Bactericidal and sterilizing activity of novel regimens combining bedaquiline or TBAJ-587 with GSK2556286 and TBA-7371 in a mouse model of tuberculosis.

The combination of bedaquiline, pretomanid, and linezolid (BPaL) has become a preferred regimen for treating multidrug- and extensively drug-resistant tuberculosis (TB). However, treatment-limiting toxicities of linezolid and reports of emerging bedaquiline and pretomanid resistance necessitate efforts to develop new short-course oral regimens. We recently found that the addition of GSK2556286 increases the bactericidal and sterilizing activity of BPa-containing regimens in a well-established BALB/c mouse model of tuberculosis. Here, we used this model to evaluate the potential of new regimens combining bedaquiline or the more potent diarylquinoline TBAJ-587 with GSK2556286 and the DprE1 inhibitor TBA-7371, all of which are currently in early-phase clinical trials. We found the combination of bedaquiline, GSK2556286, and TBA-7371 to be more active than the first-line regimen and nearly as effective as BPaL in terms of bactericidal and sterilizing activity. In addition, we found that GSK2556286 and TBA-7371 were as effective as pretomanid and the novel oxazolidinone TBI-223 when either drug pair was combined with TBAJ-587 and that the addition of GSK2556286 increased the bactericidal activity of the TBAJ-587, pretomanid, and TBI-223 combination. We conclude that GSK2556286 and TBA-7371 have the potential to replace pretomanid, an oxazolidinone, or both components, in combination with bedaquiline or TBAJ-587.

Identification of 2-Aryl-Quinolone Inhibitors of Cytochrome bd and Chemical Validation of Combination Strategies for Respiratory Inhibitors against Mycobacterium tuberculosis.

Mycobacterium tuberculosis cytochrome bd quinol oxidase (cyt bd), the alternative terminal oxidase of the respiratory chain, has been identified as playing a key role during chronic infection and presents a putative target for the development of novel antitubercular agents. Here, we report confirmation of successful heterologous expression of M. tuberculosis cytochrome bd. The heterologous M. tuberculosis cytochrome bd expression system was used to identify a chemical series of inhibitors based on the 2-aryl-quinolone pharmacophore. Cytochrome bd inhibitors displayed modest efficacy in M. tuberculosis growth suppression assays together with a bacteriostatic phenotype in time-kill curve assays. Significantly, however, inhibitor combinations containing our front-runner cyt bd inhibitor CK-2-63 with either cyt bcc-aa3 inhibitors (e.g., Q203) and/or adenosine triphosphate (ATP) synthase inhibitors (e.g., bedaquiline) displayed enhanced efficacy with respect to the reduction of mycobacterium oxygen consumption, growth suppression, and in vitro sterilization kinetics. In vivo combinations of Q203 and CK-2-63 resulted in a modest lowering of lung burden compared to treatment with Q203 alone. The reduced efficacy in the in vivo experiments compared to in vitro experiments was shown to be a result of high plasma protein binding and a low unbound drug exposure at the target site. While further development is required to improve the tractability of cyt bd inhibitors for clinical evaluation, these data support the approach of using small-molecule inhibitors to target multiple components of the branched respiratory chain of M. tuberculosis as a combination strategy to improve therapeutic and pharmacokinetic/pharmacodynamic (PK/PD) indices related to efficacy.

Lysyl-tRNA synthetase, a target for urgently needed M. tuberculosis drugs.

Tuberculosis is a major global cause of both mortality and financial burden mainly in low and middle-income countries. Given the significant and ongoing rise of drug-resistant strains of Mycobacterium tuberculosis within the clinical setting, there is an urgent need for the development of new, safe and effective treatments. Here the development of a drug-like series based on a fused dihydropyrrolidino-pyrimidine scaffold is described. The series has been developed against M. tuberculosis lysyl-tRNA synthetase (LysRS) and cellular studies support this mechanism of action. DDD02049209, the lead compound, is efficacious in mouse models of acute and chronic tuberculosis and has suitable physicochemical, pharmacokinetic properties and an in vitro safety profile that supports further development. Importantly, preliminary analysis using clinical resistant strains shows no pre-existing clinical resistance towards this scaffold.

