PDB under the accession number 5YIO.

Network analysis identifies Rv0324 and Rv0880 as regulators of bedaquiline tolerance in Mycobacterium tuberculosis.

The resilience of Mycobacterium tuberculosis (MTB) emerges from its ability to effectively counteract immunological, environmental and antitubercular challenges. Here, we demonstrate that MTB can tolerate drug treatment by adopting a tolerant state that can be deciphered through systems analysis of its transcriptional responses. Specifically, we demonstrate how treatment with the antitubercular drug bedaquiline activates a regulatory network that coordinates multiple resistance mechanisms to push MTB into a tolerant state. Disruption of this network, by knocking out its predicted transcription factors, Rv0324 and Rv0880, significantly increased bedaquiline killing and enabled the discovery of a second drug, pretomanid, that potentiated killing by bedaquiline. We demonstrate that the synergistic effect of this combination emerges, in part, through disruption of the tolerance network. We discuss how this network strategy also predicts drug combinations with antagonistic interactions, potentially accelerating the discovery of new effective combination drug regimens for tuberculosis.

Efflux pump inhibitors: targeting mycobacterial efflux systems to enhance TB therapy.

The emergence of drug resistance continues to plague TB control, with a global increase in the prevalence of MDR-TB. This acts as a gateway to XDR-TB and thus emphasizes the urgency for drug development and optimal treatment options. Bedaquiline is the first new anti-TB drug approved by the FDA in 40 years and has been shown to be an effective treatment option for MDR Mycobacterium tuberculosis infection. Bedaquiline has also recently been included in clinical trials for new regimens with the aim of improving and shortening treatment periods. Alarmingly, efflux-mediated bedaquiline resistance, as well as efflux-mediated cross-resistance to clofazimine, has been identified in treatment failures. This mechanism of resistance results in efflux of a variety of anti-TB drugs from the bacterial cell, thereby decreasing the intracellular drug concentration. In doing so, the bacillus is able to render the antibiotic treatment ineffective. Recent studies have explored strategies to reverse the resistance phenotype conferred by efflux pump activation. It was observed that the addition of efflux pump inhibitors partially restored drug susceptibility in vitro and in vivo. This has significant clinical implications, especially in MDR-TB management where treatment options are extremely limited. This review aims to highlight the current efflux pump inhibitors effective against M. tuberculosis, the effect of efflux pump inhibitors on mycobacterial growth and the clinical promise of treatment with efflux pump inhibitors and standard anti-TB therapy.

Bedaquiline metabolism: enzymes and novel metabolites.

Bedaquiline is a recently approved drug for the treatment of multidrug-resistant tuberculosis. Adverse cardiac and hepatic drug reactions to bedaquiline have been noted in clinical practice. The current study investigated bedaquiline metabolism in human hepatocytes using a metabolomic approach. Bedaquiline N-demethylation via CYP3A4 was confirmed as the major pathway in bedaquiline metabolism. In addition to CYP3A4, we found that both CYP2C8 and CYP2C19 contributed to bedaquiline N-demethylation. The Km values of CYP2C8, CYP2C19, and CYP3A4 in bedaquiline N-demethylation were 13.1, 21.3, and 8.5 µM, respectively. We also identified a novel metabolic pathway of bedaquiline that produced an aldehyde intermediate. In summary, this study extended our knowledge of bedaquiline metabolism, which can be applied to predict and prevent drug-drug interactions and adverse drug reactions associated with bedaquiline.