Relative contributions of the novel diarylquinoline TBAJ-876 and its active metabolite to the bactericidal activity in a murine model of tuberculosis.

BACKGROUND: TBAJ-876 is a next-generation diarylquinoline. In vivo, diarylquinoline metabolites are formed with activity against Mycobacterium tuberculosis. Species-specific differences in parent drug-to-metabolite ratios might impact the translational value of animal model-based predictions. This study investigates the contribution of TBAJ-876 and its major active metabolite, TBAJ-876-M3 (M3), to the total bactericidal activity in a mouse tuberculosis model. METHODS: In vitro activity of TBAJ-876 and M3 was investigated and compared to bedaquiline. Subsequently, a dose-response study was conducted in M. tuberculosis-infected BALB/c mice treated with TBAJ-876 (1.6/6.3/25 mg/kg) or M3 (3.1/12.5/50 mg/kg). Colony-forming units in the lungs and TBAJ-876 and M3 plasma concentrations were determined. M3's contribution to TBAJ-876's bactericidal activity was estimated based on M3-exposure following TBAJ-876 treatment and corresponding M3-activity observed in M3-treated animals. RESULTS: TBAJ-876 and M3 demonstrated profound bactericidal activity. Lungs of mice treated for 4 weeks with 50 mg/kg M3 were culture-negative. Following TBAJ-876 treatment, M3-exposures were 2.2-3.6x higher than for TBAJ-876. TBAJ-876 activity was substantially attributable to M3, given its high exposure and potent activity. CONCLUSION: These findings emphasize the need to consider metabolites and their potentially distinct exposure and activity profiles compared to parent drugs to enhance the translational value of mouse model-driven predictions.

Next-Generation Diarylquinolines Improve Sterilizing Activity of Regimens with Pretomanid and the Novel Oxazolidinone TBI-223 in a Mouse Tuberculosis Model.

A regimen comprised of bedaquiline (BDQ, or B), pretomanid, and linezolid (BPaL) is the first oral 6-month regimen approved by the U.S. Food and Drug Administration and recommended by the World Health Organization for the treatment of extensively drug-resistant tuberculosis. We used a well-established BALB/c mouse model of tuberculosis to evaluate the treatment-shortening potential of replacing bedaquiline with either of two new, more potent diarylquinolines, TBAJ-587 and TBAJ-876, in early clinical trials. We also evaluated the effect of replacing linezolid with a new oxazolidinone, TBI-223, exhibiting a larger safety margin with respect to mitochondrial toxicity in preclinical studies. Replacing bedaquiline with TBAJ-587 at the same 25-mg/kg dose significantly reduced the proportion of mice relapsing after 2 months of treatment, while replacing linezolid with TBI-223 at the same 100-mg/kg dose did not significantly change the proportion of mice relapsing. Replacing linezolid or TBI-223 with sutezolid in combination with TBAJ-587 and pretomanid significantly reduced the proportion of mice relapsing. In combination with pretomanid and TBI-223, TBAJ-876 at 6.25 mg/kg was equipotent to TBAJ-587 at 25 mg/kg. We conclude that replacement of bedaquiline with these more efficacious and potentially safer diarylquinolines and replacement of linezolid with potentially safer and at least as efficacious oxazolidinones in the clinically successful BPaL regimen may lead to superior regimens capable of treating both drug-susceptible and drug-resistant TB more effectively and safely.

Comparative Efficacy of the Novel Diarylquinoline TBAJ-876 and Bedaquiline against a Resistant Rv0678 Mutant in a Mouse Model of Tuberculosis.

Bedaquiline (BDQ, B) is the first-in-class diarylquinoline to be approved for treatment of tuberculosis (TB). Recent guidelines recommend its use in treatment of multidrug- and extensively drug-resistant tuberculosis (MDR/XDR-TB). The newly approved regimen combining BDQ with pretomanid and linezolid is the first 6-month oral regimen proven to be effective against MDR/XDR-TB. However, the emergence of BDQ resistance, primarily due to inactivating mutations in the Rv0678 gene encoding a repressor of the MmpS5-MmpL5 transporter, threatens to undermine the efficacy of new BDQ-containing regimens. Since the shift in MIC due to these mutations is relatively small (2-8×), safer, and more potent, diarylquinoline analogues may be more effective than BDQ. TBAJ-876, which is in phase 1 trials, has more potent in vitro activity and a superior pre-clinical safety profile than BDQ. Using a murine model of TB, we evaluated the dose-dependent activity of TBAJ-876 compared to BDQ against the wild-type H37Rv strain and an isogenic Rv0678 loss-of-function mutant. Although the mutation affected the MIC of both drugs, the MIC of TBAJ-876 against the mutant was 10-fold lower than that of BDQ. TBAJ-876 at doses ≥6.25 mg/kg had greater efficacy against both strains compared to BDQ at 25 mg/kg, when administered alone or in combination with pretomanid and linezolid. Likewise, no selective amplification of BDQ-resistant bacteria was observed at TBAJ-876 doses ≥6.25 mg/kg. These results indicate that replacing BDQ with TBAJ-876 may shorten the duration of TB treatment and be more effective in treating and preventing infections caused by Rv0678 mutants.