"Upcycling" known molecules and targets for drug-resistant TB.

Despite reinvigorated efforts in Tuberculosis (TB) drug discovery over the past 20 years, relatively few new drugs and candidates have emerged with clear utility against drug resistant TB. Over the same period, significant technological advances and learnings around target value have taken place. This has offered opportunities to re-assess the potential for optimization of previously discovered chemical matter against Mycobacterium tuberculosis (M.tb) and for reconsideration of clinically validated targets encumbered by drug resistance. A re-assessment of discarded compounds and programs from the "golden age of antibiotics" has yielded new scaffolds and targets against TB and uncovered classes, for example beta-lactams, with previously unappreciated utility for TB. Leveraging validated classes and targets has also met with success: booster technologies and efforts to thwart efflux have improved the potential of ethionamide and spectinomycin classes. Multiple programs to rescue high value targets while avoiding cross-resistance are making progress. These attempts to make the most of known classes, drugs and targets complement efforts to discover new chemical matter against novel targets, enhancing the chances of success of discovering effective novel regimens against drug-resistant TB.

The Novel Oxazolidinone TBI-223 Is Effective in Three Preclinical Mouse Models of Methicillin-Resistant Staphylococcus aureus Infection.

Staphylococcus aureus is an important cause of various infections in humans, including bacteremia, skin and soft tissue infections, and infections associated with implanted medical devices. The emergence of hospital- and community-acquired methicillin-resistant Staphylococcus aureus (MRSA) underscores the urgent and unmet need to develop novel, safe, and effective antibiotics against these multidrug-resistant clinical isolates. Oxazolidinone antibiotics such as linezolid have excellent oral bioavailability and provide coverage against MRSA infections. However, their widespread and long-term use is often limited by adverse effects, especially myelosuppression. TBI-223 is a novel oxazolidinone with potentially reduced myelosuppression, compared to linezolid, but its efficacy against MRSA infections is unknown. Therefore, the preclinical efficacy of TBI-223 (80 and 160 mg/kg twice daily) was compared with that of linezolid (40 and 80 mg/kg twice daily) and sham treatment in mouse models of MRSA bacteremia, skin wound infection, and orthopedic-implant-associated infection. The dosage was selected based on mouse pharmacokinetic analysis of both linezolid and TBI-223, as well as measurement of the MICs. In all three models, TBI-223 and linezolid had comparable dose-dependent efficacies in reducing bacterial burden and disease severity, compared with sham-treated control mice. Taken together, these findings indicate that TBI-223 represents a novel oxazolidinone antibiotic that may provide an additional option against MRSA infections. Future studies in larger animal models and clinical trials are warranted to translate these findings to humans. IMPORTANCE Staphylococcus aureus is the predominant cause of bloodstream, skin, and bone infections in humans. Resistance to commonly used antibiotics is a growing concern, making it more difficult to treat staphylococcal infections. Use of the oxazolidinone antibiotic linezolid against resistant strains is hindered by high rates of adverse reactions during prolonged therapy. Here, a new oxazolidinone named TBI-223 was tested against S. aureus in three mouse models of infection, i.e., bloodstream infection, skin infection, and bone infection. We found that TBI-223 was as effective as linezolid in these three models. Previous data suggest that TBI-223 has a better safety profile than linezolid. Taken together, these findings indicate that this new agent may provide an additional option against MRSA infections. Future studies in larger animal models and clinical trials are warranted to translate these findings to humans.

GSK2556286 Is a Novel Antitubercular Drug Candidate Effective In Vivo with the Potential To Shorten Tuberculosis Treatment.

