Redirecting raltitrexed from cancer cell thymidylate synthase to Mycobacterium tuberculosis phosphopantetheinyl transferase.

There is a compelling need to find drugs active against Mycobacterium tuberculosis (Mtb). 4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme in Mtb that has attracted interest as a potential drug target. We optimized a PptT assay, used it to screen 422,740 compounds, and identified raltitrexed, an antineoplastic antimetabolite, as the most potent PptT inhibitor yet reported. While trying unsuccessfully to improve raltitrexed's ability to kill Mtb and remove its ability to kill human cells, we learned three lessons that may help others developing antibiotics. First, binding of raltitrexed substantially changed the configuration of the PptT active site, complicating molecular modeling of analogs based on the unliganded crystal structure or the structure of cocrystals with inhibitors of another class. Second, minor changes in the raltitrexed molecule changed its target in Mtb from PptT to dihydrofolate reductase (DHFR). Third, the structure-activity relationship for over 800 raltitrexed analogs only became interpretable when we quantified and characterized the compounds' intrabacterial accumulation and transformation.

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.

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.

Discovery of natural-product-derived sequanamycins as potent oral anti-tuberculosis agents.

The emergence of drug-resistant tuberculosis has created an urgent need for new anti-tubercular agents. Here, we report the discovery of a series of macrolides called sequanamycins with outstanding in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb). Sequanamycins are bacterial ribosome inhibitors that interact with the ribosome in a similar manner to classic macrolides like erythromycin and clarithromycin, but with binding characteristics that allow them to overcome the inherent macrolide resistance of Mtb. Structures of the ribosome with bound inhibitors were used to optimize sequanamycin to produce the advanced lead compound SEQ-9. SEQ-9 was efficacious in mouse models of acute and chronic TB as a single agent, and it demonstrated bactericidal activity in a murine TB infection model in combination with other TB drugs. These results support further investigation of this series as TB clinical candidates, with the potential for use in new regimens against drug-susceptible and drug-resistant TB.

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.

"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.

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.

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.

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.

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.

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.

Dual mTORC1/mTORC2 Inhibition as a Host-Directed Therapeutic Target in Pathologically Distinct Mouse Models of Tuberculosis.

Efforts to develop more effective and shorter-course therapies for tuberculosis have included a focus on host-directed therapy (HDT). The goal of HDT is to modulate the host response to infection, thereby improving immune defenses to reduce the duration of antibacterial therapy and/or the amount of lung damage. As a mediator of innate and adaptive immune responses involved in eliminating intracellular pathogens, autophagy is a potential target for HDT in tuberculosis. Because Mycobacterium tuberculosis modulates mammalian target of rapamycin (mTOR) signaling to impede autophagy, pharmacologic mTOR inhibition could provide effective HDT. mTOR exists within two distinct multiprotein complexes, mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2). Rapamycin and its analogs only partially inhibit mTORC1. We hypothesized that novel mTOR kinase inhibitors blocking both complexes would have expanded therapeutic potential. We compared the effects of two mTOR inhibitors, rapamycin and the orally available mTOR kinase domain inhibitor CC214-2, which blocks both mTORC1 and mTORC2, as adjunctive therapies against murine TB when added to the first-line regimen (isoniazid, rifampin, pyrazinamide, and ethambutol [RHZE]) or the novel bedaquiline-pretomanid-linezolid (BPaL) regimen. Neither mTOR inhibitor affected lung CFU counts after 4 to 8 weeks of treatment when combined with BPaL or RHZE. However, addition of CC214-2 to BPaL and RHZE was associated with significantly fewer relapses in C3HeB/FeJ mice compared to addition of rapamycin and, in RHZE-treated mice, resulted in fewer relapses than RHZE alone. Therefore, CC214-2 and related mTOR kinase inhibitors may be more effective candidates for HDT than rapamycin analogs and may have the potential to shorten the duration of TB treatment.

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.