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.

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).

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.

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.

3,5-Dialkoxypyridine analogues of bedaquiline are potent antituberculosis agents with minimal inhibition of the hERG channel.

Bedaquiline is a new drug of the diarylquinoline class that has proven to be clinically effective against drug-resistant tuberculosis, but has a cardiac liability (prolongation of the QT interval) due to its potent inhibition of the cardiac potassium channel protein hERG. Bedaquiline is highly lipophilic and has an extremely long terminal half-life, so has the potential for more-than-desired accumulation in tissues during the relatively long treatment durations required to cure TB. The present work is part of a program that seeks to identify a diarylquinoline that is as potent as bedaquiline against Mycobacterium tuberculosis, with lower lipophilicity, higher clearance, and lower risk for QT prolongation. Previous work led to the identification of compounds with greatly-reduced lipophilicity compounds that retain good anti-tubercular activity in vitro and in mouse models of TB, but has not addressed the hERG blockade. We now present compounds where the C-unit naphthalene is replaced by a 3,5-dialkoxy-4-pyridyl, demonstrate more potent in vitro and in vivo anti-tubercular activity, with greatly attenuated hERG blockade. Two examples of this series are in preclinical development.

Structure-activity relationships for analogs of the tuberculosis drug bedaquiline with the naphthalene unit replaced by bicyclic heterocycles.

Replacing the naphthalene C-unit of the anti-tuberculosis drug bedaquiline with a range of bicyclic heterocycles of widely differing lipophilicity gave analogs with a 4.5-fold range in clogP values. The biological results for these compounds indicate on average a lower clogP limit of about 5.0 in this series for retention of potent inhibitory activity (MIC90s) against M.tb in culture. Some of the compounds also showed a significant reduction in inhibition of hERG channel potassium current compared with bedaquiline, but there was no common structural feature that distinguished these.

Role of the methylcitrate cycle in Mycobacterium tuberculosis metabolism, intracellular growth, and virulence.

Growth of bacteria and fungi on fatty acid substrates requires the catabolic beta-oxidation cycle and the anaplerotic glyoxylate cycle. Propionyl-CoA generated by beta-oxidation of odd-chain fatty acids is metabolized via the methylcitrate cycle. Mycobacterium tuberculosis possesses homologues of methylcitrate synthase (MCS) and methylcitrate dehydratase (MCD) but not 2-methylisocitrate lyase (MCL). Although MCLs share limited homology with isocitrate lyases (ICLs) of the glyoxylate cycle, these enzymes are thought to be functionally non-overlapping. Previously we reported that the M. tuberculosis ICL isoforms 1 and 2 are jointly required for growth on fatty acids, in macrophages, and in mice. ICL-deficient bacteria could not grow on propionate, suggesting that in M. tuberculosis ICL1 and ICL2 might function as ICLs in the glyoxylate cycle and as MCLs in the methylcitrate cycle. Here we provide biochemical and genetic evidence supporting this interpretation. The role of the methylcitrate cycle in M. tuberculosis metabolism was further evaluated by constructing a mutant strain in which prpC (encoding MCS) and prpD (encoding MCD) were deleted. The DeltaprpDC strain could not grow on propionate media in vitro or in murine bone marrow-derived macrophages infected ex vivo; growth under these conditions was restored by complementation with a plasmid containing prpDC. Paradoxically, bacterial growth and persistence, and tissue pathology, were indistinguishable in mice infected with wild-type or DeltaprpDC bacteria.