As a result of a high-throughput compound screening campaign using Mycobacterium tuberculosis-infected macrophages, a new drug candidate for the treatment of tuberculosis has been identified. GSK2556286 inhibits growth within human macrophages (50% inhibitory concentration [IC50] = 0.07 µM), is active against extracellular bacteria in cholesterol-containing culture medium, and exhibits no cross-resistance with known antitubercular drugs. In addition, it has shown efficacy in different mouse models of tuberculosis (TB) and has an adequate safety profile in two preclinical species. These features indicate a compound with a novel mode of action, although still not fully defined, that is effective against both multidrug-resistant (MDR) or extensively drug-resistant (XDR) and drug-sensitive (DS) M. tuberculosis with the potential to shorten the duration of treatment in novel combination drug regimens. (This study has been registered at ClinicalTrials.gov under identifier NCT04472897).

Delamanid or pretomanid? A Solomonic judgement!

Given the low treatment success rates of drug-resistant tuberculosis (TB), novel TB drugs are urgently needed. The landscape of TB treatment has changed considerably over the last decade with the approval of three new compounds: bedaquiline, delamanid and pretomanid. Of these, delamanid and pretomanid belong to the same class of drugs, the nitroimidazoles. In order to close the knowledge gap on how delamanid and pretomanid compare with each other, we summarize the main findings from preclinical research on these two compounds. We discuss the compound identification, mechanism of action, drug resistance, in vitro activity, in vivo pharmacokinetic profiles, and preclinical in vivo activity and efficacy. Although delamanid and pretomanid share many similarities, several differences could be identified. One finding of particular interest is that certain Mycobacterium tuberculosis isolates have been described that are resistant to either delamanid or pretomanid, but with preserved susceptibility to the other compound. This might imply that delamanid and pretomanid could replace one another in certain regimens. Regarding bactericidal activity, based on in vitro and preclinical in vivo activity, delamanid has lower MICs and higher mycobacterial load reductions at lower drug concentrations and doses compared with pretomanid. However, when comparing in vivo preclinical bactericidal activity at dose levels equivalent to currently approved clinical doses based on drug exposure, this difference in activity between the two compounds fades. However, it is important to interpret these comparative results with caution knowing the variability inherent in preclinical in vitro and in vivo models.

Predictive Modeling to Study the Treatment-Shortening Potential of Novel Tuberculosis Drug Regimens, Toward Bundling of Preclinical Data.

BACKGROUND: Given the persistently high global burden of tuberculosis, effective and shorter treatment options are needed. We explored the relationship between relapse and treatment length as well as interregimen differences for 2 novel antituberculosis drug regimens using a mouse model of tuberculosis infection and mathematical modeling. METHODS: Mycobacterium tuberculosis-infected mice were treated for up to 13 weeks with bedaquiline and pretomanid combined with moxifloxacin and pyrazinamide (BPaMZ) or linezolid (BPaL). Cure rates were evaluated 12 weeks after treatment completion. The standard regimen of isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE) was evaluated as a comparator. RESULTS: Six weeks of BPaMZ was sufficient to achieve cure in all mice. In contrast, 13 weeks of BPaL and 24 weeks of HRZE did not achieve 100% cure rates. Based on mathematical model predictions, 95% probability of cure was predicted to occur at 1.6, 4.3, and 7.9 months for BPaMZ, BPaL, and HRZE, respectively. CONCLUSION: This study provides additional evidence for the treatment-shortening capacity of BPaMZ over BPaL and HRZE. To optimally use preclinical data for predicting clinical outcomes, and to overcome the limitations that hamper such extrapolation, we advocate bundling of available published preclinical data into mathematical models.

Synthesis and structure-activity relationships for a new class of tetrahydronaphthalene amide inhibitors of Mycobacterium tuberculosis.

