*3.6. PMF*

A wide range of compounds currently exists that target the PMF in bacteria, including rotenone which inhibits major proton pumps and protonophores (e.g., carbonyl cyanide m-chlorophenyl hydrazone, also known as CCCP), which translocate protons across the cell membranes [14] (Table 5). More specifically to mycobacteria, pyrazinoic acid, the active form of the first-line TB drug PZA, was demonstrated to decrease PMF and ATP levels in *M. bovis* BCG [182]. Several other compounds active against *M. tb* including SQ109, BDQ and CFZ were also found to be multi-targeting by behaving as uncouplers in addition to targeting enzymes [183]. While such an intrinsic and critical characteristic of the ETC is highly attractive therapeutically, such an approach would necessitate identifying compounds that are specific to the perturbation of mycobacterial PMF.

Recently, 2-aminoimidazoles (2-AI), a class of molecules with anti-biofilm activity, was shown to revert drug tolerance in an in vitro *M. tb* biofilm model [184]. This class of compounds potentiate the activity of β-lactams by altering protein secretion and lipid export, suggesting that 2-AI may perturb membrane energization [185]. Derivative 2B8 rapidly depolarized the membrane potential of live *M*. *smeg*, and collapsed the ΔpH generated by *M*. *smeg* IMVs energized with NADH, similar to CCCP and other mycobacterial uncouplers. In addition, a decrease in the OCR and intracellular ATP levels were observed in *M. tb* upon exposure to 2B8. Taken together, the perturbations of 2B8 on the PMF, OCR and ATP synthesis validates its uncoupling activity in mycobacteria [184].


**Table 5.** Inhibitors of the mycobacterial proton motive force (PMF) discuss in this review.

## *3.7. Respiratory Poisoning*

NO is a key component of the innate immune response against intracellular pathogens like *M. tb* [189]. Two drugs, PA-824 and DEL (OPC-67683), were shown to release NO when activated by the deazaflavin-dependant nitroreductase Ddn (rv3547) of *M. tb* [10,190]. PA-824 and DEL are bicyclic nitroimidazoles that are approved for the treatment of DR-TB as part of a drug combination. Both drugs kill replicating and non-replicating *M. tb*. Under aerobic conditions, PA-824 and DEL inhibit mycolic acid synthesis [191,192], whereas, under anaerobic conditions, Singh et al. identified that the release of NO correlated with the formation of des-nitroimidazole metabolites, leading to the antimicrobial activity of PA-824 [10]. Transcriptomic analysis of *M. tb* treated with DEL and PA-824 revealed that respiratory poisoning by NO is fundamental for the activity of the drug in mycobacteria [193]. The transcriptomic profile of bacteria exposed to DEL and PA-824 is similar to potassium cyanide, a cytochrome c oxidase-specific inhibitor, which suggests that NO poisoning in *M. tb* may lead to the inhibition of the terminal oxidases [190].

#### **4. Combinations Including ETC Inhibitors**

TB treatment relies on the combination of several antibacterial agents. New regimens for TB need to be (1) effective against DS- and DR-TB, (2) contain drugs with new mechanisms of action, (3) are suitable for oral administration, and (4) do not interfere with drugs used to treat chronic conditions or chronic infections [194]. An effective regimen should combine drugs that preserve or even potentiate their activity (additivity or synergism) when given as a regimen. Due to their ability to perturb the energy metabolism of replicating and non-replicating *M. tb*, including drugs that target the ETC may shorten treatments against DS- and DR-TB. The recent approval of the BPaL regimen (BDQ-PA824-Linezolid; Nix-TB trial) for DR-TB highlights that inhibiting components of the ETC is key in developing new regimens against *M. tb*, even though the relative contribution of each drug to the sterilizing potency of the BPaL regimen remains to be further investigated in humans.

Several inhibitors in lead optimisation were tested in combination with other anti-TB drugs. PAB, a Cyt-bcc oxidase inhibitor, resulted in the synergistic killing of *M. tb* under both replicating and non-replicating conditions when combined with CFZ [195]. PABs in combination with BDQ demonstrated antagonism at early time points, particularly under non-replicating conditions. However, this antagonistic effect disappeared within three weeks, with PAB-BDQ combinations becoming highly bactericidal [195]. The specificity of the PAB series needs to be further studied since it kills nutrient-starved *M. tb* while remaining bacteriostatic against replicating mycobacteria, a property

not shared with other specific Cyt-bcc inhibitors [130,153]. A MenA inhibitor, NM-4, was synergistic even at low doses together with sub-bactericidal concentrations of BDQ, CFZ, and the QcrB inhibitor ND-10885 [196], causing enhanced and efficient killing of *M. tb* in a time-kill curve assay [117]. These preliminary results underline the need to assess the efficacy of candidates in combination for TB therapy in an early stage of development.

