*3.3. Inhibitors of the Cyt-bcc-aa3 Complex*

A number of chemically-diverse and distinct scaffolds target the *M. tb* cytochrome bcc oxidase by binding at the Qp site of the QcrB subunit, otherwise known as the stigmatellin pocket. The Qp site is one of two quinol catalytic sites on QcrB. The promiscuous nature of this target has been attributed to its localization in the bacterial membrane, as with other membrane-associated targets [54,68]. In this section, we provide an overview of chemical classes reported to inhibit the Cyt-bcc-aa3 terminal oxidase (Table 3).


**Table 3.** Inhibitors of the Cyt-bcc-aa3 complex and the bd oxidase discussed in this review.


**Table 3.** *Cont*.

**Table 3.** *Cont*.

Several promising hits belonging to the chemical class of imidazopyridines (IPs) have been identified in independent screening programs [12,75,137–139]. Extensive efforts have been undertaken to optimise and to explore the SAR of this scaffold [126,140–143]. To date, the most advanced QcrB inhibitor is Q203, an IP derivative in Phase 2 clinical trials. The drug candidate was initially discovered in a phenotypic high-content screen in *M. tb*-infected macrophages and further optimised to achieve an MIC50 of 2.7 nM against *M. tb* H37Rv in vitro. Its activity is comparable to that of BDQ and INH in a mouse model of chronic TB infection [12]. In addition to its potency, the positive results from the Phase 1 and Phase 2a early bactericidal activity clinical trial indicate its safety and tolerability in TB patients, emphasizing the specificity of bacterial inhibition without affecting the human counterpart [144]. Resistant mutants generated to Q203 revealed amino acid mutations T313A or T313I in QcrB [12]. This interaction between Q203 and QcrB has recently been biochemically demonstrated by in-cell NMR using whole *M. smeg* cells expressing the *M. tb* QcrCAB complex and a Q203 derivative, indicating target engagement at the Qp site of QcrB [145]. While bacteriostatic on its own in *M. tb*, Q203 becomes highly bactericidal when the alternate Cyt-bd oxidase is absent, as demonstrated in *M. tb*-infected macrophages and in a mouse model of acute TB infection [65,146]. Q203 is also highly bactericidal and potent against *Mycobacterium ulcerans*, reducing bacterial load in a mouse footpad infection model of Buruli ulcer by 99.99% at a dose of 0.5 mg/kg administered three times per week for four weeks [147]. This highlights additional opportunities for repurposing QcrB inhibitors against other organisms that do not harbour functional alternative terminal oxidases, such as *M. ulcerans* and *Mycobacterium leprae*.

Extended SAR studies of IPs imidazopyridine carboxamides led to the novel scaffold imidazothiazole carboxamides (ITA), of which analogues have nanomolar potency (MIC < 10 nM) against replicating and drug-resistant *M. tb* [143]. Derivatives of this class also demonstrate intracellular activity in macrophages and in vivo [143]. Additionally, cross-resistance studies with strains harbouring QcrB mutations and dose-response studies in a *M. tb* strain without Cyt-bd oxidase indicate that these compounds target QcrB [143]. This series is promising for its tolerability and good oral bioavailability in mice [126].

Imidazopyridine ethers (IPEs) were identified from a biochemical screen of AstraZeneca's corporate compound collection aimed at identifying inhibitors of ATP homeostasis [127]. The extent of ATP synthesis inhibition, as measured in IMV of *M. smeg*, correlated well with anti-tubercular potency in *M. tb*, with a MIC of 0.03 μM for some optimised derivatives [127]. While generating resistant mutants to the series was unsuccessful, target site deconvolution through a series of biochemical tests and cell-based assays indicated that these chemical entities target the Cyt-bcc-aa3 complex. These series have been deprioritised by the group due to their poor solubility and poor PK profile [127].

Structurally similar to the IPs, the pyrazolopyridine carboxamide series were designed as novel anti-tubercular agents through a scaffold hopping strategy [148,149]. The lead compound, TB-47, is currently in early-stage development. It has activity against replicating *M. tb* in vitro with MICs of 0.016 to 0.5 μg/mL when tested against a panel of *M. tb* drug-susceptible and drug-resistant

clinical isolates, and a high-selective index. Furthermore, TB-47 is well-tolerated and has good oral bioavailability in rats. While it did not display bactericidal activity alone in mouse models of acute and chronic TB infection, TB-47 potentiated the activity of pyrazinamide (PZA) and RIF during an acute TB infection in mice [128]. The authors speculated that alteration of the NADH/NAD<sup>+</sup> ratio triggered by QcrB inhibition sensitized the bacteria to the front-line drugs. While this hypothesis is interesting, it remains to be further explored since no positive interactions between RIF and the QcrB inhibitor Q203 in vitro have been observed [150]. Resistant mutants to TB-47 were generated in a *M. smeg* strain with a deletion of Cyt-bd oxidase, and whole-genome sequencing of the isolates revealed mutation H190Y in QcrB of *M. smeg* [128]. This residue, equivalent to H195 in *M. tb*, is located in the cd2 loop of QcrB, which is thought to interact with the Qp site [128].