Drug resistant tuberculsosis (TB) is global health crisis that demands novel treatment strategies. Bacterial ATP synthase inhibitors such as bedaquiline and next-generation analogues (such as TBAJ-876) have shown promising efficacy in patient populations and preclinical studies, respectively, suggesting that selective targeting of this enzyme presents a validated therapeutic strategy for the treatment of TB. In this work, we report tetrahydronaphthalene amides (THNAs) as a new class of ATP synthase inhibitors that are effective in preventing the growth of Mycobacterium tuberculosis (M.tb) in culture. Design, synthesis and comprehensive structure-activity relationship studies for approximately 80 THNA analogues are described, with a small selection of compounds exhibiting potent (in some cases MIC90 <1 µg/mL) in vitro M.tb growth inhibition taken forward to pharmacokinetic and off-target profiling studies. Ultimately, we show that some of these THNAs possess reduced lipophilic properties, decreased hERG liability, faster mouse/human liver microsomal clearance rates and shorter plasma half-lives compared with bedaquiline, potentially addressing of the main concerns of persistence and phospholipidosis associated with bedaquiline.

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.

Comparative Efficacy of the Novel Diarylquinoline TBAJ-587 and Bedaquiline against a Resistant Rv0678 Mutant in a Mouse Model of Tuberculosis.

Since its conditional approval in 2012, bedaquiline (BDQ) has been a valuable tool for treatment of drug-resistant tuberculosis. More recently, a novel short-course regimen combining BDQ with pretomanid and linezolid won approval to treat highly drug-resistant tuberculosis. Clinical reports of emerging BDQ resistance have identified mutations in Rv0678 that derepress the expression of the MmpL5/MmpS5 efflux transporter as the most common cause. Because the effect of these mutations on bacterial susceptibility to BDQ is relatively small (e.g., 2 to 8× MIC shift), increasing the BDQ dose would increase antibacterial activity but also pose potential safety concerns, including QTc prolongation. Substitution of BDQ with another diarylquinoline with superior potency and/or safety has the potential to overcome these limitations. TBAJ-587 has greater in vitro potency than BDQ, including against Rv0678 mutants, and may offer a larger safety margin. Using a mouse model of tuberculosis and different doses of BDQ and TBAJ-587, we found that against wild-type M. tuberculosis H37Rv and an isogenic Rv0678 mutant, TBAJ-587 has greater efficacy against both strains than BDQ, whether alone or in combination with pretomanid and either linezolid or moxifloxacin and pyrazinamide. TBAJ-587 also reduced the emergence of resistance to diarylquinolines and pretomanid.

Synthesis and structure-activity relationships for tetrahydroisoquinoline-based inhibitors of Mycobacterium tuberculosis.

A series of 5,8-disubstituted tetrahydroisoquinolines were shown to be effective inhibitors of M. tb in culture and modest inhibitors of M. tb ATP synthase. There was a broad general trend of improved potency with higher lipophilicity. Large substituents (e.g., Bn) at the tetrahydroquinoline 5-position were well-tolerated, while N-methylpiperazine was the preferred 8-substituent. Structure-activity relationships for 7-linked side chains showed that the nature of the 7-linking group was important; -CO- and -COCH2- linkers were less effective than -CH2- or -CONH- ones. This suggests that the positioning of a terminal aromatic ring is important for target binding. Selected compounds showed much faster rates of microsomal clearance than did the clinical ATP synthase inhibitor bedaquiline, and modest inhibition of mycobacterial ATP synthase.

Synergistic Activity of Nitroimidazole-Oxazolidinone Conjugates against Anaerobic Bacteria.

The introductions of the bicyclic 4-nitroimidazole and the oxazolidinone classes of antimicrobial agents represented the most significant advancements in the infectious disease area during the past two decades. Pretomanid, a bicyclic 4-nitroimidazole, and linezolid, an oxazolidinone, are also part of a combination regimen approved recently by the US Food and Drug Administration for the treatment of pulmonary, extensively drug resistant (XDR), treatment-intolerant or nonresponsive multidrug-resistant (MDR) Mycobacterium tuberculosis (TB). To identify new antimicrobial agents with reduced propensity for the development of resistance, a series of dual-acting nitroimidazole-oxazolidinone conjugates were designed, synthesized and evaluated for their antimicrobial activity. Compounds in this conjugate series have shown synergistic activity against a panel of anaerobic bacteria, including those responsible for serious bacterial infections.

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.

Variations in the C-unit of bedaquiline provides analogues with improved biology and pharmacology.