TBAJ-876 Displays Bedaquiline-Like Mycobactericidal Potency without Retaining the Parental Drug's Uncoupler Activity.

The diarylquinoline F1FO-ATP synthase inhibitor bedaquiline (BDQ) displays protonophore activity. Thus, uncoupling electron transport from ATP synthesis appears to be a second mechanism of action of this antimycobacterial drug. Here, we show that the new BDQ analogue TBAJ-876 did not retain the parental drug's protonophore activity. Comparative time-kill analyses revealed that both compounds exert the same bactericidal activity. These results suggest that the uncoupler activity is not required for the bactericidal activity of diarylquinolines.

TBAJ-876, a 3,5-Dialkoxypyridine Analogue of Bedaquiline, Is Active against Mycobacterium abscessus.

Lung disease caused by Mycobacterium abscessus is very difficult to cure, and treatment failure rates are high. The antituberculosis drug bedaquiline (BDQ) is used as salvage therapy against this dreadful disease. However, BDQ is highly lipophilic, displays a long terminal half-life, and presents a cardiotoxicity liability associated with QT interval prolongation. Recent medicinal chemistry campaigns resulted in the discovery of 3,5-dialkoxypyridine analogues of BDQ which are less lipophilic, have higher clearance, and display lower cardiotoxic potential. TBAJ-876, a clinical development candidate of this series, shows attractive in vitro antitubercular activity and efficacy in a murine tuberculosis model. Here, we asked whether TBAJ-876 is active against M. abscessus TBAJ-876 displayed submicromolar in vitro activity against reference strains representing the three subspecies of M. abscessus and against a collection of clinical isolates. Drug-drug potency interaction studies with commonly used anti-M. abscessus antibiotics showed no antagonistic effects, suggesting that TBAJ-876 could be coadministered with currently used drugs. Efficacy studies, employing a mouse model of M. abscessus infection, demonstrated potent activity in vivo In summary, we demonstrate that TBAJ-876 shows attractive in vitro and in vivo activities against M. abscessus, similar to its BDQ parent. This suggests that next-generation BDQ, with improved tolerability and pharmacological profiles, may be useful for the treatment of M. abscessus lung disease in addition to the treatment of tuberculosis.

Synthetic Studies to Help Elucidate the Metabolism of the Preclinical Candidate TBAJ-876-A Less Toxic and More Potent Analogue of Bedaquiline.

Bedaquiline is a novel drug approved in 2012 by the FDA for treatment of drug-resistant tuberculosis (TB). Although it shows high efficacy towards drug-resistant forms of TB, its use has been limited by the potential for significant side effects. In particular, bedaquiline is a very lipophilic compound with an associated long terminal half-life and shows potent inhibition of the cardiac potassium hERG channel, resulting in QTc interval prolongation in humans that may result in cardiac arrhythmia. To address these issues, we carried out a drug discovery programme to develop an improved second generation analogue of bedaquiline. From this medicinal chemistry program, a candidate (TBAJ-876) has been selected to undergo further preclinical evaluation. During this evaluation, three major metabolites arising from TBAJ-876 were observed in several preclinical animal models. We report here our synthetic efforts to unequivocally structurally characterize these three metabolites through their independent directed synthesis.

TBAJ-876 Retains Bedaquiline's Activity against Subunits c and ε of Mycobacterium tuberculosis F-ATP Synthase.