A host cell-based screen of FDA-approved drugs of the Prestwick chemical library for compounds that protect lung fibroblasts from *M. tb*-induced cytotoxicity identified the gastric proton-pump inhibitor, lansoprazole (LPZ, Prevacid), as a potent hit compound [129]. LPZ is a prodrug that requires the host cell environment for conversion into lansoprazole sulphide (LPZS). Oral administration of LPZS significantly reduced bacterial burden during an acute TB infection in mice, and the compound lacks cytotoxicity, being well-tolerated in mice at a dose of 300 mg/kg. Whole-genome sequencing of resistant mutants generated to LPZS revealed mutation L176P in QcrB, and cross-resistance studies with imidazopyridine amide compounds and the T313A mutant strain indicated a distinct binding mode [129]. A follow-up study demonstrated that when LPZ is administered orally or intraperitoneally (i.p.) to rats, conversion to the active metabolite LPZS is undetectable in plasma and lung tissue, whereas LPZS was highly stable after i.p. administration [151]. In spite of this, LPZ intake has been found to significantly protect against TB incidence in individuals based on a cohort study conducted using the United Kingdom Clinical Practice Research Datalink [152].

The phenoxyalkylbenzimidazole (PAB) series, with an excellent growth-inhibitory potency and low cytotoxicity, is another promising QcrB inhibitor [130,153]. The lead compound, 54, demonstrated good efficacy against intracellular *M. tb*. Resistant mutants to PAB isolated in *M. tb* revealed mutations in qcrB and rv1339, a gene of unknown function. Through additional cross-resistance studies, QcrB was implicated as the main target [130]. The mutations identified in QcrB were A179P, M342T, W312C, and W312G, residues of the Qp binding site. Interestingly, while PAB compounds are bacteriostatic against actively growing *M. tb*, as with other QcrB inhibitors such as Q203 and LPZS, they are bactericidal against *M. tb* grown under conditions of nutrient starvation, in contrast to the lack of activity of other QcrB inhibitors in non-replicating models [129]. This discrepancy may be due to a potential involvement of a secondary target of PAB, for instance, rv1339, the second gene in which mutations were identified from resistant mutants.

The screening of a library of small polar molecules in a distinctive chemical space conducted at the Novartis Institute for Tropical Diseases led to the identification of hit compound pyrrolo[3,4-c]pyridine-1,3(2H)-dione, which was further optimised to increase its stability by replacing the ester moiety with a methyl oxadiazole bioisostere [131]. Lead compound 5h has an MIC90 below 0.156 μM in replicating *M. tb*, and is non-cytotoxic. The high clearance and poor plasma exposure of 5h in mouse PK studies could be improved by co-dosing with a pan-CYP inhibitor, such that a compound concentration above that of the MIC could be achieved in the blood for 6 h at a dose of 20 mg/kg. While attempts at isolating resistant mutants to this novel chemical class were unsuccessful, *M. tb* with a Cyt-bd oxidase deletion was hypersusceptible to the analogues. Moreover, the presence of mutation A317T in QcrB in this strain rendered *M. tb* resistant to the compounds, thus implicating QcrB in the mechanism of action of this class. Further studies on the pyrrolo[3,4-c]pyridine-1,3(2H)-diones will be aimed at improving their plasma concentration in vivo.

In 2013, GlaxoSmithKline published the results of a large phenotypic screening campaign against *M. bovis* BCG and *M. tb*, in which 177 hits belonging to several structurally distinct groups were identified [125]. To date, these chemical starting points have led to the discovery of two new classes of QcrB inhibitors, the 2-quinolin-4-yloxyacetamides (QOA) and the arylvinylpiperazine amides. Several SAR studies of the QOAs have been reported and are in agreement regarding the potency of this chemical entity against *M. tb*, the lack of toxicity in a zebrafish model and the narrow-spectrum activity specific for *M. tb* [132,154,155]. Independent work through cross-resistance studies and isolation of resistance mutants harbouring the mutation T313A in QcrB confirmed cytochrome bcc as the target of QOAs [155,156]. The solubility and stability of this series, as well as their in vivo potency, remain to be addressed [133,154]. The arylvinylpiperazine amides were generated through lead optimisation and SAR studies conducted with GW861072X from the GSK screen, which was an attractive starting point for its structural simplicity and potent activity against *M. bovis* and *M. tb* [76]. The most potent analogues have improved MICs compared to the parent scaffold, displaying low cytotoxicity and activity against intracellular *M. tb*. The lead compound, AX-35 and two other analogues, AX-37 and AX-39, were also active in an acute TB infection model in mice. While isolation of *M. tb-*resistant mutants on agar proved unsuccessful, continual and increasing exposure of *M. tb* to AX-35 over several passages in liquid culture resulted in the selection of resistant clones harbouring the mutations S182P, M342V and M342I in QcrB. Cross-resistance studies provided insight on residues which were important for the binding of the compound to QcrB, indicating a slightly different interaction with the quinol binding pocket than Q203 and LPZS. Improving the metabolic stability of this series would likely improve the in vivo activity in mice, although these compounds appear to be more stable in human microsomes.