Analogues of the anti-tuberculosis drug bedaquiline, bearing a 3,5-dimethoxy-4-pyridyl C-unit, retain high anti-bacterial potency yet exert less inhibition of the hERG potassium channel, in vitro, than the parent compound. Two of these analogues (TBAJ-587 and TBAJ-876) are now in preclinical development. The present study further explores structure-activity relationships across a range of related 3,5-disubstituted-4-pyridyl C-unit bedaquiline analogues of greatly varying lipophilicity (clogP from 8.16 to 1.89). This broader class shows similar properties to the 3,5-dimethoxy-4-pyridyl series, being substantially more potent in vitro and equally active in an in vivo (mouse) model than bedaquiline, while retaining a lower cardiovascular risk profile through greatly attenuated hERG inhibition.

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 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.

Contribution of Pretomanid to Novel Regimens Containing Bedaquiline with either Linezolid or Moxifloxacin and Pyrazinamide in Murine Models of Tuberculosis.

Novel regimens combining bedaquiline and pretomanid with either linezolid (BPaL regimen) or moxifloxacin and pyrazinamide (BPaMZ regimen) shorten the treatment duration needed to cure tuberculosis (TB) in BALB/c mice compared to that of the first-line regimen and have yielded promising results in initial clinical trials. However, the independent contribution of the investigational new drug pretomanid to the efficacy of BPaMZ has not been examined, and its contribution to BPaL has been examined only over the first 2 months of treatment. In the present study, the addition of pretomanid to BL increased bactericidal activity, prevented emergence of bedaquiline resistance, and shortened the duration needed to prevent relapse with drug-susceptible isolates by at least 2 months in BALB/c mice. Addition of pretomanid to bedaquiline, moxifloxacin, and pyrazinamide (BMZ) resulted in a 1-log10 greater CFU reduction after 1 month of treatment and/or reduced the number of mice relapsing in each of 2 experiments in BALB/c mice and in immunocompromised nude mice. Bedaquiline-resistant isolates were found at relapse in only one BMZ-treated nude mouse. Treatment of infection with a pyrazinamide-resistant mutant in BALB/c mice with BPaMZ prevented selection of bedaquiline-resistant mutants and reduced the proportion of mice relapsing compared to that for BMZ treatment alone. Among severely ill C3HeB/FeJ mice with caseous pneumonia and cavitation, BPaMZ increased median survival (≥60 versus 21 days) and reduced median lung CFU by 2.4 log10 at 1 month compared to the level for BMZ. In conclusion, in 3 different mouse models, pretomanid contributed significantly to the efficacy of the BPaMZ and BPaL regimens, including restricting the selection of bedaquiline-resistant mutants.

Structure-activity relationships for unit C pyridyl analogues of the tuberculosis drug bedaquiline.

The ATP-synthase inhibitor bedaquiline is effective against drug-resistant tuberculosis but is extremely lipophilic (clogP 7.25) with a very long plasma half-life. Additionally, inhibition of potassium current through the cardiac hERG channel by bedaquiline, is associated with prolongation of the QT interval, necessitating cardiovascular monitoring. Analogues were prepared where the naphthalene C-unit was replaced with substituted pyridines to produce compounds with reduced lipophilicity, anticipating a reduction in half-life. While there was a direct correlation between in vitro inhibitory activity against M. tuberculosis (MIC90) and compound lipophilicity, potency only fell off sharply below a clogP of about 4.0, providing a useful lower bound for analogue design. The bulk of the compounds remained potent inhibitors of the hERG potassium channel, with notable exceptions where IC50 values were at least 5-fold higher than that of bedaquiline. Many of the compounds had desirably higher rates of clearance than bedaquiline, but this was associated with lower plasma exposures in mice, and similar or higher MICs resulted in lower AUC/MIC ratios than bedaquiline for most compounds. The two compounds with lower potency against hERG exhibited similar clearance to bedaquiline and excellent efficacy in vivo, suggesting further exploration of C-ring pyridyls is worthwhile.