Several ETC inhibitors are currently in ongoing pre-clinical and clinical trials to assess their efficacy against TB as part of new regimens [11]. These include BDQ, TBAJ-587 and TBAJ-876 (ATP synthase inhibitors), the first-line anti-TB drug PZA and SQ109 (inhibitors of the PMF), CFZ and its analogue TBI-166, and Q203 (inhibitor of Cyt-bcc-aa3 oxidase) [11]. SQ109 was reported to enhance the activity of anti-tuberculosis drugs INH, RIF and BDQ, and to shorten the time required to cure *M. tb*-infected mice [197]. Additionally, several inhibitors of the ETC compounds have a multi-target activity against the bacilli. The uncoupling effects of SQ109, PZA, BDQ, and CFZ suggest that these inhibitors may have a critical role due to their multi-targeting activity and should be taken into consideration for the further development of regimens against *M. tb*, particularly against DR-TB [183]. Assessing the in vitro or in vivo combinatory effects with repurposed or approved drugs for the treatment of TB has been another fruitful avenue to find new potential regimens comprising drug candidates that target the ETC. An example is TBI-166, an analogue of clofazimine with excellent potency alone or in combination with BDQ, PZA and linezolid in vitro and in vivo [198].

Q203 and BDQ were the first-in-class, orally-available representatives of the Cyt-bcc-aa3 and ATP synthase inhibitors, respectively. Q203 is currently in Phase 2b clinical trials and demonstrated potency in a 14-day, proof-of-concept design study of early bactericidal activity. It was safe and well-tolerated throughout the different dose strengths (100, 200 and 300 mg). Preliminary studies in vivo using a mouse footpad infection model of Buruli ulcer (*M. ulcerans*) showed that the addition of Q203 to the two-drug regimen of RIF and CFZ, or the three-drug regimen rifamycin, CFZ, and BDQ can decrease the treatment duration from four to two weeks, without any relapse after 12 weeks from the completion of treatment [199]. A similar approach could be developed to find an effective Q203-based regimen against *M. tb* with the addition of a chemical inhibitor of the Cyt-bd. Nonetheless, the plasticity and the possible re-routing of the mycobacterial ETC through chemical inhibition can be used against mycobacteria to conceive an effective regimen. Bioenergetics and *ex vivo* efficacy studies revealed that a combination of BDQ, CFZ and Q203 killed *M. tb* synergistically, with BDQ and Q203 potentiating CFZ's ROS production [158]. These results suggest that the potentiation of Q203 can be achieved without a Cyt-bd oxidase inhibitor as well.

With the number of BDQ-containing regimens undergoing clinical trials, BDQ seems a drug of choice for further development of TB therapy. Several Phase 1 and 2 clinical trials which include BDQ are currently in progress [194]. However, the emergence of resistance and its high cardiotoxicity may jeopardize the potency of this antitubercular drug. The development of new ATP synthase inhibitors that harbour less cardiotoxicity and are potentially less prone to the development of resistance, such as TBAJ-876 and TBAJ-587, will most likely lead to the development of new regimens including these second generation of diaryquinolines [179,180].

### **5. Conclusions and Perspectives**

This review has highlighted the current efforts made to find new inhibitors against components of the ETC. To date, tackling the mycobacterial OxPhos pathway has been a prolific avenue in finding new inhibitors against *M. tb*, as well as other mycobacteria such as *M. ulcerans*. Deciphering the role of each new scaffold targeting ETC components will enable a further understanding of this pivotal pathway in *M. tb* survival and metabolism. Several components are still poorly exploited as potential targets for chemical inhibition, either due to their homology to eukaryotic components or their regulation as a specific bacterial response to the environment/stress (e.g., Cyt-bd). Redundancy of several components of the ETC further complicates the establishment of a background regimen comprising of ETC inhibitors. A deeper understanding of the intricately-linked energy metabolism processes would be required to

target the various metabolic pathways which *M. tb* can reroute to, such that a successful regimen can be developed. Most screens to identify inhibitors of the OxPhos have been conducted using whole-cell assays, target-based assays or phenotypic screening using ATP as a readout. However, these screening methods may not be suitable for the identification of inhibitors of some components such as the Cyt-bd, which are conditionally essential under specific conditions. Therefore, a better understanding of the modulation of the ETC under the host physiological conditions encountered by *M. tb* would be of great interest for further development of energy metabolism inhibitors. Lastly, ETC inhibitors have the potential to revolutionise future TB treatments by contributing to efficacious regimens which are simpler and shorter, as evident from BDQ's role in the novel, three-drug, all-oral BPaL regimen.

**Author Contributions:** Conceptualization, C.S.-Y.F., K.P. and A.L.; writing—original draft preparation, C.S.-Y.F., K.P. and A.L.; writing—review and editing, C.S.-Y.F., K.P. and A.L.; visualization, C.S.-Y.F. and A.L.; project administration, K.P. and A.L.; funding acquisition, K.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported in part by the National Research Foundation (NRF) Singapore, NRF Competitive Research Programme (CRP), Grant Award Number NRF–CRP18–2017–01 (K.P.)

**Acknowledgments:** We would like to thank Marcel Behr and Jean-Yves Dubé for their contributions to the revision of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results