The antituberculosis drug bedaquiline (BDQ) inhibits Mycobacterium tuberculosis F-ATP synthase by interfering with two subunits. Drug binding to the c subunit stalls the rotation of the c ring, while binding to the ε subunit blocks coupling of c ring rotation to ATP synthesis at the catalytic α3:ß3 headpiece. BDQ is used for the treatment of drug-resistant tuberculosis. However, the drug is highly lipophilic, displays a long terminal half-life, and has a cardiotoxicity liability by causing QT interval prolongation. Recent medicinal chemistry campaigns have resulted in the discovery of 3,5-dialkoxypyridine analogues of BDQ that are less lipophilic, have higher clearance, and display lower cardiotoxic potential. TBAJ-876, which is a new developmental compound of this series, shows attractive antitubercular activity and efficacy in a murine tuberculosis model. Here, we asked whether TBAJ-876 and selected analogues of the compound retain BDQ's mechanism of action. Biochemical assays showed that TBAJ-876 is a potent inhibitor of mycobacterial F-ATP synthase. Selection of spontaneous TBAJ-876-resistant mutants identified missense mutations at BDQ's binding site on the c subunit, suggesting that TBAJ-876 retains BDQ's targeting of the c ring. Susceptibility testing against a strain overexpressing the ε subunit and a strain harboring an engineered mutation in BDQ's ε subunit binding site suggest that TBAJ-876 retains BDQ's activity on the ε subunit. Nuclear magnetic resonance (NMR) titration studies confirmed that TBAJ-876 binds to the ε subunit at BDQ's binding site. We show that TBAJ-876 retains BDQ's antimycobacterial mode of action. The developmental compound inhibits the mycobacterial F-ATP synthase via a dual-subunit mechanism of interfering with the functions of both the enzyme's c and ε subunits.

Binding properties of the anti-TB drugs bedaquiline and TBAJ-876 to a mycobacterial F-ATP synthase.

Tuberculosis (TB), the deadly disease caused by Mycobacterium tuberculosis (Mtb), kills more people worldwide than any other bacterial infectious disease. There has been a recent resurgence of TB drug discovery activities, resulting in the identification of a number of novel enzyme inhibitors. Many of these inhibitors target the electron transport chain complexes and the F1FO-ATP synthase; these enzymes represent new target spaces for drug discovery, since the generation of ATP is essential for the bacterial pathogen's physiology, persistence, and pathogenicity. The anti-TB drug bedaquiline (BDQ) targets the Mtb F-ATP synthase and is used as salvage therapy against this disease. Medicinal chemistry efforts to improve the physio-chemical properties of BDQ resulted in the discovery of 3,5-dialkoxypyridine (DARQ) analogs to which TBAJ-876 belongs. TBAJ-876, a clinical development candidate, shows attractive in vitro and in vivo antitubercular activity. Both BDQ and TBAJ-876 inhibit the mycobacterial F1FO-ATP synthase by stopping rotation of the c-ring turbine within the FO domain, thereby preventing proton translocation and ATP synthesis to occur. While structural data for the BDQ bound state are available, no structural information about TBAJ-876 binding have been described. In this study, we show how TBAJ-876 binds to the FO domain of the M. smegmatis F1FO-ATP synthase. We further calculate the binding free energy of both drugs bound to their target and predict an increased affinity of TBAJ-876 for the FO domain. This approach will be useful in future efforts to design new and highly potent DARQ analogs targeting F-ATP synthases of Mtb, nontuberculosis mycobacteria (NTM) as well as the M. leprosis complex.

Re-Understanding the Mechanisms of Action of the Anti-Mycobacterial Drug Bedaquiline.

Bedaquiline (BDQ) inhibits ATP generation in Mycobacterium tuberculosis by interfering with the F-ATP synthase activity. Two mechanisms of action of BDQ are broadly accepted. A direct mechanism involves BDQ binding to the enzyme's c-ring to block its rotation, thus inhibiting ATP synthesis in the enzyme's catalytic α3ß3-headpiece. An indirect mechanism involves BDQ uncoupling electron transport in the electron transport chain from ATP synthesis at the F-ATP synthase. In a recently uncovered second direct mechanism, BDQ binds to the enzyme's ε-subunit to disrupt its ability to link c-ring rotation to ATP synthesis at the α3ß3-headpiece. However, this mechanism is controversial as the drug's binding affinity for the isolated ε-subunit protein is moderate and spontaneous resistance mutants in the ε-subunit cannot be isolated. Recently, the new, structurally distinct BDQ analogue TBAJ-876 was utilized as a chemical probe to revisit BDQ's mechanisms of action. In this review, we first summarize discoveries on BDQ's mechanisms of action and then describe the new insights derived from the studies of TBAJ-876. The TBAJ-876 investigations confirm the c-ring as a target, while also supporting a functional role for targeting the ε-subunit. Surprisingly, the new findings suggest that the uncoupler mechanism does not play a key role in BDQ's anti-mycobacterial activity.