The morpholino-thiophenes were identified from the Lilly corporate library that was screened in an aerobic whole-cell phenotypic setting against *M. tb*. The series was extensively profiled and optimised, with the lead of this series having an MIC90 of 0.24 μM against *M. tb*, a high-selective index and improved microsomal stability [134]. In an acute model of TB infection, the lead compound reduced the bacterial burden by 0.8 log CFUs in mouse lungs. Cross-resistance studies of the compounds to *M. tb* strains harbouring QcrB T313I and M342T mutations indicated that the compounds interact with the quinol binding site of QcrB.

4-amino-thieno[2,3-d] pyrimidines were found to inhibit the growth of *M. smeg* in a screen of small-molecule nucleotide mimetics from ChemBridge Corporation. The most potent compound of the series, CWHM-1023, had an IC50 of 0.083 μM against *M. tb* [135]. Resistant mutants raised to CWHM-1023 in *M. tb* had mutations A178T, A178V, V338G, G175S or G315S in QcrB, and this target was further confirmed by the increased susceptibility of a *M. tb* strain lacking Cyt-bd oxidase to the compounds.

The most recent class of Cyt-bcc-aa3 inhibitors identified are the quinazoline derivatives, 2-ethylthio-4-methylaminoquinazolines [77]. The most potent derivatives have activity against in vitro and intracellular *M. tb* in the micromolar range, and have low cytotoxicity in human hepatocytes. The lead compound 11726148 has a low clearance in human microsomes and was active in an acute TB infection model. Whole-genome sequencing of *M. tb* resistant mutants to the quinazoline derivatives revealed mutations Trp312Gly and Gly175Ser in QcrB. Interestingly, one escape mutant has a SNP in qcrA leading to the substitution of Leu356Val in QcrA, the Rieske iron-sulphur protein of the Cyt-bcc-aa3. Mapping of the mutated residues on a model of the *M. smeg* Cyt-bcc-aa3 complex [58] indicated that all of the mutated residues, including Leu356Val in QcrA, map to the Qp site of QcrB. This is in line with the Q-cycle model, in which quinol oxidation occurs at the interface of cytochrome b and the 2Fe-2S cluster domain of the Rieske protein, which make up the catalytic Qp site [57,157]. Cross-resistance studies confirmed the target of QcrB, and also reveal the role of Leu356Val in QcrA in its interaction with other QcrB inhibitors, namely Q203, AX-35, and LPZS. With the implication of QcrA in the pharmacological inhibition of Cyt-bcc-aa3, novel inhibitors can be generated which target both QcrB and QcrA subunits using structure-assisted drug-design to decrease the likelihood of resistance to QcrB inhibitors. Within this novel class, lead compound 11726148 appears the most amenable for further optimisation, as its phenyl moiety can be changed on different heterocycles to improve pharmacological properties.

The Qp site of the Cyt-bcc-aa3 complex is particularly susceptible to chemical inhibition, as evident from the multitude of structurally diverse and distinct compounds detailed above. It has been consistently observed across several of the studies that characteristic consequences of cytochrome bcc inhibition include a general depletion of intracellular bacterial ATP levels, an upregulation of Cyt-bd oxidase, and an increase in OCR and bacterial respiration due to Cyt-bd [12,76,77,128–130,134,135,155, 158]. This compensation by the alternate terminal oxidase leads to an incomplete respiratory shutdown in *M. tb*, resulting in the bacteriostatic nature of cytochrome bcc inhibitors alone. However, with the deletion of Cyt-bd, mycobacterial respiration is effectively blocked, and cytochrome bcc inhibitors become bactericidal [65,76,77]. Taken altogether, these concerted efforts have generated a potential pool of backup cytochrome bcc inhibitors as Q203 continues in clinical trials. Additionally, these findings have highlighted the extreme vulnerability of *M. tb* without both of its terminal oxidases, and the immense potential of both respiratory branches as drug targets.
